BRAF Melanoma Research Articles

BRAF as a positive predictive biomarker: Focus on lung cancer and melanoma patients.

In the era of personalized medicine, BRAF mutational assessment is mandatory in advanced-stage melanoma and non-small cell lung cancer (NSCLC) patients. The identification of actionable mutations is crucial for the adequate management of these patients. To date various drugs have been implemented in clinical practice. Similarly, various methods may be adopted for the identification of BRAF mutations. Here, we briefly review the current literature on BRAF in melanoma and NSCLC, focusing attention in particular on the different methods and drugs adopted in these patients. In addition, an overview of the real-world practice in different Italian laboratories with high expertise in molecular predictive pathology testing is provided.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Five-Year Outcomes With Nivolumab in Patients With Wild-Type BRAF Advanced Melanoma

PURPOSEThe CheckMate 066 trial investigated nivolumab monotherapy as first-line treatment for patients with previously untreated BRAF wild-type advanced melanoma. Five-year results are presented herein.PATIENTS AND METHODSIn this multicenter, double-blind, phase III study, 418 patients with previously untreated, unresectable, stage III/IV, wild-type BRAF melanoma were randomly assigned 1:1 to receive nivolumab 3 mg/kg every 2 weeks or dacarbazine 1,000 mg/m2 every 3 weeks. The primary end point was overall survival (OS), and secondary end points included progression-free survival (PFS), objective response rate (ORR), and safety.RESULTSPatients were followed for a minimum of 60 months from the last patient randomly assigned (median follow-up, 32.0 months for nivolumab and 10.9 months for dacarbazine). Five-year OS rates were 39% with nivolumab and 17% with dacarbazine; PFS rates were 28% and 3%, respectively. Five-year OS was 38% in patients randomly assigned to dacarbazine who had subsequent therapy, including nivolumab (n = 37). ORR was 42% with nivolumab and 14% with dacarbazine; among patients alive at 5 years, ORR was 81% and 39%, respectively. Of 42 patients treated with nivolumab who had a complete response (20%), 88% (37 of 42) were alive as of the 5-year analysis. Among 75 nivolumab-treated patients alive and evaluable at the 5-year analysis, 83% had not received subsequent therapy; 23% were still on study treatment, and 60% were treatment free. Safety analyses were similar to the 3-year report.CONCLUSIONResults from this 5-year analysis confirm the significant benefit of nivolumab over dacarbazine for all end points and add to the growing body of evidence supporting long-term survival with nivolumab mono-therapy. Survival is strongly associated with achieving a durable response, which can be maintained after treatment discontinuation, even without subsequent systemic therapies.

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.

MiR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators

BackgroundTargeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance.MethodsThe miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy.ResultsmiR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor.ConclusionsDeregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment.4ZiY3nqT8xQ_yvPXVejWjwVideo Graphical

Essential Oils and Their Main Chemical Components: The Past 20 Years of Preclinical Studies in Melanoma.

Simple SummaryIn the last years, targeted therapy and immunotherapy modified the landscape for metastatic melanoma treatment. These therapeutic approaches led to an impressive improvement in patients overall survival. Unfortunately, the emergence of drug resistance and side effects occurring during therapy strongly limit the long-term efficacy of such treatments. Several preclinical studies demonstrate the efficacy of essential oils as antitumoral agents, and clinical trials support their use to reduce side effects emerging during therapy. In this review we have summarized studies describing the molecular mechanism through which essential oils induce in vitro and in vivo cell death in melanoma models. We also pointed to clinical trials investigating the use of essential oils in reducing the side effects experienced by cancer patients or those undergoing anticancer therapy. From this review emerged that further studies are necessary to validate the effectiveness of essential oils for the management of melanoma.The last two decades have seen the development of effective therapies, which have saved the lives of a large number of melanoma patients. However, therapeutic options are still limited for patients without BRAF mutations or in relapse from current treatments, and severe side effects often occur during therapy. Thus, additional insights to improve treatment efficacy with the aim to decrease the likelihood of chemoresistance, as well as reducing side effects of current therapies, are required. Natural products offer great opportunities for the discovery of antineoplastic drugs, and still represent a useful source of novel molecules. Among them, essential oils, representing the volatile fraction of aromatic plants, are always being actively investigated by several research groups and show promising biological activities for their use as complementary or alternative medicine for several diseases, including cancer. In this review, we focused on studies reporting the mechanism through which essential oils exert antitumor action in preclinical wild type or mutant BRAF melanoma models. We also discussed the latest use of essential oils in improving cancer patients’ quality of life. As evidenced by the many studies listed in this review, through their effect on apoptosis and tumor progression-associated properties, essential oils can therefore be considered as potential natural pharmaceutical resources for cancer management.

Activation of CD8+ T Cells Contributes to Antitumor Effects of CDK4/6 Inhibitors plus MEK Inhibitors.

Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients. Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8+ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8+ T cells or blockade of the CD137 ligand-receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo Together, our data outline an antitumor immune-based mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.

Abstract 1095: Comprehensive pan-cancer analyses of RAS genomic diversity

Abstract The RAS family of small GTPases represents the most commonly activated oncogenes and mutations in KRAS, NRAS and HRAS are found in approximately one third of all human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that have RAS-dependent co-mutation frequencies in an unbiased pan-cancer manner, we analyzed targeted next-generation sequence data from the AACR Project GENIE Registry (GENIE version 6.1-public). Our analyses utilized 644,757 mutations from 64,217 tumors in 97 cancer types. A Bayesian hierarchical model was implemented to estimate cancer-specific prevalence of RAS and non-RAS somatic mutations. To evaluate RAS co-mutations, we compared the observed co-mutation frequencies of RAS and non-RAS somatic mutations to posterior predictive distributions of their expected co-mutation frequencies under independence. The prevalence of RAS codon 12, 13 and 61 mutations varied among cancer types, ranging from 77.7% in pancreatic cancer-PAC, 44.8% in colorectal cancer-CRC, 31.2% in non-small cell lung cancer-NSCLC, 1.1% in breast cancer, and 0.5% in mesotheliomas. A cancer type-specific distribution was observed, such that KRAS was frequently mutated in >25% of the tumors, NRAS was frequently mutated in melanoma, hematologic malignancies and thyroid cancer, while HRAS was overall less frequently mutated. KRAS codon 12 mutations predominated in pancreatic cancers (69.8%), GI cancers (30.9-48.5%), NSCLC (26.3%), endometrial cancer (14.7%) and germ cell tumors (11.6%). KRAS G12C mutations were predominantly seen in NSCLC (12.4%, 95% CI: 11.8-13.1%) with lower frequencies in GI cancers (3.2% in CRC). RAS mutations co-occurred with mutations in STK11, ATM, KEAP1, RBM10 and EPHA5 in NSCLC, TERT in melanoma and PIK3CA in CRC. Patterns of mutual exclusivity emerged between RAS mutations and BRAF in melanoma, thyroid and NSCLC, EGFR and RB1 in NSCLC, GNA11 and GNAQ in melanoma, FGFR3 in bladder cancer, MEN1 in PAC, RNF43 in CRC, BAP1 in melanoma and BAP1 and TERT2 in hepatobiliary carcinoma. A cancer type-specific co-occurrence pattern was noted for RAS and TP53 genomic alterations, with a significant enrichment in TP53 mutations in RAS-mutant PAC but a significant under-representation of TP53 mutations in RAS-mutant endometrial cancer, NSCLC, ovarian cancer and CRC. Analysis of codon specific RAS variants revealed additional co-mutation patterns, including an enrichment in NTRK3 mutations in KRAS G12C-mutant NSCLC and mutual exclusivity between KRAS G12C mutations and EGFR and BRAF in NSCLC. The tissue-distinct differential enrichment in RAS mutations and co-mutation patterns may be reflective of selection of oncogenic RAS signaling under the tumor type-specific modifying effect of other driver mutations. The RAS genetic architecture points to tissue-specific therapeutic vulnerabilities that may be translated to targeted treatment strategies. Citation Format: Robert Scharpf, Gregory Riely, Mark Awad, Michele Lenoue-Newton, Biagio Ricciuti, Julia Rudolph, Leon Raskin, Andrew Park, Jocelyn Lee, Christine Lovly, Valsamo Anagnostou. Comprehensive pan-cancer analyses of RAS genomic diversity [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 1095.

Abstract 2979: The effect of interferon gamma on melanoma progression under MAPK inhibitor therapy

Abstract About half of metastatic cutaneous melanoma harbor a mutation in the BRAF protein, causing constitutive activation of the MAPK pathway. Currently, patients with such mutation are treated with MAPK pathway inhibitors (MAPKi) targeting the mutant BRAF and its downstream MEK proteins. However, within a span of 6-12 months, these patients will relapse, and their tumors will acquire resistance to this treatment. In this project, we aim to understand the role of IFN gamma (IFNg) to help us determine if the resistance phenotype differs between cells exposed with MAPKi alone versus cells treated with MAPKi and IFNg. Previous literature has reported on the development of a dedifferentiated resistance phenotype which was characterized by a downregulation of MITF and an upregulation of AXL proteins. However, the patient-derived transcriptomic data comparing paired tumors pre- and post-MAPKi therapy did not show such changes in MITF or AXL levels. We posit that the discrepancy may be because most of the earlier studies on MAPKi resistance were done in vitro or immune-suppressed in vivo models. Melanoma biopsies from patients responding to MAPKi treatment showed a substantial increase in the tumor-infiltrating T cell and other immune cells. One of the most dominant immune factors produced by the T cells is the interferon gamma (IFNg). Intriguingly, our data show that IFNg, in the presence of MAPKi, can suppress the dedifferentiation switch that is induced by MAPKi-alone. Specifically, through serial western blots, we discovered that MAPKi and IFNg reduced the previously reported AXL upregulation and MITF downregulation in four different mutant BRAF melanoma lines. RNA sequencing from the same time points revealed that melanoma treated with MAPKi and IFNg upregulate IFN-stimulated genes that are typically expressed by immune cells; this leads us to believe that some novel transcriptomic programs have been activated by the combination of MAPKi and IFNg. By solely examining one immune factor, IFNg, we discovered one potential explanation for the discrepancy in the prevalence of MAPKi-induced dedifferentiation phenotype in the patient-derived melanoma samples. We are in the midst of performing drug screening experiments on MAPKi and IFNg-resistant melanoma lines in order to discover novel therapeutic vulnerabilities that were not observed with MAPKi-alone. Finally, by taking into account the secreted factors in the tumor microenvironment, our study can provide a more accurate depiction of MAPKi resistance evolution in mutant BRAF melanoma patients. Citation Format: Kevin Chen, Rachel McMillan, Mona Chatrizeh, Willy Hugo. The effect of interferon gamma on melanoma progression under MAPK inhibitor therapy [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 2979.

NTRK gene fusions in melanoma: detection, prevalence and potential therapeutic implications.

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) are known drivers of oncogenesis and also occur in melanoma, although very rarely. A particularly high incidence of NTRK gene fusions is reported in infantile fibrosarcoma (>90%) or the secretory type of breast cancer (>90%). Recently, larotrectinib (a tropomyosin receptor kinase [TRK] inhibitor) was approved, and we wondered whether TRK inhibitors might also be helpful for melanoma patients. We therefore screened the literature and obtained relevant results. NTRK fusions are relatively common in spitzoid melanoma, with a prevalence of 21-29% compared to <1% in cutaneous or mucosal melanoma and 2.5% in acral melanoma. It appears that fusion proteins are mutually exclusive for most common oncogenic drivers such as BRAF or NRAS. A further indicator of an increased probability of detecting NTRK-positive tumors could be a low mutation load. Since TRK inhibitors are already available for patients with NTRK fusions, the challenge will be to implement screening for NTRK gene fusions in clinical practice. A possible approach could be to screen BRAF, NRAS and KIT wild-type melanoma patients with next-generation sequencing as soon as they need systemic treatment or at the latest when they have no tumor control on checkpoint inhibitors.

BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases

The development of mutant BRAF inhibitors has improved the outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes resulting in complete tumor regression, the majority of melanomas become resistant. To study resistance to BRAF inhibition, we developed a novel mouse model of melanoma using a tetracycline/doxycycline-regulated system that permits control of mutant BRAF expression. Treatment with doxycycline leads to loss of mutant BRAF expression and tumor regression, but tumors recur after a prolonged period of response to treatment. Vemurafenib, encorafenib and dabrafenib induce cell cycle arrest and apoptosis in BRAF melanoma cell lines; however, a residual population of tumor cells survive. Comparing gene expression in human cell lines and mouse tumors can assist with the identification of novel mechanisms of resistance. Accordingly, we conducted RNA sequencing analysis and immunoblotting on untreated and doxycycline-treated dormant mouse melanomas and human mutant BRAF melanoma cell lines treated with 2 μM vemurafenib for 20 days. We found conserved expression changes in histone methyltransferase genes ASH2, EZH2, PRMT5, SUV39H1, SUV39H2, and SYMD2 in P-ERK low, p-38 high melanoma cells following prolonged BRAF inhibition. Quantitative mass spectrometry, determined a corresponding reduction in histone Lys9 and Lys27 methylation and increase in Lys36 methylation in melanoma cell lines treated with 2 μM vemurafenib for 20 days. Thus, these changes as are part of the initiate response to BRAF inhibition and likely contribute to the survival of melanoma cells.

Establishing the six-month resource utilization and cost-of-care for the treatment of first-line metastatic BRAF (V600) melanoma with combination BRAF and MEK inhibitors.

e19396 Background: Comparative data on cancer therapy health care resource utilization (HCRU) and associated cost will be helpful as value-based healthcare moves forward. BRAF &amp; MEK inhibitor combinations are considered first-line treatment for BRAF (V-600) metastatic melanoma (MM), although head-to-head trials are lacking. We aimed to establish the real-world HCRU and 6-month (mo) cost-of-care in V-600 MM treated with BRAF &amp; MEK inhibitor therapy. Methods: A single data team in 2018 performed a multicenter, retrospective chart audit of adult patients with BRAF V-600 MM. Four institutions from across the US with patients who had received either dabrafenib + Trametinib (DT) or vemurafenib + cobimetinib (VC) were enrolled. In the most recent 12 mo period, data was captured from the start of therapy for 6 mo or until therapy was stopped. Dose change or stoppage was accessed for cause (toxicity, disease, death, other). Variables included hospitalization, emergency room (ER), all clinic visits (routine + extra), scans, labs, and treatment drug (AWP). Medicare reimbursed rates were applied for cost estimates. Utilization and costs were measured on per patient per month (PPPM) bases and the total cost over 6 mo for each combination. Results: Of the 42 patients included, 34 and 8 were initiated on DT and VC, respectively. Proportions of patients with extra clinic visits and hospital admissions were 79%, 15% and 75%, 13%, respectively for DT and VC. PPPM hospitalization was the lowest among the resources utilized 0.24 for DT and 0.17 for VC. A higher proportion of VC patients (75%) had a dose reduction due to drug toxicity compared with 29% of patients treated with DT (P &lt; 0.05). Discontinuation rates were the same between both combinations (0.26). 32 patients had completed 6 mo of treatment (26 DT and 6 VC). For those DT, the mean total costs including drug and the mean monthly total costs were $157,253 and $26,209 compared to $107,240 and $17,873 for VC, respectively. The mean total costs for hospitalization were $10,562 for DT and $7,456 for VC. The mean total costs for the drug were $145,012 for DT and $97,924 for VC. Conclusions: The 6-month total cost-of-care for the treatment of first-line V-600 MM with DT was $157,253 and $107,240 for VC, mostly attributable to drug cost. In a value-based healthcare system, total 6-month cost-of-care may help distinguish between equally effective regimens.

Targeted therapies in melanoma beyond BRAF: targeting NRAS-mutated and KIT-mutated melanoma.

Melanoma treatment have been revolutionized since 2010 by the development of immune checkpoint inhibitors, and, for BRAF-mutated melanoma, targeted therapies based on BRAF and MEK inhibitors, which is a model of effective targeted therapy in cancer. However, patients with BRAF wild type cannot benefit for such treatments. In this review, we will focus on the current clinical development of targeted therapies beyond BRAF, in NRAS-mutated and KIT-altered melanoma. In NRAS-mutated melanoma, targeted therapies based on MEK inhibition are being developed as monotherapy or in combination with MAPK, PI3K or CDK4/6 inhibitor. Targeted therapies of KIT-altered melanoma patients is based in KIT inhibitor (mostly imatinib, nilotinib), although for both melanoma subtypes, results are for now disappointing as compared with BRAF and MEK inhibitors in BRAF-mutated melanoma. Combined therapeutic targeted strategies are awaited in NRAS-mutated and KIT-altered melanoma and could provide additional benefit.

Melanoma Persister Cells are Tolerant to BRAF/MEK Inhibitors Via ACOX1-Mediated Fatty Acid Oxidation

Emerging evidences indicate that non-mutational drug tolerance mechanisms underlie the survival of residual cancer “persister” cells. Here, we find that BRAF(V600E)-mutant melanoma persister cells tolerant to BRAF/MEK inhibitors switch their metabolism from glycolysis to oxidative respiration supported by peroxisomal fatty acid β-oxidation (FAO) that is transcriptionally regulated by PPARα. Knockdown of the key peroxisomal FAO enzyme, ACOX1, as well as treatment with the peroxisomal FAO inhibitor thioridazine specifically suppresses the oxidative respiration of persister cells and significantly decreased their emergence. Consistently, combination treatment of BRAF/MEK inhibitors with thioridazine in human melanoma-bearing mice results in a durable anti-tumor response. In BRAF(V600E) melanoma samples from patients treated with BRAF/MEK inhibitors, higher baseline expression of FAO-related genes and PPARα correlate with patients’ outcomes. These results pave the way for a new metabolic strategy to overcome drug resistance.

Aldo-keto reductases protect metastatic melanoma from ER stress-independent ferroptosis

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients. Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use. Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells. Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level. Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution. At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance. However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution. Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process. However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis. Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

RARE-18. CLINICAL EXPERIENCE OF DABRAFENIB IN COMBINATION WITH TRAMETINIB TREATMENT FOR ANAPLASTIC PXA CASES

Abstract Anaplastic PXA is classed as grade III tumor because of its aggressive behavior in 2016 WHO classification. Typical treatment of PXA is gross total resection followed by radiotherapy and cytotoxic chemotherapy at recurrence. Despite aggressive behavior of anaplastic PXA, there is some evidence for the use of BRAF inhibitors in BRAF V600 mutant PXA. We report two cases of successful treatment with BRAF-MEK inhibitor combination therapy (dabrafenib plus trametinib). A 19 year-old man initially diagnosed as PXA for rt temporal tumor. Subtotal removal and following 30Gy radiation was performed. After eight years, the recurrent tumor was removed subtotally and diagnosed as anaplastic PXA. Residual tumor and distant metastasis region were completely disappeared by Dabrafenib-Trametinib therapy. Tumor was diagnosed as BRAF V600 mutant and TERT wt. A 30 year-old female initially diagnosed as PXA for rt temporal cystic tumor. She was treated with subtotal removal followed by stereotactic radiosurgery. Instead of Gd-total removal, tumor recurred six times. Chemotherapy with alkyl-agent did not respond. Tumor at first surgery was diagnosed as grade II PXA. Tissues collected after SRS showed malignant feature and diagnosed as anaplastic PXA. All five specimens showed BRAF V600 mutation and additional TERT mutation was confirmed after SRS treatment. She showed paraparesis because of thoracic metastases then Dabrafenib-Trametinib therapy was started. Thoracic tumor showed PR and no remarkable recurrence in intracranial region for more than eight months, so far. BRAF and MEK inhibitor combination treatment are thought to have potential for BRAF V600 mutant NSCLC and melanoma. Anaplastic PXA with BRAF V600 mutation could be another target for the treatment.

Assessing melanoma BRAF status through ddPCR of cfDNA

Abstract Background Targeted therapy has revolutionised the systemic treatment of melanoma. Patients with stage III/IV melanoma with a mutation in BRAF are treated with BRAF inhibitors, with BRAF mutation status being assessed by tissue analysis. Cell-free DNA (cfDNA) released from tumours provides a method for detecting BRAF mutations. This project analysed BRAF status in cfDNA through digital droplet PCR (ddPCR). We aimed to assess the relationship between cfDNA BRAF status and disease relapse and progression. Methods Plasma from 100 patients with active or recently resected melanoma was obtained during previous work. 85 samples had cfDNA extracted. Tissue BRAF status was known for 57 samples. cfDNA was extracted from 1-2ml plasma with the QIAamp circulating nucleic acid kit (QIAGEN®) following manufacturer protocol, eluting cfDNA into 100µL. cfDNA was quantified with SYBR green quantitative real-time PCR (Life Technologies), based on an 87bp GAPDH gene amplicon. ddPCR™ was performed using the Bio-Rad QX200 Droplet Generator™ and Droplet Reader as per manufacturer protocol. Analysis was performed with Bio-Rad QuantaSoft Version 1.7.4. Results Median yield of cfDNA was 1.97ng/ml. This correlated well with other yields from this sample set (Pearson’s r = 0.6687, p 10,000 droplets and were included for analysis. 12 samples contained BRAF mutant positive droplets. Concordance rate between tissue BRAF status and the presence/absence of cfDNA BRAF mutant droplets was 74%. 7/18 tissue BRAF mutant samples were cfDNA BRAF mutant positive, in contrast to 2/32 tissue BRAF wild-type samples. The presence of BRAF mutant positive droplets in cfDNA was significantly different between tissue BRAF mutant and wild-type groups (p = 0.004). The presence of BRAF mutant droplets did not confer any significant difference in rate of relapse (p = 0.758) or mortality rate (p = 0.654). Conclusions A very low volume of cfDNA can be used to detect BRAF mutations in patients with melanoma through ddPCR. Having used small yields of cfDNA, it is possible that some samples fell below the limit of detection for ddPCR. Longitudinal study is warranted to monitor cfDNA BRAF status, and whether this correlates with relapse and disease progression. Legal entity responsible for the study The authors. Funding Weston Park Cancer Charity. Disclosure All authors have declared no conflicts of interest.

Biomarkers of Targeted Therapy and Immuno-Oncology in Cancers Metastatic to the Breast.

The breast is a rare site for metastases, and their molecular characteristics have not been studied yet. Intrinsic molecular genetics, cancer characteristics, and breast tissue immune responses in diverse metastases to the breast have not been previously studied. We identified 64 patients with cancers metastatic to the breast: 51 carcinomas and 13 melanomas. Programmed death ligand 1 (PD-L1), steroid receptors, and HER2/neu expressions were evaluated using immunohistochemistry. Gene sequencing, copy number alterations, microsatellite instability, and tumor mutational burden were performed using next-generation sequencing platforms. The 3 most common primary sites for metastatic carcinomas were lung (37%), ovary (29%), and fallopian tubes/peritoneum (14%). TP53 mutations were commonly (50%) observed among the carcinoma cases, while other mutations were characteristic for the primary cancers (VHL in renal, BRCA1 in the fallopian tube, and BRAF in melanomas). High tumor mutational burden was detected in 5/14 carcinomas and 3/7 melanomas. Tumor cell PD-L1 expression was detected in 6 carcinomas, but not in any of the melanomas, whereas immune cells' expression of PD-L1 was seen in 17 carcinomas and 6 melanomas. Estrogen receptor status was positive in 13/49 carcinomas including 12 adenocarcinomas originating from the ovary and fallopian tube or peritoneum and 1 duodenal neuroendocrine carcinoma. No carcinoma was HER2/neu positive. Intrinsic genetic characteristics of the metastases to the breast followed the pattern commonly seen in primary tumors. Biomarkers of potential benefit to immune checkpoint inhibition therapy were limited to PD-L1-positive non-small cell lung cancer. No common characteristics of the heterogeneous group of tumor metastases to this organ were identified.

Dynamic and unpredictable changes in mutant allele fractions of BRAF and NRAS during visceral progression of cutaneous malignant melanoma

BackgroundData indicate that primary cutaneous melanomas are characterized by clonal heterogeneity associated with oncogenic drivers. Less data are available on the clonal changes occurring during melanoma progression. We therefore wished to analyse these changes in skin melanomas in common sites of visceral metastases as compared to the primary tumor.MethodsAn autopsy cohort of 50 patients with BRAF- and NRAS-mutant cutaneous metastatic melanomas including 139 visceral metastases was analysed for mutant allele fractions (MAF), determined by pyrosequencing and corrected for tumor/normal ratio. MAF levels were also classified as high (> 40%), medium (15–40%) or low (< 15%).ResultsContrary to NRAS mutant cases, in BRAF-mutant melanomas MAFs were found to be significantly increased in visceral metastases compared to the primary due to the significantly higher levels in lung-, adrenal gland-, intestinal- and kidney metastases. The incidence of the three MAF variants in BRAF-mutant primaries was similar, whereas the high MAF cases were found to be increased in metastases. On the other hand, medium MAF levels were more common in case of NRAS-mutant tumors. Only 31.3% of BRAF mutant- and 50% of NRAS mutant cases maintained the MAF profile of the primary in metastasis. In the majority of multiple metastatic tumors, (BRAF:71.8%, NRAS:75%) metastases were relatively homogeneous regarding MAF. However, in 6/32(18.7%) of BRAF mutant cases low MAF primaries switched to high MAF in metastases. In heterogeneous BRAF mutant metastatic cases low to high or high to low MAF conversions occurred in a further 4/32(12.5%) cases in individual metastases as compared to the primary tumors. At lower frequency, in NRAS mutant tumor such changes also observed (2/12,16.7%).ConclusionWe provided evidence for the selection of BRAF-mutant melanoma cells during metastatic progression to the lung, intestine, adrenal gland and kidney. Our findings suggest that in visceral metastases of malignant melanoma BRAF- or NRAS-MAFs are rather heterogeneous and cannot be predicted from data of the primary tumor. These data may have clinical significance when using targeted therapies.

Abstract 928: Preventing melanoma from healing itself after therapy-induced immune attack through IPRES blockade

Abstract Over the past few years, therapeutics development for melanoma has led the oncology field, including the development of mutant BRAF-targeted therapies (e.g., BRAF inhibitor (BRAFi)) and immune checkpoint blockade therapies (e.g., anti-PD-1 or aPD-1, aPD-L1, etc). Based on the complementary qualities of aPD-1/L1 immunotherapy (long duration of response) and MAPKi (high rate of initial response), there are currently clinical trials testing the combination of the two. However, despite a more durable tumor suppression, resistance still eventually develops in patients treated by the combination (ref: KEYNOTE-022). My earlier study reported significant intra-patient and intra-tumoral heterogeneity which affect patient-specific responses to therapy. Furthermore, I also observed complex transcriptomic and tumor microenvironment reprogramming associated with resistance to targeted- or immuno-therapies in melanoma. However, the increased immune/stromal cells in the tumor under therapy has prevented us from definitively accounting the source of such reprogramming. To address this need, I utilized the novel single cell transcriptomic analysis to discover therapy-response-associated gene programs across different types of cells within a tumor. In murine mutant BRAF melanoma YUMM1.7, regressing/responding tumors on the 6th day of BRAFi treatment showed significant infiltration of the T cells and other immune cell population and, by the 15th day, the tumor showed activation of IPRES related processes. Combination of BRAFi and aPD-L1 in YUMM1.7 increased the proportion of lymphoid population in the tumor on the 7th day but only incrementally delayed the development of BRAFi resistance. Inhibition of IPRES-related processes (anti-VEGF and/or anti-TGFB) further delayed BRAFi resistance but no complete responses were observed. However, when we employed a UV-mutated subline of the same model, named the YUMMER (Bosenberg lab, Yale), combination of BRAFi, aPD-L1 and IPRES inhibition started to result in durable complete responses in our mouse model. We are now in midst of analyzing the differential response that we observed with this model; our current working hypothesis is that YUMMER induces higher level of antigen-specific antitumor response, which is augmented by the addition of immune checkpoint blockade and IPRES suppression. The results from our study will eventually pave the way to a novel therapeutic combination to overcome melanoma. Citation Format: Marco Piva, Lu Sun, Gatien Moriceau, Zhentao Yang, Antoni Ribas, Willy Hugo, Roger S. Lo. Preventing melanoma from healing itself after therapy-induced immune attack through IPRES blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 928.