Wild-type BRAF Research Articles

Association of BRAFV600E Mutation with the Aggressive Behavior of Papillary Thyroid Microcarcinoma: A Meta-Analysis of 33 Studies

An association between the BRAFV600E mutation and the clinicopathological progression of papillary thyroid microcarcinoma (PTMC) has been suggested. We aimed to summarize the relevant literature and determine the predictive value of BRAFV600E mutation in predicting clinical outcomes and risk stratification in patients with PTMC. A systematic search using PubMed, Cochrane, and Embase up to February 2020 was performed. A total of 33 studies met the inclusion criteria, resulting in a pool of 8838 patients, of whom 5043 (57.1%) patients were positive for BRAFV600E mutation. Tumors with positive BRAFV600E mutation had a higher tendency for multifocality (RR = 1.09, 95%CI = 1.03-1.16), extrathyroidal extension (RR = 1.79, 95%CI = 1.37-2.32), and lymph node metastasis (RR = 1.43, 95%CI = 1.19-1.71). Patients with BRAFV600E mutation were at increased risk of disease recurrence (RR = 1.90, 95%CI = 1.43-2.53). PTMC in patients positive for the BRAFV600E mutation is more aggressive than wild-type BRAF PTMC. Since BRAF-mutated PTMC is generally more resistant to radioiodine treatment, patients with BRAFV600E-mutated PTMC may require earlier management, such as a minimally invasive ablative intervention. Conservative management by active surveillance may be suitable for patients with wild-type BRAFV600E PTMC.

Predictive biomarkers for response to trametinib in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer deaths. Current companion diagnostics use driver mutation sequencing to select patients for molecularly targeted agents (MTA), even though most patients lack actionable mutations. These diagnostics utilize static biomarkers, ignoring real-time tumor cell biology. Trametinib is FDA-approved in combination with dabrafenib for BRAF V600E-positive NSCLC, however, it has plausible utility beyond these patients. We sought to identify novel biomarkers for maximizing trametinib application. Trametinib responses were evaluated in 12 EGFR/BRAF wild-type (WT) NSCLC cell lines with diverse RAS mutational status. We identified three response categories by colony assay. Trametinib-induced molecular dynamics were studied using immunoassays and apoptosis/necrosis assays, to identify predictive response biomarkers. p27 accumulation and cyclin D1 downregulation suggested universal cell cycle arrest with trametinib. However, 4 cell lines showed PARP cleavage and 8 showed increased phospho-4E-BP1, suggesting varied cellular outcomes from apoptosis, necrosis, senescence to autophagy. Cleaved PARP, phospho-4E-BP1 and phospho-AKT expression can predict these outcomes. Trametinib monotherapy outcome may depend upon cellular context more than oncogenic mutation status. In BRAF WT NSCLC, trametinib may be best suited for combination therapy and dynamic biomarkers could select combinations and predict responses.

Body mass index and molecular subtypes of colorectal cancer.

Obesity is an established risk factor for colorectal cancer (CRC), but the evidence for the association is inconsistent across molecular subtypes of the disease. We pooled data on body mass index (BMI), tumor microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, and Jass classification types for 11 872 CRC cases and 11 013 controls from 11 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) adjusted for covariables. Higher BMI was associated with increased CRC risk (OR per 5 kg/m2 = 1.18, 95% CI = 1.15 to 1.22). The positive association was stronger for men than women but similar across tumor subtypes defined by individual molecular markers. In analyses by Jass type, higher BMI was associated with elevated CRC risk for types 1-4 cases but not for type 5 CRC cases (considered familial-like/Lynch syndrome microsatellite instability-H, CpG island methylator phenotype-low or negative, BRAF-wild type, KRAS-wild type, OR = 1.04, 95% CI = 0.90 to 1.20). This pattern of associations for BMI and Jass types was consistent by sex and design of contributing studies (cohort or case-control). In contrast to previous reports with fewer study participants, we found limited evidence of heterogeneity for the association between BMI and CRC risk according to molecular subtype, suggesting that obesity influences nearly all major pathways involved in colorectal carcinogenesis. The null association observed for the Jass type 5 suggests that BMI is not a risk factor for the development of CRC for individuals with Lynch syndrome.

Ensemble Pharmacophore Meets Molecular Docking: A Novel Screening Approach for the Identification of B-Raf Kinase Inhibitors

In US about 106110 diagnosed as melanomas, 7,180 people expected to die due to melanoma. B-RAF is a cytoplasmic serine - threonine kinase that is found in a mutated form in melanoma and colorectal cancer. Sorafenib was initially introduced as a B-RAF inhibitor in melanoma. Hence it is taken as a pivot molecule in our study. Four potent B-Raf kinase inhibitors (Sorafenib, Regorafenib, N-desmethyl sorafenib & Donafenib) are used to build a pharmacophore model with ‘PharmaGist webserver’ which generated a 5-point hypothesis. The best model with score of 27.780, was used to screen the Zinc database of ZINCPharmer web server to obtain similar pharmacophore hits. By applying filters like Lipinski rule, RMSD criteria in ZINCPharmer top ten hits were identified. Subsequently, molecular docking was performed on wild (1UWH) and mutated (3IDP) B-Raf kinase protein targets by using GLIDE 5.6 (Schrödinger), to prioritize top lead molecules. Further these molecules are subjected to ADME Properties Prediction by Qik Prop module. Among ten, nine molecules have glide scores in the range nearer to the standard molecule i.e., Sorafenib. Finally, we conclude that ZINC02853810 may act as a powerful inhibitor against both wild and mutant type B-Raf kinase as it has highest glide scores than the Standard. 
 GRAPHICAL ABSTRACT

Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

Anaplastic thyroid cancer is an extremely lethal malignancy without reliable treatment. BRAFV600E point mutation is common in ATCs, which leads to MAPK signaling activation and is regarded as a therapeutic target. Resveratrol inhibits ATC cell growth, while its impact on BRAF-MAPK signaling remains unknown. This study aims to address this issue by elucidating the statuses of BRAF-MAPK and STAT3 signaling activities in resveratrol-treated THJ-11T, THJ-16T, and THJ-21T ATC cells and Nthyori 3-1 thyroid epithelial cells. RT-PCR and Sanger sequencing revealed MKRN1-BRAF fusion mutation in THJ-16T, BRAF V600E point mutation in THJ-21T, and wild-type BRAF genes in THJ-11T and Nthyori 3-1 cells. Western blotting and immunocytochemical staining showed elevated pBRAF, pMEK, and pERK levels in THJ-16T and THJ-21T, but not in THJ-11T or Nthyori 3-1 cells. Calcein/PI, EdU, and TUNEL assays showed that compared with docetaxel and doxorubicin and MAPK-targeting dabrafenib and trametinib, resveratrol exerted more powerful inhibitory effects on mutant BRAF-harboring THJ-16T and THJ-21T cells, accompanied by reduced levels of MAPK pathway-associated proteins and pSTAT3. Trametinib- and dabrafenib-enhanced STAT3 activation was efficiently suppressed by resveratrol. In conclusion, resveratrol acts as dual BRAF-MAPK and STAT3 signaling inhibitor and a promising agent against ATCs with BRAF mutation.

High RPMB predicts poor disease-free survival of male N1 papillary thyroid cancer after adjuvant radioiodine therapy

The current recommendation for the use of adjuvant radioactive iodine (RAI) therapy in papillary thyroid cancer (PTC) after radical surgery is based on clinicopathological factors; however, this recommendation remains controversial. Our present study established a new biomarker, RPMB (promotor methylation burden of DNA repair genes (DRGs)), to identify a patient subgroup suitable for adjuvant RAI therapy. We defined RPMB as the ratio of methylated DRGs to the total number of DRGs. Methylation profiles of 498 PTC tumors and their clinical data were retrieved from the Cancer Genome Atlas (TCGA) database. DRGs of PTC subjects were found to be much more hypomethylated than controls across the whole profile (all p < 0.001). PTC patients with higher RPMBs tended to be ≥45 years old and female, and these PTCs were commonly unifocal, with N0 disease, wild-type BRAF, and mutated RAS. The subgroup analysis indicated that high RPMBs were significantly associated with poor disease-free survival (DFS) in male patients with PTC (HR = 4.855, 95% CI: 1.527–15.433, p = 0.007). Moreover, Kaplan–Meier analysis showed that high RPMBs could significantly predict poor DFS in male patients after R0 resection for N1 disease (HR: 5.431, 95% CI: 1.045–28.219, p = 0.024), and the p-value was very close to significance in these patients after adjuvant RAI therapy (HR: 6.269, 95% CI: 0.693–56.714, p = 0.062). Multivariate analysis indicated that both male sex (HR = 14.565, 95%CI: 2.153–98.507, p = 0.006) and high RPMBs (HR = 11.206, 95%CI: 1.622–77.405, p = 0.014) were independent unfavorable factors for DFS after adjuvant RAI therapy. Therefore, RPMB might be a potential predictor for identifying suitable male patients with PTC who can benefit from adjuvant RAI therapy.

The diagnostic utility of BRAF VE1 mutation-specific immunohistochemistry in ameloblastoma

AbstractAmeloblastoma is a benign, locally aggressive odontogenic neoplasm with variable solid and cystic morphology. On account of its histologic variety, diagnostically challenging cases can bear resemblance to odontogenic keratocyst/keratocystic odontogenic tumor (KCOT) or dentigerous cyst (DC). BRAFV600E mutation has been reported to be specific for and frequent in ameloblastoma, and this study evaluated the usefulness of immunohistochemistry (IHC) using the BRAF VE1 mutant-specific antibody as a diagnostic adjunct in this setting. We investigated 46 ameloblastomas, 30 KCOTs, and 30 DCs. BRAF VE1 IHC was performed on all cases and allele-specific polymerase chain reaction (AS-PCR) for BRAFV600E mutation was performed on 30 ameloblastomas and any IHC-positive KCOT/DC. BRAF VE1 IHC was positive in 31/37 (83.8%) mandibular ameloblastomas but not in any maxillary ameloblastomas (0/9), KCOT (0/30), or DC (0/30). Equivocal staining was seen in 1/37 (3.3%) mandibular ameloblastomas. Of the 30 ameloblastomas subjected to AS-PCR, BRAFV600E mutation was identified in 19/23 (82.6%) mandibular ameloblastomas and 0/7 (0.0%) maxillary ameloblastomas. BRAFV600E mutant ameloblastomas were positive by IHC in 18/19 (94.7%) cases and equivocal in 1/19 (5.3%) cases. All 11 (100.0%) BRAF-wild type ameloblastomas were negative by IHC. BRAF VE1 is an excellent tool for the diagnosis of mandibular ameloblastoma but of limited utility in the maxilla, where it less commonly occurs and where BRAFV600E mutation is considerably less frequent.

Establishment and Characterisation of Human Clear Cell Sarcoma and Malignant Melanoma Cell Lines.

Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

436TiP COLSTAR: Randomized, open-label, multicentre, phase III study comparing futuximab/modotuximab plus trifluridine/tipiracil to trifluridine/tipiracil in KRAS/NRAS and BRAF wild type (wt) metastatic colorectal cancer (mCRC) previously treated with both standard and anti-EGFR treatment

Approved treatments for 3rd-line mCRC are limited and outcomes remain poor. Trifluridine/tipiracil (TT) is a standard of care option for patients previously treated/not considered candidates for available therapies. Futuximab/modotuximab (FuMo) is a biological composed of a 1:1 mixture of two monoclonal antibodies, futuximab and modotuximab, that bind to non-overlapping epitopes on the extracellular domain (ECD) of EGFR. FuMo has demonstrated activity in RAS/BRAF wt mCRC EGFR-pretreated patients in the Sym004-05 phase II study. Anti-EGFR Mabs have been safely combined with TT. COLSTAR (NCT05223673) is a randomized, open-label, phase III study comparing FuMo + TT with TT alone with a safety lead-in (SLI) part. Main eligibility criteria include KRAS/NRAS (exons 2, 3 and 4) and BRAF wt (V600E mutation) (double negative; DN) assessed by centralized analysis of ctDNA at screening, and previous treatment with at least 2 regimens including fluoropyrimidine, irinotecan, oxaliplatin, at least one anti-VEGF (bevacizumab, aflibercept, ramucirumab, regorafenib) and at least one anti-EGFR mAb (cetuximab or panitumumab) for ≥4 months. Randomization will be done in a 1:1 ratio to TT (35 mg/m2 BID on days 1-5 and days 8-12 of a 28-day cycle) or to the same dose of TT + FuMo (9 mg/kg loading dose on day 1 of cycle 1 followed by 6 mg/kg weekly). Stratification will be done according to performance status (ECOG 0 vs 1), previous regimens of treatment (2 vs ≥3), and EGFR ECD mutations (presence vs absence). Approximately 25 patients will be enrolled in the SLI part and 500 in the randomized part of the study. Primary objectives are: safety and tolerability of FuMo + TT according to CTCAE v5.0 (part 1) and superiority of the combination in terms of OS in the DN population (part 2). The key secondary endpoint is OS in the triple-negative (KRAS/NRAS, BRAF, and EGFR-ECD wt) population (part 2). A group sequential design with 3 interim analyses (IA) will allow to stop the trial prematurely for futility at each IA as well as for efficacy at last IA. NCT05223673. Institut de Recherches Internationales Servier.

351P Prevalence of ERBB alterations in colorectal cancer patients using genomic profiling

In colorectal cancer (CRC) resistance to EGFR inhibitors such as cetuximab or panitumumab can involve activating alterations in human epidermal growth factor receptor 2 (ERBB2/HER2). Anti-ERBB2 therapy is indicated in ERBB2-amplified CRC that is RAS and BRAF wild type. ERBB3/4 alterations can also activate HER2 signaling and thus be effective therapy targets. Our study investigates the prevalence of ERBB alterations in patients with CRC, as well as their importance for treatment. The tumor-normal pairing based OncomapTM ExTra assay was employed to sequence CRC samples by whole-exome DNA sequencing to detect single base substitutions, indels, copy number alterations, and structural variants, and calculate tumor mutational burden (TMB) and microsatellite instability (MSI). Clinically actionable alterations, defined as associated with FDA-approved drugs or clinical trial enrollment, were identified. BRAF mutations, and RAS (KRAS, NRAS, HRAS) and ERBB (ERBB2/3/4) amplification or gain-of-function missense mutations were summarized. Of 757 CRC patients assayed, 38 ERBB alterations were found in 37 (4.9%) patients; 32% of the alterations were not in ERBB2 (26 ERBB2, 11 ERBB3, 1 ERBB4). Of the 38 ERBB alterations, 16 (42%) were ERBB2 amplifications and the rest were gain-of-function missense mutations capable of activating the ERBB2 signaling pathway; 1 patient had alterations in two ERBB genes. There were 56 (7.4%) BRAF mutant samples and 375 (49.5%) RAS mutant samples (348 KRAS, 27 NRAS, 0 HRAS), which included 5 (1.3%) KRAS amplifications. Of the 37 patients with an ERBB alteration, 22 (59%) had no RAS or BRAF alteration (17 ERBB2, 3 ERBB3, 1 ERBB4, 1 ERBB2 ERBB3) identifying a subset in which anti-ERBB2 therapy is likely to respond. Further, 15 (39%) had a RAS but no BRAF alteration (8 ERBB2, 7 ERBB3), which may be targeted by a combination of anti-ERBB2 and MEK inhibitors. Presence of both a BRAF and ERBB mutation, which may be targeted by anti-ERBB2 and RAF/MEK inhibitors, was not observed. The results of whole-exome genomic profiling of CRC samples highlight that combinations of gene alterations are critical in the management of CRC and guidance towards appropriate clinical trials.

848P Second-line systemic treatment for patients with advanced melanoma: Results from the prospective real-world study GEM1801

Information regarding charatcteristics and outcomes of second line treatment for patients with advanced melanoma is scarce since no randomized clinical trials are published in the post-targeted and immunotherapy era. Real-world data studies are capital for benchmarking patients´ outcomes in order to design clinical trials in this scenario. GEM1801 is a prospective observational real-world study that currently has included 551 patients with advanced melanoma treated in 37 centers from the Spanish Melanoma Group (GEM). This is a descriptive analysis of basal characteristics, progression free survival (PFS) and overall survival (OS) from patients that received second line treatment in GEM1801. 186 (33.8%) patients received a second line (2L) treatment. For the 186 patients receiving 2L, with a median follow up of 6.4 months (m) (95% CI: 4.9-9.8), median PFS was 4.7 m (95% CI: 3.5-5.8) and median OS 11.3 m (95% CI: 8.6-13.4) from the beginning of 2L. Additionally, 63 (33.9%) patients received treatment beyond 2L. Table summarizes the patients characteristics and their outcomes according to BRAF status and treatments. For the 365 (66.2%) patients that did not receive 2L, the reasons were: 249 (68.2%) no progression with first line; 110 (30.1%) death before 2L; 6 (1.6%) loss of follow up.Table: 848PCHARACTERISTIC AT 2LBRAF Mutated N= 99BRAF wildtype N= 82SEX female N (%)47 (47.5)33 (40.2)age (median, Rank)59 (23-90)69 (29-93)ECOG 0-1 N (%)87 (87.9)78 (95.1)LDH >UNL N (%)34 (34.3)26 (31.7)M1C-D N (%)62 (62.6)40 (48.8)2L targeted therapy (TT): N, (%)44 (44.4)2 (2.4)TT mPFS, m (95% CI)12.4 (6.1-NR)2 (NA-NA)TT mOS, m (95% CI)20 (11.5-NR)2.7 (NA-NA)2L immunotherapy (IT): N (%)46 (46.5)34 (41.5)IT mPFS, m (95% CI)5.3 (2.9-10.8)3.1 (2.3-8.2)IT mOS, m (95% CI)10.9 (3.7-NR)12.5 (8.1-NR)2L Chemotherapy (CT): N, (%)4 (4)29 (35.4)CT mPFS, m (95% CI)0.4 (0-NR)3.2 (2.7-5.5)CT mOS, m (95% CI)0.6 (0.2-NR)5.6 (3.9-15.2)2L non specified: N (%)5 (5.1)13 (15.9)Treatment beyond 2L N (%)27 (27.8)33 (39.3)There are 5 patients with unknown BRAF status. Open table in a new tab There are 5 patients with unknown BRAF status. Second line treatment is still a difficult clinical scenario, especially for patients with BRAF wildtype melanoma and with BRAF mutated that progress after targeted therapy. The results, remarkably for PFS, need to be improved, thereby clinical trials participation is encouraged.

The Genomic Environment of BRAF Mutated and BRAF/PIK3CA Double Mutated Colorectal Cancers.

Background: Colorectal cancer represents the most prevalent gastrointestinal malignancy. Prognosis of metastatic disease has improved in recent years with the introduction of effective systemic therapies, but mean survival remains in the range of two to three years. Targeted therapies based on specific molecular alterations in sub-sets of colorectal cancers have the potential of contributing to therapeutic progress. BRAF and PIK3CA are oncogenic kinases commonly mutated in colorectal cancers and can be targeted through small molecule kinase inhibitors. Methods: Clinical and genomic data from two extensive series of colorectal cancers were interrogated to define the molecular characteristics of cancers with BRAF mutations with and without concomitant mutations in PIK3CA. Results: Colorectal cancers that are BRAF and PIK3CA double mutants represent a small minority of about 5% of colorectal cancers in the two examined series of mostly localized disease. They also represent about one third of all BRAF mutated colorectal cancers. Most mutations in BRAF are classic V600E mutations. A high prevalence of MSI and CIMP is observed in BRAF mutated colorectal cancers with or without PIK3CA mutations. Mutations in tumor suppressors FBXW7 and ATM display a higher prevalence in BRAF mutated cancers. The prognosis of BRAF mutated colorectal cancers with or without PIK3CA mutations is not significantly different than counterparts with wild type BRAF. This contrasts with the known adverse prognostic effect of BRAF in metastatic disease and relates to the different prevalence of MSI in mutant BRAF localized versus metastatic colorectal cancers. Conclusions: BRAF mutations are the defining molecular alterations in double mutant BRAF and PIK3CA colorectal cancers as determined by increased MSI and CIMP in BRAF subsets with and without PIK3CA mutations. Moreover, BRAF mutated cancers with and without PIK3CA mutations are characterized by the absence of KRAS mutations and a lower prevalence of APC mutations than BRAF wild type counterparts. Mismatch-repair-associated gene mutations display higher frequencies in BRAF mutated colorectal cancers. Despite the absence of prognosis implications of BRAF mutations in the studied cohorts of mostly localized cancers, such mutations could be prognostic in certain subsets. The presence of mutations in other genes, such as ATM and high MSI status present opportunities for combination therapies.

Proteasomal down-regulation of the proapoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma.

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase-independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wild-type BRAF protein, binds to MST2 inhibiting its proapoptotic signalling. Down-regulation of MST2 reduces BRAFi-induced apoptosis. In BRAFi-resistant cell lines, MST2 pathway proteins are down-regulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway-induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAFi resistance in melanoma.

MR Imaging Characteristics and ADC Histogram Metrics for Differentiating Molecular Subgroups of Pediatric Low-Grade Gliomas.

BRAF and type 1 neurofibromatosis status are distinctive features in pediatric low-grade gliomas with prognostic and therapeutic implications. We hypothesized that DWI metrics obtained through volumetric ADC histogram analyses of pediatric low-grade gliomas at baseline would enable early detection of BRAF and type 1 neurofibromatosis status. We retrospectively evaluated 40 pediatric patients with histologically proved pilocytic astrocytoma (n = 33), ganglioglioma (n = 4), pleomorphic xanthoastrocytoma (n = 2), and diffuse astrocytoma grade 2 (n = 1). Apart from 1 patient with type 1 neurofibromatosis who had a biopsy, 11 patients with type 1 neurofibromatosis underwent conventional MR imaging to diagnose a low-grade tumor without a biopsy. BRAF molecular analysis was performed for patients without type 1 neurofibromatosis. Eleven patients presented with BRAF V600E-mutant, 20 had BRAF-KIAA rearrangement, and 8 had BRAF wild-type tumors. Imaging studies were reviewed for location, margins, hemorrhage or calcifications, cystic components, and contrast enhancement. Histogram analysis of tumoral diffusivity was performed. Diffusion histogram metrics (mean, median, and 10th and 90th percentiles) but not kurtosis or skewness were different among pediatric low-grade glioma subgroups (P < .05). Diffusivity was lowest in BRAF V600E-mutant tumors (the 10th percentile reached an area under the curve of 0.9 on receiver operating characteristic analysis). There were significant differences between evaluated pediatric low-grade glioma margins and cystic components (P = .03 and P = .001, respectively). Well-defined margins were characteristic of BRAF-KIAA or wild-type BRAF rather than BRAF V600E-mutant or type 1 neurofibromatosis tumors. None of the type 1 neurofibromatosis tumors showed a cystic component. Imaging features of pediatric low-grade gliomas, including quantitative diffusion metrics, may assist in predicting BRAF and type 1 neurofibromatosis status, suggesting a radiologic-genetic correlation, and might enable early genetic signature characterization.

MODUL cohort 2: an adaptable, randomized, signal-seeking trial of fluoropyrimidine plus bevacizumab with or without atezolizumab maintenance therapy for BRAFwt metastatic colorectal cancer

BackgroundMODUL is an adaptable, signal-seeking trial designed to test novel agents in predefined patient subgroups in first-line metastatic colorectal cancer (mCRC).Patients and methodsPatients with measurable, unresectable, previously untreated mCRC received induction with ≤8 cycles of FOLFOX + bevacizumab followed by randomization to maintenance treatment comprising control [fluoropyrimidine (FP)/bevacizumab: 5-fluorouracil 1600-2400 mg/m2 46-h intravenous (i.v.) infusion day 1 q2 weeks plus leucovorin 400 mg/m2 2-h infusion i.v. day 1 q2 weeks or capecitabine 1000 mg/m2 b.i.d. orally days 1-14 every 21 days; bevacizumab 5 mg/kg 15-30-min i.v. infusion q2 weeks] or experimental treatment in one of four biomarker-driven cohorts. In patients with BRAF wild-type (BRAFwt) tumors (cohort 2), experimental treatment was FP/bevacizumab + atezolizumab (800 mg 60-min i.v. infusion q2 weeks). Primary efficacy endpoint was progression-free survival (PFS; intent-to-treat population). Enrollment is complete; efficacy and safety findings from cohort 2 are presented.ResultsFour hundred and forty-five patients with BRAFwt mCRC were randomized (2 : 1) to maintenance in cohort 2. At a median follow-up of 10.5 months, PFS outcome hypothesis was not met [hazard ratio (HR) 0.92; 95% confidence interval (CI) 0.72-1.17; P = 0.48]; overall survival (OS) was immature. At a median follow-up of 20.3 months (2-year survival follow-up), PFS benefit was also not met (HR 0.95; 95% CI 0.77-1.18; P = 0.666); OS HR with nearly two-thirds of patients with events was 0.83 (95% CI 0.65-1.05; P = 0.117). No new safety signals were identified. The most common grade ≥3 treatment-emergent adverse events (TEAEs) for experimental versus control arms were hypertension (6.1% versus 4.2%), diarrhea (3.1% versus 2.1%), and palmar-plantar erythrodysesthesia syndrome (1.0% versus 2.5%). Four patients experienced TEAEs with fatal outcome, two were study treatment-related: hepatic failure (experimental arm) and large intestine perforation (control arm; bevacizumab-related).ConclusionsAdding atezolizumab to FP/bevacizumab as first-line maintenance treatment after FOLFOX + bevacizumab induction for BRAFwt mCRC did not improve efficacy outcomes.

PD-L1+ neutrophils as novel biomarkers for stage IV melanoma patients treated with nivolumab.

Melanoma displays a rising incidence, and the mortality associated with metastatic form remains high. Monoclonal antibodies that block programmed death (PD-1) and PD Ligand 1 (PD-L1) network have revolutionized the history of metastatic disease. PD-L1 is expressed on several immune cells and can be also expressed on human neutrophils (PMNs). The role of peripheral blood PMNs as predictive biomarkers in anti-PD-1 therapy of melanoma is largely unknown. In this study, we aimed to determine activation status and PD-L1 expression on human neutrophils as possible novel biomarkers in stage IV melanoma patients (MPs). We found that PMNs from MPs displayed an activated phenotype and increased PD-L1 levels compared to healthy controls (HCs). Patients with lower PD-L1+ PMN frequencies displayed better progression-free survival (PFS) and overall survival (OS) compared to patients with high PD-L1+ PMN frequencies. Multivariate analysis showed that PD-L1+ PMNs predicted patient outcome in BRAF wild type MP subgroup but not in BRAF mutated MPs. PD-L1+ PMN frequency emerges as a novel biomarker in stage IV BRAF wild type MPs undergoing anti-PD-1 immunotherapy. Our findings suggest further evaluation of the role of neutrophil subsets and their mediators in melanoma patients undergoing immunotherapy.

Discovery of BRAF/HDAC Dual Inhibitors Suppressing Proliferation of Human Colorectal Cancer Cells.

The combination of histone deacetylase inhibitor and BRAF inhibitor (BRAFi) has been shown to enhance the antineoplastic effect and reduce the progress of BRAFi resistance. In this study, a series of (thiazol-5-yl)pyrimidin-2-yl)amino)-N-hydroxyalkanamide derivatives were designed and synthesized as novel dual inhibitors of BRAF and HDACs using a pharmacophore hybrid strategy. In particular, compound 14b possessed potent activities against BRAF, HDAC1, and HDAC6 enzymes. It potently suppressed the proliferation of HT-29 cells harboring BRAFV600E mutation as well as HCT116 cells with wild-type BRAF. The dual inhibition against BRAF and HDAC downstream proteins was validated in both cells. Collectively, the results support 14b as a promising lead molecule for further development and a useful tool for studying the effects of BRAF/HDAC dual inhibitors.

Cytotoxicity, acute and sub-chronic toxicities of the fruit extract of Tetrapleura tetraptera (Schumm. & Thonn.) Taub. (Fabaceae)

BackgroundTetrapleura tetraptera is a medicinal spice traditionally used to treat cancer, diabetes, and several other ailments. This study analyzed the cytotoxicity of the dichloromethane methanol extract of T. tetraptera fruits (TTF) and its constituents. The toxicity profile of the TTF extract was also evaluated in rats.MethodsThe Cytotoxicity of this extract was evaluated using the resazurin reduction assay (RRA). Acute and sub-chronic toxicity studies were performed according to the protocol described by the Organisation for Economic Cooperation, and Development (OECD). Hematological, serum, and urine biochemical parameters, as well as histological sections of the liver and kidney, were also evaluated based on standard methods.ResultsThe TTF extract, compound 5, and the reference drug doxorubicin were active in all 9 tested cancer cell lines. The recorded IC50 ranged from 18.32 μM (against B16-F1 murine melanoma cells) to 36.18 μM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 μM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 μM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 μM (against B16-F1 cells) to 9.39 μM (against SKMel-505 cells) for doxorubicin. The study of acute toxicity test showed that the lethal dose (LD50) of this extract was greater than 5000 mg/kg body weight. In the sub-chronic toxicity studies, variations were observed in some biochemical parameters, especially at higher doses.ConclusionTTF and its most active compound (5) are found to be potential cytotoxic agents, meanwhile, TTF was safe when given a single oral dose of 5000 mg/kg. However, caution is necessary in case of prolonged oral administration due to potential alterations of renal function at high doses (> 1000 mg/kg).

Efficacy of Nivolumab and Ipilimumab Combined Therapy as a First-Line Therapy for Patients with Advanced Melanoma and the Urgent Need for an Effective Second-Line Therapy for Patients with Wild-Type BRAF in Japan: A Single Center Retrospective Study.

Therapeutic advantages of immune checkpoint inhibitors, anti-programmed death-1 (PD-1), and anticytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in melanoma have been reported recently. In this study, we conducted a retrospective study to evaluate the clinical efficacy and safety of the combined use of nivolumab and ipilimumab as a first-line therapy for Japanese patients with advanced melanoma. Moreover, we examined the effects of second-line treatment. Seven patients were enrolled in this study. The median progression-free survival (PFS) and median overall survival (OS) were 7 months (95%CI, 1.868-12.132) and 12 months (95%CI, 0.000- 27.397), respectively. The objective response rate (ORR) and the disease control rate (DCR) were 42.9 % and 85.7 %. Three patients chose pembrolizumab monotherapy as second-line therapy after the combination therapy due to their BRAF wild-type status, which resulted in progressive disease. ORR and DCR were 0% and 33.3%, respectively, with pembrolizumab. Grade 3 or 4 immune-related adverse events occurred in 71.4% of the patients treated with the combined-therapy. All irAEs were treated with corticosteroid or hormone replacement therapy. Although this single center retrospective study had some limitations, it demonstrated good efficacy for the combined use of nivolumab and ipilimumab as a first-line therapy for Japanese patients with advanced melanoma. Moreover, poor efficacy was observed for the second-line therapy after the combined therapy. These findings suggest that a novel second-line therapy is required for patients with advanced melanoma in Japan, particularly for patients with wildtype BRAF.

Knockdown of 15-bp Deletion-Type v-raf Murine Sarcoma Viral Oncogene Homolog B1 mRNA in Pancreatic Ductal Adenocarcinoma Cells Repressed Cell Growth In Vitro and Tumor Volume In Vivo.

Simple SummaryThe v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene containing a 15-base pair (bp) deletion at L485-P490 is the cause of several cancers. We generated siRNA to specifically knock down BRAF mRNA containing the 15-bp deletion. This siRNA suppressed the expression of BRAF, harboring the deletion without affecting wild-type BRAF expression in BxPC-3 pancreatic ductal adenocarcinoma cells in vitro and in vivo. Cell growth and phosphorylation of downstream extracellular-signal-regulated kinase proteins were also repressed. An off-target effect is the most common side effect of siRNA therapy. In this study, we reveal that siRNA with a 2′-O-methyl chemical modification in the seed region of the siRNA guide strand reduced seed-dependent off-target effects.Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second-most common cause of death within the next 10 years. Due to the limited efficacy of available therapies, the survival rate of PDAC patients is very low. Oncogenic BRAF mutations are one of the major causes of PDAC, specifically the missense V600E and L485–P490 15-bp deletion mutations. Drugs targeting the V600E mutation have already been approved by the United States Food and Drug Administration. However, a drug targeting the deletion mutation at L485–P490 of the BRAF gene has not been developed to date. The BxPC-3 cell line is a PDAC-derived cell line harboring wild-type KRAS and L485–P490 deleted BRAF genes. These cells are heterozygous for BRAF, harboring both wild-type BRAF and BRAF with the 15-bp deletion. In this study, siRNA was designed for the targeted knockdown of 15-bp deletion-type BRAF mRNA. This siRNA repressed the phosphorylation of extracellular-signal-regulated kinase proteins downstream of BRAF and suppressed cell growth in vitro and in vivo. Furthermore, siRNAs with 2′-O-methyl modifications at positions 2–5 reduce the seed-dependent off-target effects, as confirmed by reporter and microarray analyses. Thus, such siRNA is a promising candidate therapy for 15-bp deletion-type BRAF-induced tumorigenesis.