BRAFV600-mutated Metastatic Melanoma Research Articles

Theragnostic significance of tumor-infiltrating lymphocytes and Ki67 in BRAFV600-mutant metastatic melanoma (BRIM-3 trial)

We conducted a retrospective tumor tissue analysis as part of the BRIM3 trial to evaluate the theragnostic significance of tumor-infiltrating lymphocytes (TILs) and melanoma cell proliferation. Using manual semi-quantitative analyses, we assessed the density of TILs by pathology review of tissue sections stained with hematoxylin and eosin (H&E TIL score) and by immunohistochemistry (IHC) with an anti–CD8 antibody (CD8 TIL score); also, the melanoma cell proliferation by IHC with an anti–Ki67 antibody. Three hundred and fifty-three, 280, and 172 patients’ tumor tissue samples were available for H&E, CD8, and Ki67 IHC analysis, respectively. There was no association between high (2+, 3+) peritumoral and intratumoral H&E and/or TIL CD8 score or high Ki67 proliferation index (>15%) with serum LDH level and stage IV melanoma. Neither high Ki67 proliferation, nor high peritumoral and/or intratumoral TIL score was significantly associated with objective antitumor response in any treatment arm. High intratumoral and high peritumoral CD8 TIL score was significantly associated with progression-free survival (PFS) only in DTIC-treated patients (P = 0.002 and 0.037, respectively); in vemurafenib-treated patients, high intratumoral and/or peritumoral CD8 TIL score was not significant (log-rank P = 0.053 and 0.062, respectively). Nevertheless, a high peritumoral CD8 TIL score was a significant predictor of PFS and overall survival after adjustment for age, sex, serum LDH, ECOG performance status, and treatment arm in a Cox regression model. Vemurafenib does not only benefit patients bearing brisk TILs; even vemurafenib-treated patients with absent and/or non–brisk TILs tend to have longer PFS compared to DTIC-treated patients with brisk TILs. High peritumoral CD8 TIL score is a favorable prognostic factor independent of well-established AJCC staging factors.

STARBOARD: encorafenib+binimetinib+ pembrolizumab for first-line metastatic/unresectable BRAF V600-mutant melanoma.

Despite the significant progress in the treatment of unresectable or metastatic BRAF V600-mutant melanoma, there remains two primary treatment options: targeted therapy and immunotherapy. Targeted therapy or immunotherapy alone is associated with efficacy limitations including efficacy limited to select patient subsets. With separate mechanisms of action and different response patterns, the combination of targeted agents and immunotherapy to treat patients with BRAF V600-mutant melanoma may further improve patient outcomes. Current treatment guidelines recommend treatment with targeted agents alone, immunotherapy, or the combination of targeted agents and immunotherapy. The randomized, double-blind STARBOARD trial aims to evaluate efficacy and safety of encorafenib, binimetinib and pembrolizumab in treatment-naive patients with metastatic or unresectable locally advanced BRAF V600-mutant melanoma in comparison to pembrolizumab.

DREAMseq (Doublet, Randomized Evaluation in Advanced Melanoma Sequencing): A phase III trial—ECOG-ACRIN EA6134.

356154 Background: Combinations of immune checkpoint inhibitors (CPI) blocking PD-1 and CTLA-4 or BRAF/MEK inhibitors have both shown significant antitumor efficacy and overall survival (OS) benefit in patients (pts) with BRAFV600-mutant metastatic melanoma (MM), leading to broad regulatory approval. Little prospective data exists to guide the choice of one over the other as initial therapy or the preferred treatment sequence in this population. The DREAMseq Trial was designed to compare the efficacy and toxicity of the sequence of nivolumab/ipilimumab (N/I) followed by dabrafenib/trametinib (D/T) to the converse sequence.Methods: Eligible pts with treatment-naive BRAFV600-mutant MM were stratified by ECOG Performance Status (PS) 0 or 1 and LDH level and randomized 1:1 to receive Step 1 with either N/I (Arm A) or D/T (Arm B) and at disease progression (PD) were enrolled in Step 2 receiving the alternate therapy, D/T (Arm C) or N/I (Arm D), respectively. Pts received N (1mg/kg)/I (3 mg/kg) q3 wks x 4 doses followed by N 240 IV q2 wks for up to 72 wks (Arms A and D) or D 150 mg po BID and T 2 mg po qD until PD (Arms B and C). In 2019, investigators were given the option to use alternate induction dosing of N (3mg/kg)/I (1 mg/kg) q3 wks x 4 doses for Arms A and D. Cycles were every 6 wks and imaging was obtained at baseline and q12 wks on each arm. Primary endpoint was 2-year OS. At the 4th Interim Analysis with 59% of pts being 2 yrs from enrollment, the DSMC and NCI CTEP recommended halting accrual and releasing the data.Results: Beginning 7/2015, 265 out of a proposed 300 pts were enrolled (133 Arm A and 132 Arm B). Median age was 61 (25-85) and 63% were male. Demographics for Arm A and B were balanced with 67% PS 0 and 60% with normal LDH. As of 7/16/21, at a median follow-up of 27.7 mos, 27 pts had switched to Arm C and 46 to Arm D. Overall Grade 3+ toxicity was 60% in Arm A and 52% in Arm B. Grade 5 treatment-related AEs included 2 on Arm A and 1 on Arm C. ORR to date is: Arm A 46% (52/113), Arm B 43% (49/114), Arm C 48% (11/23) and Arm D 30% (8/27). 37/42 assessed pts in Arm A and 19/37 in Arm B remain in response. Median DOR: Arm A- Not reached; Arm B-12.7 mos (95% CI: 8.2, -) (p <0.001). There were 100 deaths (Arm A to C- 38/Arm B to D- 62). 2-yr OS rate for those starting with Arm A was 72% (95% CI: 62-81%) and for Arm B 52% (95% CI: 42-62%) (log-rank p= 0.0095). PFS showed a trend in favor of Arm A (log-rank p=0.054). Both the PFS and OS curves show a biphasic pattern with Arm B being above Arm A until 6 and 10 mos, respectively. For the 115 pts with documented progression on Step 1 (Arm A-44/Arm B-71), 60 (52%) had registered for Step 2. The principal reason for not enrolling on Step 2 was death from PD within 6 mos (Arm A:15/23; Arm B: 25/32). Conclusions: For pts with advanced BRAFV600-mutant MM, the treatment sequence beginning with the CPI combination of N/I resulted in superior OS, which became evident at 10 mos, with longer Step 1 DOR and more ongoing responses than the treatment sequence beginning with D/T. Clinical trial information: NCT02224781.

Évolution du statut braf dans le melanome : mythe ou Réalité ?

Knowledge of the BRAF mutational status has become essential for melanoma therapeutic management. B-Raf inhibitors are associated with significant overall survival in patients with BRAFV600-mutated metastatic melanoma. Although the BRAF mutation appears to be an early and driver mutation, some authors hypothesized that its expression was not stable during melanoma progression, suggesting a molecular heterogeneity. This argument is often used to explain discrepancy in molecular status among patients with melanoma, discrepancies that we occasionally met during our practice. We retrospectively compared BRAF mutational status on matched melanoma samples (primary & metastatic lesions), thus 150 samples from 56 patients were analysed through immunohistochemistry anti-BRAF, PCR-HRM and Sanger sequencing, Next Generation Sequencing (NGS) and digital PCR. Seven cases presented an apparent tumor heterogeneity. The analysis of these discrepancies by a technique of increasing sensitivity made it possible to identify 1 false-negative result for the immunohistochemistry, 1 false-negative result for the NGS sequencing and 5 (3%) false-negative results by PCR-HRM SANGER. Our results are consistent with the most recent data, demonstrating the stability of the BRAF mutation during the course of melanoma. Immunohistochemistry shows excellent sensitivity for detecting the main BRAF mutation. In our study, the mutational heterogeneity was actually misleading, a result of imperfect sensitivity of some older molecular approaches.

BRAF and MEK Inhibitors Affect Dendritic-Cell Maturation and T-Cell Stimulation.

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAFV600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8+ and CD4+ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8+ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.

Retrospective Chart Review of Dabrafenib Plus Trametinib in Patients with Metastatic BRAF V600-Mutant Melanoma Treated in the Individual Patient Program (DESCRIBE Italy).

BackgroundReal-world data on extended follow-up of patients with BRAF V600-mutant metastatic melanoma are limited. We investigated dabrafenib plus trametinib (dab + tram) outside of a clinical trial setting (Individual Patient Program; DESCRIBE Italy).ObjectiveTo describe the baseline features, treatment patterns, efficacy, and safety outcomes in patients with BRAF V600-mutant unresectable or metastatic melanoma who had received dab + tram as part of the Managed Access Program (MAP) in Italy.Patients and methodsAn observational, retrospective chart review was conducted in Italian patients with BRAF V600-mutant unresectable stage III/IV melanoma receiving dab + tram as part of the MAP. Baseline features, treatment patterns, efficacy, and safety outcomes were evaluated.ResultsOverall, 499 patients were included in this analysis. BRAF V600E mutation was seen in 81.4% of patients. Overall response rate achieved in 243 of the 390 evaluable patients was 62.3% (95% CI 57.5–67.1). Median progression-free survival (PFS) was 9.3 months (95% CI 8.6–10.6). Subgroup analyses revealed that patients with normal lactate dehydrogenase (LDH) and ≤ three metastatic sites without brain metastases at baseline had better outcomes. With normal LDH at baseline, median PFS for patients with one or two metastatic sites other than cerebral was 18 months. No new safety signals were observed. Treatment was permanently discontinued because of treatment-emergent adverse events (TEAEs) in 9.2% of patients, and pyrexia (27.3%) was the most common TEAE, with a lower incidence than that in the phase 3 studies of dab + tram.ConclusionTreatment of BRAF V600E-mutant metastatic melanoma with dab + tram in the real-world setting was effective and safe, including the unselected population with several patients having a high tumor burden – concordant with the results of the pivotal phase 3 studies of dab + tram.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11523-021-00850-1.

1045P Comparison between first-line target therapy and immunotherapy in different prognostic categories of BRAF mutant metastatic melanoma patients

BRAF and MEK inhibitors target therapies (TT) and AntiPD1 immunotherapies (IT) are available first-line treatments for BRAF v600 mutant metastatic melanoma patients (pts). ECOG PS (E), baseline LDH (L), baseline number of metastatic sites (N)are well known clinical prognostic markers that identify different prognostic categories of pts. Direct comparison between first-line TT and IT in different prognostic categories could help in first-line treatment decision.

Plasma proteome alterations by MAPK inhibitors in BRAFV600-mutated metastatic cutaneous melanoma

Approximately half of metastatic cutaneous melanomas (CM) harbor a mutation in the BRAF protooncogene, upregulating the mitogen-activated protein kinase (MAPK)-pathway. The development of inhibitors targeting the MAPK pathway (MAPKi), i.e., BRAF- and MEK-inhibitors (BRAFi and MEKi), have substantially improved the survival in BRAFV600E/K-mutated stage IV metastatic CM. However, most patients develop resistance to treatment and no predictive biomarkers exist in practice. This study aimed at discovering plasma proteome changes during treatment MAPKi in patients with metastatic (stage IV) CM. Matched plasma samples before (pre) and during treatment (trm) from 23 patients with stage IV CM, treated with BRAF-inhibitors (BRAFi) alone or BRAF- and MEK- inhibitors combined (BRAFi and MEKi), were collected and analyzed with targeted proteomics by proximity extension assays. Additionally, plasma from 9 patients treated with BRAFi and MEKi was analyzed with in-depth high-resolution isoelectric focusing liquid-chromatography mass-spectrometry proteomics. Alterations of plasma proteins involved in granzyme and interferon gamma pathways were detected in patients treated with BRAFi, and cell adhesion-, neutrophil degranulation-, and proteolysis pathways in patients treated with BRAFi and MEKi. Several proteins were associated with progression-free survival after MAPKi treatment. We show that the majority of the altered plasma proteins were traceable to BRAFV600E-mutant metastatic CM tissue at mRNA level in 154 patients from the TCGA, further strengthening their involvement in tumoral response to treatment. This wide screen of plasma proteins unravels proteins that may serve as predictive and/or prognostic biomarkers of MAPKi treatment, opening a window of opportunity for plasma biomarker discovery in MAPKi-treatment of BRAFV600-mutant metastatic CM.

Role of Tumor-Infiltrating B Cells in Clinical Outcome of Patients with Melanoma Treated With Dabrafenib Plus Trametinib.

Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients. There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type. Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells. We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening. Baseline cell-cycle gene expression signature was associated with progression-free survival (P = 0.007). Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months; 95% CI, 13.4-38.6 months). Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased MAPK activity and increased expression of immunosuppressive markers. B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib. As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection. Further validation in a larger patient cohort is needed.

Sarcoid-like reaction in a BRAF V600E-mutated metastatic melanoma patient during treatment with BRAF/MEK-targeted therapy.

Treatment with combined BRAF and MEK inhibition is widely accepted as a first-line treatment option for patients with advanced BRAF V600E mutant melanoma. It is generally well-tolerated and has limited side-effects. However, we report a case of a sarcoid-like syndrome induced by treatment with dabrafenib/trametinib (D/T) in a patient with stage IV-M1d melanoma. Sarcoid-like syndrome is a known side-effect of immune checkpoint-inhibition therapy but has only rarely been described in BRAF/MEK inhibition. However, recognizing this side-effect is important because of potential misinterpretation as progressive disease and influence on treatment. We describe a 48-year-old female patient who initially presented with solitary brain metastasis and diffuse lung lesions. She was treated with D/T to which she had an initial response in all lesions. One year later, new hilar and mediastinal lymphadenopathies were detected. Imaging was suggestive of the sarcoid-like syndrome. An endoscopic biopsy of the enlarged lymph node showed no melanoma cells. Treatment was continued. Three months later, the patient experienced a drop in hemoglobin, which prompted further investigations into possible occult intestinal metastasis. Video capsule examination revealed a metastatic lesion in the small intestine. A treatment switch to the combination of checkpoint inhibitors nivolumab and ipilimumab successfully treated both lung and small intestine lesions. After the third dose of this combination therapy, she developed an immune-related pneumonitis. Treatment with corticosteroids resolved the pneumonitis and decreased metabolism in the sarcoid-like syndrome. The treatment was not restarted afterward. She remains free of the disease up to today, 2.5 years after diagnosis.

A Retrospective Analysis of Dabrafenib and/or Dabrafenib Plus Trametinib Combination in Patients with Metastatic Melanoma to Characterize Patients with Long-Term Benefit in the Individual Patient Program (DESCRIBE III).

Simple SummaryCompassionate-use programs provide an opportunity to retrospectively evaluate the treatment patterns and clinical outcomes in a real-world setting to validate the results derived from controlled randomized clinical trials. The COMBI-d and COMBI-v studies established the superior efficacy of dabrafenib + trametinib (dab + tram) versus BRAF inhibitor monotherapy in patients with BRAF V600–mutant metastatic melanoma. In light of their five-year results demonstrating long-term benefit with first-line dab + tram, it is important to get a real-world perspective of the long-term treatment duration for dab + tram. DESCRIBE III was designed to retrospectively evaluate the impact of patient characteristics on the long-term outcomes of dab + tram in a real-world setting based on the duration of clinical benefit. Consistent with the findings from the pooled analysis of COMBI-d and COMBI-v, lower LDH level and <3 metastatic sites at baseline were associated with a longer duration of treatment benefit in a real-world setting.The dabrafenib plus trametinib (dab + tram) combination has demonstrated durable long-term efficacy in patients with BRAF V600–mutant metastatic melanoma. However, real-world data characterizing patients with long-term benefit are limited. DESCRIBE III was a global, observational, retrospective, chart review study in patients with unresectable or metastatic melanoma treated with dab monotherapy and/or dab + tram combination therapy as part of the Named Patient Program or Individual Patient Program. Overall, 509 patients were enrolled. Patients were categorized into three groups based on their observed treatment duration: long-term (on therapy ≥12 months), intermediate (on therapy ≥6 months and <12 months), and short-term (on therapy <6 months) duration of benefit. More patients in the short-term duration of benefit group had baseline characteristics associated with poor prognosis compared with the other two groups. Median lactate dehydrogenase (LDH) levels (368 U/L) at baseline were also higher in the short-term duration of benefit group. No new safety signals were identified. DESCRIBE III identified baseline characteristics associated with long-term benefit of dab + tram. Lower LDH level and <3 metastatic sites at baseline were associated with a longer duration of benefit, confirming that the findings from COMBI-d and COMBI-v are relevant to patients treated in a real-world setting.

Immune-Related Erythema Nodosum Mimicking in Transit Melanoma Metastasis on [18F]-FDG PET/CT.

Early detection of immune-related adverse events (irAEs) with immune checkpoint inhibitors (ICIs) is crucial, particularly when these are likely to mimic tumor progression, as well as sarcoid-like reactions. Here, we report the case of a 68-year woman, with a history of four primary cutaneous melanomas (thickest lesion with BRAF mutation removed from the left axilla 2 years before), who was diagnosed with BRAF V600E-mutant metastatic melanoma and treated by ICI targeting the PD-1 receptor. Follow-up whole-body positron emission tomography/computed tomography (PET/CT) using 18F-fluorodeoxyglucose ([18F]-FDG) was performed at 15 months, and FDG-avid subcutaneous nodules on her legs were detected. A biopsy from a lesion on her right leg was obtained, and histology strongly suggested erythema nodosum. Given the isolated nature of these lesions, the normal serum Angiotensin-Converting Enzyme and the context of ICI, an immune-related sarcoid-like reaction was retained as the most likely diagnosis. Recent literature in immune-oncology suggests that erythema nodosum could be directly related to ICI(s). Although erythema nodosum is a rare occurrence with imaging features overlapping with malignancy, it should be considered in the differential diagnosis of suspicious in-transit metastasis, especially when the patient is treated with ICIs and when lesions follow a bilateral distribution. In conclusion, nuclear medicine physicians should keep in mind this irAE when interpreting PET/CT scans in clinical practice in order to avoid false-positive findings.

Treatment combinations in BRAF-mutant metastatic melanoma.

Combinations of BRAF and MEK inhibitors are now the standard treatment in patients with BRAF V600-mutant metastatic melanoma. Three combination treatments with BRAF and MEK inhibitors are available for thetreatment of metastatic melanoma: vemurafenib + cobimetinib, dabrafenib + trametinib, encorafenib + binimetinib. These 3 combination treatments have distinct safety profiles as evidenced by the data from the phase III studies. For example, fever was observed more frequently in patients treated with dabrafenib and trametinib, photosensitivity in patients treated with vemurafenib and cobimetinib while patients treated with the encorafenib + binimetinib combination have a higher incidence of adverse events related to gastrointestinal system. Considering the similar clinical efficacy demonstrated by the three BRAFi + MEKi combinations, the therapeutic decision is often based on the side effect profile that characterizes each of the combinations.

Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAFV600E Mutant Melanoma Lung Metastasis in Mice.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

BRAF inhibitor-induced panniculitis in patients treated for stage IV metastatic melanoma: a case series

BRAF and MEK inhibitor combination therapy is the standard treatment for patients with BRAF V600E mutant metastatic melanoma. Neutrophilic panniculitis is a known rare complication of BRAF inhibitor therapy and can act as a potential mimic of melanoma metastases on 18F-FDG PET/CT. In this case series, we present three cases of BRAF inhibitor-induced panniculitis in patients being treated for BRAF-mutant metastatic melanoma and emphasize the use of ultrasound to differentiate between panniculitis lesions, which are typically ill-defined echogenic masses and subcutaneous soft tissue melanoma metastases, which present as hypoechoic vascular masses.

150 Poster - Glucocorticoid receptor antagonism overcomes resistance to BRAF inhibition in BRAFV600E-mutated metastatic melanoma

Clinical applications of glucocorticoids (GC) in Oncology are dependent on their pro-apoptotic action to treat lymphoproliferative cancers, and to alleviate side effects induced by chemotherapy and/or radiotherapy. However, the mechanism(s) by which GC may also promote tumor progression remains unclear. GC receptor (GR) knockdown decreases the antioxidant protection of highly metastatic B16-F10 melanoma cells. We hypothesize that a GR antagonist (RU486, mifepristone) could increase the efficacy of BRAF-related therapy in BRAFV600E-mutated metastatic melanoma. In vivo formed spontaneous skin tumors were reinoculated into nude mice to expand the metastases of different human BRAFV600E melanoma cells. The GR content of melanoma cell lines was measured by [3H]-labeled ligand binding assay. Nuclear Nrf2 and its transcription activity was investigated by RT-PCR, western blotting, and by measuring Nrf2- and redox state-related enzyme activities and metabolites. GR knockdown was achieved using lentivirus, and GR overexpression by transfection with the NR3C1 plasmid. shRNA-induced selective Bcl-xL, Mcl-1, AKT1 or NF-kB/p65 depletion was used to test the efficacy of vemurafenib (VMF) and RU486 against BRAFV600E-mutated metastatic melanoma. During early progression of skin melanoma metastases, RU486 and VMF induced a drastic metastases regression. However, treatment at an advanced stage of growth demonstrated the development of resistance to RU486 and VMF. This resistance was mechanistically linked to overexpression of specific proteins of the Bcl-2 family (Bcl-xL and Mcl-1 in our experimental models). We found that melanoma resistance is decreased if AKT and NF-kB signaling pathways are blocked. Our results highlight mechanisms by which metastatic melanoma cells adapt to survive.

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

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

Abstract 1794: Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted therapy

Abstract Introduction: Targeted therapies have recently revolutionized the treatment of BRAFV600-mutant metastatic cutaneous melanoma (CM). However, most patients do not achieve tumor regression at all due to the occurrence of resistance mechanisms. Mitogen-activated protein kinase (MAPK) pathway activation is a central step in BRAFV600-mutant CM pathogenesis and a better understanding of molecular mechanisms involved MAPK modulation is necessary to develop more effective therapeutic strategies. In the last years, Spry1 has been frequently described as an upstream regulator of MAPK signaling pathway, but its specific role in BRAFV600-mutant CM is still poorly defined. Materials and methods: Autologous BRAFV600-mutant CM cell cultures were generated from CM patients resected in our institution. The expression of Spry1 was evaluated by quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses. Spry1 gene was knock out using CRISPR. RNA-seq identified mRNAs commonly differentially expressed in Spry1 knock-out (Spry1KO) clones, and their expression was further validated by qRT-PCR. WB analyses evaluated the modulation of MAPK signaling pathways in Spry1KO clones. Cell proliferation was measured using xCELLigence instrument, whereas cell cycle progression, ROS induction, and apoptosis were assessed by flow cytometry. In vivo effects of Spry1 silencing were investigated in xenograft mice treated with or without BRAF inhibitor (BRAFi). Results: The expression of Spry1 in human CM was explored using publicly available cancer gene expression profiling and transcriptome sequencing data. mRNA expression of Spry1 was significantly elevated in metastatic CM respect to primary tumors and mRNA levels of Spry1 were significantly up-regulated in metastatic CM compared with primary lesions. The function of Spry1 was evaluated by comparing three BRAFV600-mutant CM cell lines and the respective Spry1KO cells. We found that Spry1KO induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. Moreover Spry1KO was found to impair the expression of several markers of epithelial-mesenchimal transition. In addition, ERK1/2 was found hyperactivated together with p38 leading to an increase in basal ROS levels in Spry1KO clones. This suggests that BRAFV600-mutant CM may restrain the activity of Spry1 to avoid oncogenic stress and to enable tumor growth. Indeed, treatment with the BRAFi vemurafenib down-regulated Spry1 levels in parental CM cell lines, indicating that Spry1 expression is sustained by MAPK/ERK signaling pathway in a positive feedback loop that safeguards cells from the potentially toxic effects of ERK1/2 hyperactivation. Disruption of this feedback loop rendered Spry1KO cells more susceptible to apoptosis and markedly improved response to BRAFi both in vitro and in vivo, as a consequence of the detrimental effect of ERK1/2 hyperactivation observed upon Spry1 abrogation. Conclusions: All together, our data strongly suggest that targeting Spry1 might offer a treatment strategy for BRAFV600-mutant CM by inducing the toxic effects of ERK-mediated signaling. Citation Format: Barbara Montico, Francesca Colizzi, Giorgio Giurato, Aurora Rizzo, Annamaria Salvati, Lorena Baboci, Dania Benedetti, Eliana Pivetta, Alessia Covre, Michele Dal Bo, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted 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 1794.

Impact of initial treatment and prognostic factors on postprogression survival in BRAF-mutated metastatic melanoma treated with dacarbazine or vemurafenib\u2009\xb1\u2009cobimetinib: a pooled analysis of four clinical trials

BackgroundWe sought to identify patient subgroups with distinct postprogression overall survival (ppOS) outcomes and investigate the impact of original treatment assignment and initial postprogression treatment (ppRx) on ppOS.MethodsRecursive partitioning analysis (RPA) was performed to model relationships between prespecified covariates and ppOS in patients with BRAFV600-mutated metastatic melanoma who had experienced progressive disease (PD) following treatment with cobimetinib plus vemurafenib, vemurafenib monotherapy, or dacarbazine in the BRIM-2, BRIM-3, BRIM-7, and coBRIM studies. Prognostic subgroups identified by RPA were then applied to pooled treatment cohorts. The primary endpoint was ppOS, defined as time from first PD to death from any cause.ResultsRPA identified baseline lactate dehydrogenase (LDH), baseline disease stage, Eastern Cooperative Oncology Group performance status at PD, and ppRx as significant prognostic factors for ppOS. Median ppOS was longest in patients with normal baseline LDH, stage M1c disease at baseline, and ppRx with immunotherapy or targeted therapy (12.2 months; 95% CI 10.3–16.1) and shortest in those with elevated baseline LDH > 2 × upper limit of normal (2.3 months; 95% CI 1.8–2.7). Original treatment assignment did not impact ppOS. Across treatment cohorts, patients treated with immunotherapy or targeted therapy after PD had better ppOS than those given other treatments.ConclusionA combination of factors at baseline (LDH, disease stage) and PD (performance status, ppRx) impact ppOS outcomes. ppRx with immunotherapy or targeted therapy is an independent prognostic factor for improved overall survival following progression regardless of original treatment.Trial registration The trials included in this analysis are registered with ClinicalTrials.gov: NCT00949702 (BRIM-2), NCT01006980 (BRIM-3), NCT01271803 (BRIM-7), and NCT01689519 (coBRIM).

Dabrafenib plus trametinib is effective in the treatment of BRAF V600-mutated metastatic melanoma patients: analysis of patients from the dabrafenib plus trametinib Named Patient Program (DESCRIBE II)

In clinical trials, dabrafenib plus trametinib improved overall survival (OS) compared with single-agent BRAF inhibitors (BRAFi) in patients with BRAF V600-mutant unresectable or metastatic melanoma. We investigated dabrafenib plus trametinib therapy in a compassionate-use setting [Named Patient Program (NPP); DESCRIBE II]. A retrospective chart review of patients with BRAF V600-mutated unresectable stage III/IV melanoma receiving dabrafenib plus trametinib as compassionate use was conducted. Treatment patterns and duration, clinical outcomes, and tolerability were evaluated. Of 271 patients, 92.6% had stage IV melanoma, including 36.5% with brain metastases. Overall, 162 patients (59.8%) were BRAFi naive and 171 (63.1%) received first-line dabrafenib plus trametinib. Among BRAFi-naive patients, the overall response rate (ORR) was 67.3%, median OS (mOS) was 20.0 months, and median progression-free survival (mPFS) was 7.5 months. In BRAFi-naive patients with known brain metastases (n = 62), ORR was 61.3%, mOS was 15.5 months, and mPFS was 6.2 months. Eighty-four patients received BRAFi monotherapy for >30 days and switched to dabrafenib plus trametinib prior to progression. Of these 84 patients, 63 had known disease status at the time of switch, and 22 improved with the combination therapy. No new safety signals were identified, and dabrafenib plus trametinib was well tolerated. Dabrafenib plus trametinib showed substantial clinical activity in NPP patients with BRAF V600-mutated unresectable or metastatic melanoma. Analysis of treatment patterns demonstrated the effectiveness of the combination in patients with brain metastases and across lines of therapy with a well tolerated and manageable safety profile.