BRAF-mutated Patients Research Articles

Bioinformatics-based analysis reveals elevated CYTL1 as a potential therapeutic target for BRAF-mutated melanoma.

Introduction: Despite many recent emerging therapeutic modalities that have prolonged the survival of melanoma patients, the prognosis of melanoma remains discouraging, and further understanding of the mechanisms underlying melanoma progression is needed. Melanoma patients often have multiple genetic mutations, with BRAF mutations being the most common. In this study, public databases were exploited to explore a potential therapeutic target for BRAF-mutated melanoma. Methods: In this study, we analyzed differentially expressed genes (DEGs) in normal tissues and melanomas, Braf wild-type and Braf mutant melanomas using information from TCGA databases and the GEO database. Subsequently, we analyzed the differential expression of CYTL1 in various tumor tissues and its effect on melanoma prognosis, and resolved the mutation status of CYTL1 and its related signalling pathways. By knocking down CYTL1 in melanoma cells, the effects of CYTL1 on melanoma cell proliferation, migration and invasion were further examined by CCK8 assay, Transwell assay and cell migration assay. Results: 24 overlapping genes were identified by analyzing DEGs common to melanoma and normal tissue, BRAF-mutated and BRAF wild-type melanoma. Among them, CYTL1 was highly expressed in melanoma, especially in BRAF-mutated melanoma, and the high expression of CYTL1 was associated with epithelial-mesenchymal transition (EMT), cell cycle, and cellular response to UV. In melanoma patients, especially BRAF-mutated melanoma patients, clinical studies showed a positive correlation between increased CYTL1 expression and shorter overall survival (OS) and disease-free survival (DFS). In vitro experiments further confirmed that the knockdown of CYTL1 significantly inhibited the migration and invasive ability of melanoma cells. Conclusion: CYTL1 is a valuable prognostic biomarker and a potentially effective therapeutic target in melanoma, especially BRAF-mutated melanoma.

Treatment management for BRAF-mutant melanoma patients with tumor recurrence on adjuvant therapy: a multicenter study from the prospective skin cancer registry ADOREG

BackgroundAdjuvant therapy with immune-checkpoint inhibitors (CPI) or BRAF/MEK-directed targeted therapy (TT) improves recurrence-free survival (RFS) for patients with advanced, BRAFV600-mutant (BRAFmut) resected melanoma. However, 40% of these patients will develop...

Single cell profiling of tumour biopsies and heterogeneity in response of dedifferentiated melanoma.

64 Background: Patients with advanced BRAFV600-mutant melanoma can be treated with immune checkpoint inhibitors or combination BRAF/MEK inhibitors. The sequence of these therapies can influence response to second-line treatment. Patients who progress on first-line PD1 blockade have poorer outcomes on BRAF/MEK inhibitors compared to patients who are treatment-naïve. Thus, defining the molecular features that are modified by therapy and that influence subsequent treatment responses require thorough characterization. Methods: In this study, tumor biopsies from five BRAF-mutant melanoma patients progressing on anti-PD1 (PROG) and five BRAF-mutant treatment-naïve (NAÏVE) patients were dissociated and treated ex vivo with BRAF/MEK inhibitors (dabrafenib+trametinib (DT)) before flow cytometry profiling and single cell transcriptome sequencing. Results: DT treatment did not alter tumor or immune cell proportions but effectively inhibited MAPK signaling by decreasing phosphorylated S6 levels in tumor cells within 8/10 melanoma samples. Single cell RNA-sequencing of the tumor dissociates identified four distinct melanoma differentiation states (e.g. melanocytic, transitory, neural crest-like and undifferentiated). The proportions of these cell states differed between tumor specimens, and were not affected by DT treatment. The neural crest-like and undifferentiated melanoma cells were enriched in PROG compared to NAIVE tumor samples and all differentiation states responded, albeit variably. For instance, MAPK inhibition after DT treatment was less pronounced in undifferentiated melanoma cells. Importantly, within the undifferentiated cell state, we identified a proportion of melanoma cells capable of responding to DT, and comparison of responding vs non-responding undifferentiated cells showed enrichment of pro-inflammatory IL-6 and TNFα signaling in the non-responding population. Elevated IL-6 and TNFα pathway activity correlated with decreased potency to MAPK inhibition, indicating that increased IL-6 and TNFα signaling in undifferentiated melanoma may contribute to resistance to MAPK inhibition. Conclusions: Ex vivo treatment of tumor dissociates coupled with single cell transcriptome sequencing identified cell state-specific responses to MAPK inhibition. Resistance to MAPK inhibition may be driven, in part, by tumor-intrinsic pro-inflammatory IL-6 and TNFα signalling.

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

Adjuvant treatment and outcome of stage III melanoma patients: Results of a multicenter real-world German Dermatologic Cooperative Oncology Group (DeCOG) study

PurposeClinical trials demonstrated significantly improved recurrence-free survival (RFS) of melanoma patients receiving adjuvant treatment. As data from controlled trials are based on selected populations, we investigated adjuvantly treated stage III melanoma patients under real-world conditions. Patients and methodsIn a prior multicenter cohort study, stage III-IV melanoma patients were analysed for their choice of adjuvant therapy. In this follow-up study, we examined RFS, overall and melanoma-specific survival (MSS) and response to the subsequent treatment of 589 stage III patients (232 BRAF-mutated) receiving adjuvant PD-1 inhibitors (PD1; n = 479) or targeted therapy (TT; n = 110). ResultsThe median follow-up of the total cohort was 25.7 months. The main reason for premature discontinuation of adjuvant therapy was disease progression in PD1- (28.8%, n = 138/479) and adverse events in TT-treated patients (28.2%, n = 31/110).Among BRAF-mutated patients, RFS at 24 months was 49% (95% CI 40.6–59.0%) for PD1- and 67% (95% CI 58–77%) for TT-treated patients. The risk of recurrence was higher for BRAF-mutated PD1 than TT (hazard ratio 1.99; 95% CI 1.34–2.96; hazard ratio adjusted for age, sex and tumour stage, 2.21; 95% CI 1.48–3.30). Twenty-four months MSS was 87% (95% CI 81.0–94.1) for PD1 and 92% (95% CI 86.6–97.0) for TT.Response to subsequent systemic treatment for unresectable disease was 22% for all PD1- and 16% for TT-treated patients. ConclusionsPD1-treated patients had more and earlier recurrences than TT patients. In BRAF-mutated patients, adjuvant TT might prevent early recurrences more effectively than PD1 treatment. Management of recurrence despite adjuvant treatment is challenging, with low response to current therapeutic options.

Nrf2 as a Therapeutic Target in the Resistance to Targeted Therapies in Melanoma.

The use of specific inhibitors towards mutant BRAF (BRAFi) and MEK (MEKi) in BRAF-mutated patients has significantly improved progression-free and overall survival of metastatic melanoma patients. Nevertheless, half of the patients still develop resistance within the first year of therapy. Therefore, understanding the mechanisms of BRAFi/MEKi-acquired resistance has become a priority for researchers. Among others, oxidative stress-related mechanisms have emerged as a major force. The aim of this study was to evaluate the contribution of Nrf2, the master regulator of the cytoprotective and antioxidant response, in the BRAFi/MEKi acquired resistance of melanoma. Moreover, we investigated the mechanisms of its activity regulation and the possible cooperation with the oncogene YAP, which is also involved in chemoresistance. Taking advantage of established in vitro melanoma models resistant to BRAFi, MEKi, or dual resistance to BRAFi/MEKi, we demonstrated that Nrf2 was upregulated in melanoma cells resistant to targeted therapy at the post-translational level and that the deubiquitinase DUB3 participated in the control of the Nrf2 protein stability. Furthermore, we found that Nrf2 controlled the expression of YAP. Importantly, the inhibition of Nrf2, directly or through inhibition of DUB3, reverted the resistance to targeted therapies.

The BEETS (JACCRO CC-18) trial: an observational and translational study of BRAF-mutated metastatic colorectal cancer.

For BRAF V600E-mutated metastatic colorectal cancer (mCRC), the BEACON phase 3 trial showed survival benefit of triplet therapy with cetuximab (anti-EGFR antibody), encorafenib (BRAF inhibitor) and binimetinib (MEK inhibitor) as well as doublet therapy with cetuximab and encorafenib over irinotecan-based chemotherapy plus anti-EGFR antibody. Both regimens are standards of care in Japan, but definite biomarkers for predicting efficacy and selecting treatment remain lacking. The mechanisms underlying resistance to these regimens also warrant urgent exploration to further evolve treatment. This prospective observational/translational study evaluated real-word clinical outcomes with cetuximab and encorafenib with or without binimetinib for BRAF-mutated mCRC patients and investigated biomarkers for response and resistance by collecting blood samples before and after treatment. Clinical Trial Registration: UMIN000045530 (https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000051983).

Phase III FIRE-4 study (AIO KRK-0114): Influence of baseline liquid biopsy results in first-line treatment efficacy of FOLFIRI/cetuximab in patients with tissue RAS-WT mCRC.

3507 Background: FIRE-4 (AIO KRK-0114) is performed in RAS wild-type (wt) mCRC patients. This randomized study tests the efficacy of early switch maintenance during 1st-line therapy (part 1) and re-challenge with cetuximab (part 2) in later-line treatment. In part 1, arm A patients continued FOLFIRI/Cet until progression or intolerable toxicity. In arm B, patients received FOLFIRI/Cet for 8-12 cycles, after which maintenance therapy with 5-FU/FA plus bevacizumab (5-FU/Bev) was applied. The first randomization evaluates the question if an early switch from cetuximab to bevacizumab may prolong PFS. Within the translational protocol, serial baseline liquid biopsy were taken to analyze RAS and BRAF mutations. Methods: Within this randomized, controlled, open-label phase-III study, patients received FOLFIRI (irinotecan plus 5-FU/FA) plus cetuximab at the standard dosing schedule. In arm A, FOLFIRI plus cetuximab was continued until progression or intolerable toxicity. In arm B, patients received 8 cycles of FOLFIRI plus cetuximab (in case of tumor response) or 12 cycles (in case of stable disease) followed by maintenance with 5-FU/FA plus bevacizumab (5mg/kg) until disease progression or intolerable toxicity. Overall survival after second randomization (part 2) is evaluated as a primary endpoint. Liquid Biopsies were analyzed by RAS BEAMing and BRAF ddPCR technology. Results: From August 2015 to January 2021, 673 patients were randomized, and liquid biopsies of 540 patients were evaluable at baseline. Of those, 70 (13%) were RAS mutant and 38 (7%) BRAFV600E mutant at baseline. Patients with a detectable RASmut had a significant shorter PFS and OS when compared to RASwt patients (PFS: 9.0mo vs. 11.5mo; p < 0.001; OS: 22.1mo vs 33.6mo; p < 0.001). Whereas, for RASwt patients no difference for both arms with respect to PFS and OS could be observed, RASmut patients (n = 70) had a clear trend towards shorter survival in the standard FOLFIRI cetuximab arm (PFS: 6.4mo vs. 10.1mo, p = 0.54; OS: 24.9mo vs. 16.3mo, p = 0.10). Patients with a BRAF mutation in liquid biopsy had median survival times as expected for BRAF mutant patients (PFS = 5.5mo; OS = 12.0mo). In the standard arm, with a continuous administration of cetuximab, the conversion rate from RASwt to RASmut was significantly higher at progression than in the switch maintenance arm. Conclusions: Liquid biopsy detected RAS mutation in 13% of patients deemed RASwt based on tissue analyses. These patients show outcome characteristics expected for RAS mutant patients (median PFS of 9.0 months and median OS of 22 months). The study thus shows the clinical relevance of liquid biopsy in the verification of RAS mutational status. Clinical trial information: NCT02934529 .

Abstract 1012: Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic melanoma

Abstract The combination of BRAF and MEK inhibitors (BRAFi/MEKi) in metastatic melanoma patients with BRAF activating mutations have resulted in dramatic responses in most patients, but unfortunately most patients progress after one year and limited subsequent treatment options are available. Although resistance mutations to BRAFi/MEKi mainly occur in the MAPK and PI3K pathways, after progression on BRAFi/MEKi drugs, targeting these mutations have been clinically unsuccessful. One possible cause of relapse is that a resistant subpopulation to BRAFi/MEKi exists prior to treatment, raising the possibility that earlier counter-resistance treatment could be administered and potentially be more successful. However, the prevalence and clinical significance of pre-existing resistance is currently unknown in melanoma. To address this, we used quantitative blocker displacement amplification (qBDA) to quantify the VAF (down to 0.01% resolution) of resistant subclones along the MAPK/PI3K pathways from FFPE biopsies on 149 metastatic melanoma patients. Due to possible intra-tumoral heterogeneity, multiple samples were taken when available per patient. Of 29 patients currently processed, 5/29 (17%) had detectable NRAS Q61L or Q61R, 5/29 (17%) had KRAS G12 or G13 mutations, and 3/29 (10.3%) had PIK3CA H1047R/Y. As expected, BRAF activating mutations were detectable in 29/29 patients, while VAF of the resistant subclones were found to be as low as 0.16%. We expect some of the putative resistance mutations to be artifacts, and all will be validated by ddPCR. To model the behavior of low-VAF resistance mutations, we conducted in vitro spike-in experiments using paired and labeled parental and BRAF/MEK inhibitor-resistant melanoma lines. Our results show that even down to 0.1%, the resistant lines can rapidly emerge as the dominant population while under drug treatment. We also tested the timing of applying a counter-resistance therapy: early vs late. Earlier intervention led to a significant delay in growth of the resistant population, as expected. However, we are currently preparing an experiment that will determine whether an earlier intervention might not simply delay resistance, but also alter its strength, at different % VAFs. We therefore anticipate mechanistic analyses of how %VAF and drug timing can optimize counter-resistance therapies. Overall, our interim data suggest a significant percentage of metastatic BRAF-mutated melanoma patients may harbor pre-existing resistant subclones, and that early treatment with counter-resistance therapy can potentially delay resistance to targeted therapy. Citation Format: Michael Nakazawa, Lauren Cheng, Mishal Ahmed, Rossana Lazcano, Khalida Wani, Omid Veiseh, Lawrence Kwong. Detecting and intervening on rare pre-existing resistant subclones to BRAF/MEK inhibitors in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1012.

Abstract 1164: Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of melanoma

Abstract Over half of BRAF mutant melanoma patients with response to targeted therapy recur within 15 months. Resistance is postulated to arise from certain clonal populations that enter a slow cycling persister state, evade treatment with relative dormancy, and repopulate the tumor when reactivated. Via longitudinal profiling we define expression states of clonal populations and track their evolutionary progression. BRAF mutant patient derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) was performed at 5 timepoints across 94 days of treatment, tracking clonal populations with intact tissue structure. Deep learning on H&E-stained images automatically detected histological features that co-localize with expression levels. A novel computational pipeline performed clustering, differential expression and pathway enrichment analysis, copy number variation detection, and pseudotemporal ordering, allowing spatial recreation of clonal phylogenies and cell fate trajectory inference during the treatment course. Three clonal categories were defined; Sensitive: predominant in treatment-naïve samples, Persister: remaining clusters in maximally shrunken specimens, Resistant: re-emergent, fast-proliferating. Analysis of clonal composition identified GAPDH and CCND1 as highly expressed in sensitive clones, suppressed in persisters, and reactivated in resistant clones; TYRP1, DCT, MITF, and NGFR showed an opposite pattern. Persister clones evolved from oxidative phosphorylation toward glycolysis and MAPK regulation. Spatial analysis showed increased glycolysis:oxidative phosphorylation ratio in centrally located sensitive clusters, with more balanced peripheral expression. Resistant clones showed upregulated DUSP6 expression and enrichment in Orexin signaling and MAPK regulation. Imaging- and expression-based clones were highly concordant. Spatial profiling during melanoma treatment identified transient persister and emergent resistant clones, defining expression profiles and phenotypic features. Altered ratio of oxidative to glycolytic metabolic pathways was a hallmark of clonal evolution in both space and time. Specific MAPK pathway re-entry points were identified as candidate resistance mechanisms, suggesting potential therapeutic targets. Deep learning features derived from histological images showed good correlation with ST profiles, providing a promising method for clonal tracking via images alone. This longitudinal experiment mimics a tumor’s clinical course, inducing persistence and resistance via treatment that parallels that seen by patients. Combining ST and imaging techniques, we provide insight into clonal dynamics with novel spatiotemporal resolution, defining an evolutionary roadmap to acquired treatment resistance. Citation Format: Jill Carol Rubinstein, Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang. Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1164.

The profile and prognostic value of circulating lymphocyte subsets in metastatic colon cancer.

Colon cancer (CC) are the most common malignant cancer in human digestive system, however, the profile and prognostic value of circulating lymphocyte subsets in CC patients has not been systemically clarified. In this study, 158 patients with metastatic CC were enrolled. Chi-square test was used to analyze the relationship between baseline peripheral blood lymphocyte subsets and clinicopathological parameters. Kaplan-Meier and Log-rank tests were used to analyze the relationship between clinicopathological parameters and baseline peripheral lymphocyte subsets and overall survival (OS) of patients with metastatic CC. Univariate/multivariate COX regression analysis was used to identify the independent factors in metastatic CC. The baseline peripheral blood CD3+T cells, CD4+T cells, NK cells and B cells of BRAF mutant patients were significantly lower than those in BRAF wild-type patients; The baseline CD8+T cells of KRAS mutation group was lower than that in KRAS wild type group. Peripheral blood CA19-9>27, left-sided colon cancer (LCC), KRAS and BRAF mutation were poor prognostic factors, and ALB>40, NK cells were protective prognostic factors for metastatic CC. In patients with liver metastases subgroup, higher NK cells also indicated a longer OS. Finally, LCC (HR=0.56), CA19-9 (HR=2.13), ALB (HR=0.46) and circulating NK cells (HR=0.55) were independent prognostic factors for metastatic CC. LCC, higher level of ALB and NK cells at baseline are protective factors, and higher CA19-9, KRAS/BRAF gene mutation are adverse prognostic factors. Sufficient circulating NK cells are independent prognostic factor for metastatic CC patients.

Treatment of Recurrent Melanoma Following Adjuvant Therapy.

In the era of effective therapies in melanoma, notably the widespread use of two types of adjuvant treatments: anti-PD-1 immunotherapies and therapies targeting the mitogen-activated protein kinase pathway, for BRAF-mutant patients, an important question arises about how to treat these patients in case of recurrent melanoma following adjuvant therapy. Prospective data are lacking in this area and might be difficult to obtain due to the constant progress being made in the field. Therefore, we reviewed available data suggesting that the initial adjuvant treatment received and the following events provide information about the biology of the disease and the probability of response to following systemic treatments. Thus, in case of relapse during or just after adjuvant anti-PD-1, immune resistance is probable, an anti-PD-1 monotherapy rechallenge has a low likelihood of clinical benefit, and escalation with a combination of immunotherapies should be given priority. In case of relapse during treatment with BRAF plus MEK inhibitors, there may be a risk of lower efficacy of immunotherapy than in naïve patients since this relapse attests not only to a resistance to BRAF-MEK inhibition, but also the introduction of immunotherapy to rescue a progression on targeted therapy. In case of relapse long after adjuvant treatment cessation, whatever the treatment received, no conclusion can be drawn about the efficacy of these drugs, and these patients can be treated like naïve patients. Thus, a combination of anti-PD-1 and anti-CTLA4 is probably the best solution, and the following line can be BRAF-MEK inhibitors in BRAF-mutated patients. Finally, in case of recurrent melanoma following adjuvant therapy, given the promising upcoming strategies, inclusion in a clinical trial should be offered as frequently as possible.

RIDR-PI-103, ROS-activated prodrug PI3K inhibitor inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor-resistant BRAF-mutant melanoma cells.

Reactive oxygen species (ROS) levels are elevated after acquisition of resistance to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors including dabrafenib and MEK inhibitors such as trametinib in BRAF-mutant melanoma. To circumvent toxicity to PI-103 (a pan PI3K inhibitor), we utilized a novel ROS-induced drug release (RIDR)-PI-103, with a self-cyclizing moiety linked to PI-103. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Previous findings demonstrate that trametinib and dabrafenib-resistant (TDR) cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on melanocytes and TDR cells. RIDR-PI-103 exhibited less toxicity compared to PI-103 at 5 µM in melanocytes. RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM. Twenty-four hour treatment with RIDR-PI-103 inhibited p-Akt, p-S6 (Ser240/244) and p-S6 (Ser235/236). We assessed the mechanism of activation of RIDR-PI-103, using glutathione or t-butyl hydrogen peroxide (TBHP) on the TDR cells in the presence or absence of RIDR-PI-103. Addition of the ROS scavenger glutathione to RIDR-PI-103 significantly rescued the cell proliferation in TDR cell lines while addition of the ROS inducer TBHP and RIDR-PI-103 inhibited cell proliferation in WM115 and WM983B TDR cell lines. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor-resistant cells will expand possible treatment options and open avenues for the development of new ROS-based treatment therapies for BRAF-mutant melanoma patients.

STRA6 regulates tumor immune microenvironment and is a prognostic marker in BRAF-mutant papillary thyroid carcinoma.

BRAF mutation is one of the most common genetic alterations contributing to the initiation and progression of papillary thyroid carcinoma (PTC). However, the prognostic value of BRAF mutation for PTC is limited. Novel markers are needed to identify BRAF-mutant patients with poor prognosis. Transcriptional expression data were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Pathway enrichment was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA). Protein-protein interaction networks were predicted by the GeneMANIA. The correlation between STRA6 expression and immune infiltration was analyzed by tumor immune estimation resource (TIMER) and tumor-immune system interaction database (TISIDB). Immunohistochemistry was used to detect the STRA6 protein expression level of PTC. Infiltration of regulatory T cells (Tregs) and CD8+ T cells in tumor samples were analyzed by fluorescent multiplex immunohistochemistry. In BRAF-mutant PTC, STRA6 was extremely upregulated and predicted unfavorable survival, which was an independent risk factor for increased mortality risk. Bioinformatic analyses indicated that STRA6 might activate the MAPK pathway synergistically with BRAFV600E. The expression of STRA6 was associated with immune infiltrates and T cell exhaustion. Fluorescent multiplex immunohistochemistry showed that STRA6 increased Tregs abundance and decreased CD8+ T cells infiltration in PTC. Moreover, STRA6 promoted epithelial-mesenchymal transition via increased cancer-associated fibroblasts infiltration. Our study demonstrates STRA6 may serve as a prognostic marker for BRAF-mutated PTC, which may drive thyroid carcinogenesis via activation of oncogenic pathway and regulation of tumor immunosuppressive microenvironment.

Neoadjuvant plus adjuvant combined or sequenced vemurafenib, cobimetinib and atezolizumab in patients with high-risk, resectable BRAF-mutated and wild-type melanoma: NEO-TIM, a phase II randomized non-comparative study.

Following the increased survival of patients with metastatic melanoma thanks to immunotherapy and targeted therapy, neoadjuvant approaches are being investigated to address the unmet needs of unresponsive and intolerant patients. We aim to investigate the efficacy of neoadjuvant plus adjuvant combined or sequenced vemurafenib, cobimetinib and atezolizumab in patients with high-risk, resectable BRAF-mutated and wild-type melanoma. The study is a phase II, open-label, randomized non-comparative trial in patients with stage IIIB/C/D surgically resectable, BRAF-mutated and wild-type melanoma, with three possible treatments: (1) vemurafenib 960 mg twice daily from day 1 to 42; (2) vemurafenib 720 mg twice daily from day 1 to 42; (3) cobimetinib 60 mg once daily from day 1 to 21 and from day 29 to 42; and (4) atezolizumab 840 mg for two cycles (day 22 and day 43).Patients will be randomized to three different arms: A) BRAF-mutated patients will receive over 6 weeks (1) + (3); B) BRAF-mutated patients will receive over 6 weeks (2) + (3) + (4); C) BRAF wild-type patients will receive over 6 weeks (3) + (4). All patients will also receive atezolizumab 1200 mg every 3 weeks for 17 cycles after surgery and after a second screening period (up to 6 weeks). Neoadjuvant therapy for regional metastases may improve operability and outcomes and facilitate the identification of biomarkers that can guide further lines of treatment. Patients with clinical stage III melanoma may especially benefit from neoadjuvant treatment, as the outcomes of surgery alone are very poor. It is expected that the combination of neoadjuvant and adjuvant treatment may reduce the incidence of relapse and improve survival. eudract.ema.europa.eu/protocol.htm, identifier 2018-004841-17.

First-line anti-EGFR or anti-VEGF therapy, tumour sidedness, and survival: Results from the South Australian (SA) Metastatic Colorectal Cancer (mCRC) Registry.

42 Background: The anti-EGFR monoclonal antibodies panitumumab (Pmab) and cetuximab (Cmab) and the anti-VEGF antibody bevacizumab (bev) have demonstrated clinical efficacy in mCRC in combination with first-line chemotherapy. Clinical and molecular characteristics, such as primary tumour sidedness, RAS, BRAF and MMR status are prognostic and predictive biomarkers for response to treatment. Our aim was to compare the survival and tumour characteristics of patients receiving first-line chemotherapy and antibody combinations using the SA mCRC Registry. Methods: This real-world registry has collected data from all patients diagnosed with mCRC in SA prospectively since 2006. We identified patients who had received first-line Pmab, Cmab or bev. Survival was analysed using the Kaplan-Meier method and log-rank test to compare outcomes based on primary tumour location. Results: Of the 5537 patients currently entered onto the registry, 1313 with RAS results available had received first-line anti-EGFR/VEGF antibodies and had colon tumour side recorded. The table summarises median overall survival (mOS) and patient characteristics. Survival was significantly shorter for those with right colon cancers (P < 0.001). There were more high-grade (P < 0.001), KRAS mutant (P = 0.004) and BRAF mutant (P = 0.036) tumours in those with right colon cancers, and a trend towards fewer curative liver resections (P = 0.059). There was no significant difference in mOS seen between Pmab, Cmab or bev for both left (P = 0.14) and right-sided (P = 0.88) primary tumours. Conclusions: There were more high-grade, BRAF and KRAS mutant patients with right colon tumours, and lower rates of liver resection, which may explain the significantly shorter mOS. The survival seen for Pmab and Cmab was almost identical for both right and left primary tumours. When comparing Pmab, Cmab and bev as first-line therapy, there was no significant difference in mOS for both left- and right-sided CRC. [Table: see text]

Targeting EphA2 and DDR signaling can overcome the BRAF and MEK inhibitors acquired resistance in melanoma cell lines

The BRAF and MEK inhibitors combined strategies have dramatically changed the outcome of BRAF-mutated metastatic melanoma patients. However, despite the initial promising results, the onset of primary or acquired resistance occurs in nearly half of the patients at about one year from the diagnosis. Understanding the mechanisms of resistance to these inhibitors is therefore critical for planning more effective therapeutic strategies able to improve patient outcomes. To this aim we generated BRAF and MEK inhibitors resistant melanoma cells starting from the SAN and A375 lines, both harboring the most common BRAF-V600 mutation and sensitive to these drugs. The obtained double-resistant cell lines were characterized by MTT cell proliferation, migration, invasion assays, phosphoarray and western blot analysis. Here we report that the overexpression of several Tyrosine Kinase Receptors (TKRs), such as EphA2 and DDRs, drives the resistance to these drugs and that this resistance can be overcome by treatment with ALW‑II‑41‑27 multikinase inhibitor. ALW‑II‑41‑27 blocks not only TKRs expression, but also the related downstream AKT and MAPK signaling pathways and its efficacy is documented by decreased cell viability and reduced cell invasion/migration of the resistant cells. Our results can delineate a novel promising therapeutic approach to overcoming the drug resistance occurring in BRAF-mutated metastatic melanoma.

Driver and targetable alterations in Chinese patients with small bowel carcinoma.

Small bowel carcinoma (SBA) is a rare gastrointestinal cancer with a poor prognosis. Recent genomic profiling studies revealed that the landscape of molecular alterations in SBA was distinct from colorectal cancer (CRC) and gastric cancer (GC). To explore driver and targetable alterations in SBA, we performed next-generation sequencing in 107 Chinese SBA patients. DNA from paraffin-embedded SBAsamples and the corresponding peripheral blood control sampleswere analyzed through a next-generation sequencing panel. Somatic alterations including point mutations, indels, copy number alterations, gene fusions as well as pathogenic germline variants were characterized. More than half of SBA cases carried KRAS mutations, including canonical (G12, G12, Q61) and atypical mutations (A146, L19, and K117). To our best knowledge, this was the first report of rare driver alterations including KRAS A146V/L19F, PIK3CA N345K/G364R/Q546E, and ZKSCAN1-MET fusion in SBA. Compared to KRAS-mutant patients, alternative activating alterations were enriched in KRAS wild-type patients, and some of them are targetable. Among BRAF-mutated SBA patients, class 1/2 BRAF mutants were mutually exclusive with RAS mutations, but class 3 BRAF mutants were not. Activating ERBB2 alternations, including amplification and activating mutations, represent the most common targetable alternation in this SBA cohort. Of note, the spectrums of BRAF and PIK3CA mutations in this Chinese SBA cohort were distinct from those of a European SBA cohort. Patients with three druggable mutations (PIK3CA, MAP2K1, KRAS G12C) had a high prevalence of concurring drivers, which may interfere with the clinical efficacy of single-target therapy. Taken together, our work provided a comprehensive analysis of driver and targetable alterations in SBA, which can facilitate the practice of precision oncology in this challenging disease.

Tumor immune microenvironment and immunotherapy efficacy in BRAF mutation non-small-cell lung cancer

Previous small-size studies reported BRAF-mutated NSCLC patients have comparable sensitivity to immune checkpoint inhibitors (ICIs). However, how BRAF mutation affects the tumor immune microenvironment (TIME) is unknown. We performed Nanostring-panel RNA sequencing to evaluate TIME in 57 BRAF mutated and wild-type (WT) NSCLC specimens (cohort A). The efficacy of ICI monotherapy or combined therapies was determined in 417 patients with WT and BRAF mutated NSCLC (cohort B). We found that BRAF-mutant tumors had similar ratios of CD8+ T cells to Tregs, the balance of cytotoxicity gene expression signatures and immune suppressive features, and similar ICI-response-related biomarkers to WT NSCLC. A similar TIME pattern was observed between the BRAF V600E and Non-V600E subgroups of NSCLC. The further retrospective study confirmed that treatment with ICI monotherapy or combined therapies resulted in similar overall survival (OS) (HR: 0.85; 95% CI, 0.56 to 1.30; p = 0.47) and progress-free survival (PFS) (HR: 1.02; 95% CI, 0.72 to 1.44; p = 0.91) of patients with WT (n = 358) and BRAF mutant (n = 59) NSCLC. Similarly, both patients with BRAF V600E or Non-V600E NSCLC had similar responses to immunotherapy. Our findings support that BRAF mutation did not modulate TIME in NSCLC and therapeutic responses to ICIs. Patients with NSCLC harboring BRAF mutation should not be denied treatment with ICIs.

63P Activity and safety of first-line treatments for advanced melanoma: A network meta-analysis

Treatment options for advanced melanoma have recently increased with the introduction of the anti-LAG3 and anti-PD-1 relatlimab/nivolumab combination. To date, ipilimumab/nivolumab is the benchmark of overall survival (OS), despite a high toxicity profile. Furthermore, in BRAF-mutant patients, BRAF/MEK inhibitors and the atezolizumab/vemurafenib/cobimetinib triplet are also available treatments, making the first-line therapy selection more complex. To address this issue, we conducted a systematic review and network meta-analysis of the available first-line treatment options in advanced melanoma.