Indolent renal involvement with BRAFV600E mutation: Erdheim‐Chester,a rare disease with a wide spectrum of clinical manifestations

We present the case of a patient with three‐year indolent bilateral ureteral and perirenal masses. Clinical presentation, radiological context, and histopathological findings with detection of BRAF^V600E mutation confirmed the diagnosis of Erdheim‐Chester disease (ECD). A review of current knowledge regarding diagnosis, clinical assessment, management, and treatment of ECD is also presented.     

A five‐gene panel refines differential diagnosis of thyroid nodules

BackgroundMolecular testing for oncogenic mutations in fine‐needle aspiration has showed high predictive value in identifying malignant lesions from thyroid nodules with indeterminate cytology.MethodsTo figure out an efficient and economical gene panel for most medical institutions in China, we designed a five‐gene panel including BRAF/NRAS/KRAS/HRAS/TERT genes and conducted a retrospective study to evaluate the role of this five‐gene diagnostic panel in differential diagnosis of thyroid nodules.ResultsA total of 665 patients with 695 thyroid nodules were investigated in the current study. The fine‐needle aspiration biopsy and surgically separated thyroid tissue specimens were harvested to test BRAF, TERT, NRAS, KRAS, and HRAS mutations. We identified 261 mutations in 665 patients, including 177 V600E mutations in BRAF. Three hundred and sixty‐nine patients who underwent thyroid surgery after completion of the initial clinical and cytological evaluation were enrolled in the final analysis. The diagnostic sensitivity, specificity, and accuracy of the combination of FNAB cytology and five‐gene detection were 74.7%, 93.8%, and 84.8%, respectively. BRAF V600E and five‐gene panel could recognize 46.4% and 53.6% of papillary thyroid carcinoma in the patients with cytologically indeterminate nodules.ConclusionThe five‐gene panel can effectively improve the sensitivity, negative predictive value, and accuracy of fine‐needle aspiration biopsy cytology, especially in the patients with cytologically indeterminate nodules.     

Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma

Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTEN^Null melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase–dependent and –independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAF^V600E-induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway–targeted inhibitors. These experiments revealed that mutationally activated PIK3CA^H1047R cooperates with BRAF^V600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAF^V600E/PTEN^Null melanomas in vivo and in tissue culture. Combined inhibition of BRAF^V600E and PI3K had more potent effects on the regression of established BRAF^V600E/PTEN^Null melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAF^V600E/PIK3CA^H1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAF^V600E/PTEN^Null melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAF^V600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway–targeted inhibition of PI3K and BRAF^V600E in the therapeutic management of BRAF^V600E/PTEN^Null melanomas.     

Kwak TI-RADS与BRAFV600E基因突变检测在不确定意义细胞学结果的甲状腺结节中的应用价值

目的：探讨Kwak TI-RADS与BRAF_^V600E基因突变检测对具有不确定意义细胞病理学结果(包含Bethesda Ⅲ、Ⅳ和Ⅴ类)的甲状腺结节良恶性诊断的应用价值。方法：回顾性分析2018年1月～ 2018年12月至我院进行细针穿刺后细胞病理学诊断结果为不确定意义的甲状腺结节患者的常规超声图像与BRAF_^V600E基因突变检测结果的资料,以手术病理结果或临床随访为诊断标准,比较分析Kwak TI-RADS与BRAF_^V600E基因突变检测对具有不确定意义细胞学结果的甲状腺结节良、恶性鉴别诊断的效能。结果：纳入的76例甲状腺结节均经手术病理或临床随访证实。术后病理结果为：甲状腺癌有50例;甲状腺良性肿瘤有26例。BRAF_^V600E基因突变检测阳性诊断甲状腺癌的灵敏度为62.0%,特异度为100%,准确率为75.0%;而与Kwak TI-RADS超声分类诊断标准单独使用时相比,Kwak TI-RADS超声分类诊断联合BRAF_^V600E基因突变检测的灵敏度从78.0%提高到88.0%、特异度从76.9%提高到84.6%、准确率从77.6%提高到86.8%,但差异不具统计学意义(P＞0.05)。 结论：对于不确定意义甲状腺结节的良、恶性鉴别诊断,Kwak TIRADS超声分类诊断具有一定的诊断价值,在Kwak TI-RADS超声分类诊断的基础上联合BRAF_^V600E基因突变检测可以提高其诊断灵敏度、特异度、准确率,为不确定意义甲状腺结节患者的进一步诊疗提供参考依据,减少甲状腺癌的漏诊率。#$NL关键词: 甲状腺结节; Kwak TI-RADS甲状腺影像学报告及数据系统; BRAF_^V600E 基因突变     

BRAFV600E detection in melanoma is highly improved by COLD-PCR

BackgroundThe BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAF^V600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues.MethodsCOLD-PCR was used to selectively amplify BRAF^V600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations.ResultsWe used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAF^V600E were found positive after COLD-PCR.ConclusionsCOLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.     

Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing

BackgroundMismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision‐making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories.MethodsCRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next‐generation sequencing (NGS) and were then used to prepare formalin‐fixed, paraffin‐embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing.ResultsWe successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples.ConclusionsWe successfully established MLH1 protein‐deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.     

Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients

BackgroundBRAF is the most frequently mutated oncogene in melanoma with BRAF^V600E mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy.MethodsWe propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAF^V600E concentration in patients affected by cutaneous melanoma (n = 55) and non-melanoma skin cancers (n = 13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma.ResultsA significant difference between the control group and invasive melanomas (p < 0.01) was evidenced in BRAF^V600E concentration, either as relative percentage or absolute values. ROC curve indicated that BRAF^V600E absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance.ConclusionsThe allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAF^V600E mutated DNA in plasma.     

Gaussian field-based 3D-QSAR and molecular simulation studies to design potent pyrimidine–sulfonamide hybrids as selective BRAFV600E inhibitors

The “RAS-RAF-MEK-ERK” pathway is an important signaling pathway in melanoma. BRAF^V600E (70–90%) is the most common mutation in this pathway. BRAF inhibitors have four types of conformers: type I (αC-IN/DFG-IN), type II (αC-IN/DFG-OUT), type I_1/2 (αC-OUT/DFG-IN), and type I/II (αC-OUT/DFG-OUT). First- and second-generation BRAF inhibitors show resistance to BRAF^V600E and are ineffective against malignancies induced by dimer BRAF mutants causing ‘paradoxical’ activation. In the present study, we performed molecular modeling of pyrimidine–sulfonamide hybrids inhibitors using 3D-QSAR, molecular docking, and molecular dynamics simulations. Previous reports reveal the importance of pyrimidine and sulfonamide moieties in the development of BRAF^V600E inhibitors. Analysis of 3D-QSAR models provided novel pyrimidine sulfonamide hybrid BRAF^V600E inhibitors. The designed compounds share similarities with several structural moieties present in first- and second-generation BRAF inhibitors. A total library of 88 designed compounds was generated and molecular docking studies were performed with them. Four molecules (T109, T183, T160, and T126) were identified as hits and selected for detailed studies. Molecular dynamics simulations were performed at 900 ns and binding was calculated. Based on molecular docking and simulation studies, it was found that the designed compounds have better interactions with the core active site [the nucleotide (ADP or ATP) binding site, DFG motif, and the phospho-acceptor site (activation segment) of BRAF^V600E protein than previous inhibitors. Similar to the FDA-approved BRAF^V600E inhibitors the developed compounds have [αC-OUT/DFG-IN] conformation. Compounds T126, T160 and T183 interacted with DIF (Leu505), making them potentially useful against BRAF^V600E resistance and malignancies induced by dimer BRAF mutants. The synthesis and biological evaluation of the designed molecules is in progress, which may lead to some potent BRAF^V600E selective inhibitors.

Design of pyrimidine–sulfonamide hybrids as selective BRAF^V600E inhibitors using 3D-QSAR, molecular docking and MD simulations.     

Metastatic papillary thyroid cancer to cerebellum with incidental medullary microcarcinoma

Thyroid cancer is the most common endocrine cancer, with papillary thyroid carcinoma (PTC) accounting for the majority of these cases. Cerebellar metastasis is rarely the presenting feature and confers poor prognosis. Genetic mutations in this setting are most commonly TERTp, in contrast to BRAF ^V600E in the majority of PTC. We report the case of an 82 year‐old male who presented with a symptomatic right cerebellar lesion and underwent surgical resection to demonstrate metastatic PTC. Extensive workup with computed tomography, neck ultrasound and FDG‐PET was suggestive of a left thyroid primary lesion, with FNA confirming PTC. However, total thyroidectomy demonstrated incidental microMTC (medullary thyroid microcarcinoma, defined as tumour <10mm) without any evidence of PTC, whereas the left level VI neck dissection demonstrated a 30mm nodule of PTC without identifiable normal thyroid or lymph node tissue.     

Novel variants underlying autosomal recessive neurodevelopmental disorders with intellectual disability in Iranian consanguineous families

BackgroundIntellectual disability (ID) is a heterogeneous group of neurodevelopmental disorders that is characterized by significant impairment in intellectual and adaptive functioning with onset during the developmental period. Whole‐exome sequencing (WES)‐based studies in the consanguineous families with individuals affected with ID have shown a high burden of relevant variants. So far, over 700 genes have been reported in syndromic and non‐syndromic ID. However, genetic causes in more than 50% of ID patients still remain unclear.MethodsWhole‐exome sequencing was applied for investigation of various variants of ID, then Sanger sequencing and in silico analysis in ten patients from five Iranian consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders, intellectual disability, performed for confirming the causative mutation within the probands. The most patients presented moderate‐to‐severe intellectual disability, developmental delay, seizure, speech problem, high level of lactate, and onset before 10 years.ResultsFiltering the data identified by WES, two novel homozygous missense variants in FBXO31 and TIMM50 genes and one previously reported mutation in the CEP290 gene in the probands were found. Sanger sequencing confirmed the homozygote variant''s presence of TIMM50 and FBXO31 genes in six patients and two affected siblings in their respective families. Our computational results predicted that the variants are located in the conserved regions across different species and have the impacts on the protein stability.ConclusionHence, we provide evidence for the pathogenicity of two novel variants in the patients which will expand our knowledge about potential mutation involved in the heterogeneous disease.     

Analysis of anti‐apoptotic PVT1 oncogene and apoptosis‐related proteins (p53, Bcl2, PD‐1, and PD‐L1) expression in thyroid carcinoma

BackgroundAn aberrant expression of long non‐coding RNA PVT1 has been associated with apoptosis in various cancer types. We aimed to explore the PVT1 and four apoptosis‐related proteins (p53, Bcl2, and PD‐1/PD‐L1) signature in thyroid cancer (TC).MethodsThe PVT1 expression level was measured in 64 FFPE TC paired samples by real‐time quantitative PCR. Overall and stratified analyses by different clinicopathological features were done. The apoptotic proteins were evaluated by immunohistochemistry staining.ResultsOverall analysis showed significant PVT1upregulation in TC tissues (p < 0.001). Similarly, subgroup analysis by BRAF ^V600E mutation showed consistent results. Lower expression of p53 was associated with mortality (p = 0.001). Bcl2 overexpression was associated with greater tumor size (p = 0.005). At the same time, HCV‐positive cases were associated with repressed Bcl2 expression levels (54.3% in HCV‐negative vs. 6.9% in HCV‐positive cases, p = 0.011). PD‐1 expression was associated with lymph node metastasis (p = 0.004). Enhanced PD‐L1 expression in the tumor was associated with a higher tumor stage, lymphovascular invasion, and mortality risk. Kaplan–Meier curves for overall survival showed that low p53 and high PD‐L1 expressions were associated with lower survival time. The p53‐positive staining is associated with a 90% decreased mortality risk (HR = 0.10, 95%CI = 0.02–0.47, p = 0.001), while patients with high PD‐L1 were five times more likely to die (HR = 4.74, 95%CI = 1.2–18.7, p = 0.027).ConclusionOur results confirm the upregulation of PVT1 in TC. The apoptosis‐related proteins (p53, Bcl2, and PD‐1/PD‐L1) showed different prognostic utility in TC patients; in particular, low p53 and high PD‐L1 expressions associated with low survival times. Further large‐scale and mechanistic studies are warranted.     

Evaluation of LAMP assay using phenotypic tests and PCR for detection of blaKPC gene among clinical samples

BackgroundCarbapenem‐resistant Enterobacteriaceae (CRE) infection constitutes a public health threat, which blaKPC was the major carbapenemases concerned in China. Timely and efficient diagnosis is of paramount importance for controlling the spread of drug‐resistant bacteria. Here, we develop an approach based on loop‐mediated isothermal amplification (LAMP) for rapid confirmation of blaKPC within 60 min from samples collected.MethodsWe designed primers specific to detect blaKPC and evaluated it for its sensitivity and specificity of detection using real‐time monitoring. Five hundred forty‐six clinical specimens were analyzed by the LAMP assay and compared with the phenotypic tests and PCR. The samples with inconsistent results were further verified by Sanger sequencing.ResultsThe LAMP assay displayed a detection limit of 1 × 10² CFU/ml, which was 10‐fold more sensitive than the PCR. No cross‐reactivity was observed for strains that produced other types of β‐lactamase. Furthermore, we demonstrated concordant results (Kappa > 0.75) between the genotypic method and phenotypic tests for the 546 clinical samples. The data presented in this study suggested that the genotypic method is a reliable assay for identifying blaKPC‐induced CRE in China. The results of the Sanger sequencing indicate that the developed method not only has high accuracy but also meets the need for rapid diagnosis, while the PCR method is prone to false negatives.ConclusionsWe successfully constructed a LAMP technique that can be used for auxiliary diagnosis of CRE, which is faster, cheaper, and more accurate than the PCR. It may therefore be routinely applied for detection of blaKPC producers in routine clinical laboratories.     

BRAF Activation Initiates but Does Not Maintain Invasive Prostate Adenocarcinoma

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF^V600E–a mutation found in ~10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF^V600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF^V600E is not required for its maintenance.     

A novel heterozygous HTRA1 mutation in an Asian family with CADASIL‐like disease

Background HTRA1 gene mutations are related to the pathogenesis of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). However, heterozygous HTRA1 mutations at specific sites can also lead to rare autosomal dominant cerebral artery disease (CADASIL‐like disease). To date, 28 heterozygous mutations in the HTRA1 gene have been reported to be related to CADASIL‐like diseases. Only one case of this disease was caused by a heterozygous mutation of c.497G>T in exon 2 of the HTRA1 gene.MethodsIn this case, we report on an Asian family with CADASIL‐like disease caused by a heterozygous mutation of c.497G>T in exon 2 of the HTRA1 gene. The clinical and imaging characteristics of the proband were summarized, and gene mutations were verified by whole‐exome sequencing (WES) and direct Sanger sequencing.ResultsThe result of the gene sequencing showed a heterozygous missense mutation at the c.497G>T locus of the HTRA1 gene in the proband of one sick family member, resulting in a change in amino acid (p.arg166leu).ConclusionThis is the first reported pathogenic mutation at the c.497G>T locus of the HTRA1 gene in an Asian population. It provides an important theoretical basis for the specific gene‐based diagnosis and treatment of CADASIL‐like diseases.     

Conventional Ultrasound,Immunohistochemical Factors and BRAFV600E Mutation in Predicting Central Cervical Lymph Node Metastasis of Papillary Thyroid Carcinoma

The study was aimed at evaluating the correlation between central cervical lymph node metastasis (CLNM) in papillary thyroid carcinoma (PTC) patients and ultrasound (US) features, immunohistochemical factors and BRAF^V600E mutation. A total of 225 consecutive patients (225 PTCs) who had undergone surgery were included. All PTCs were pre-operatively analysed by US with respect to size, components, echogenicity, shape, margins, microcalcification, multiple cancers or not, internal vascularity and capsule contact or involvement. The presence of four immunohistochemical factors, including cytokeratin 19, human bone marrow endothelial cell 1, galectin-3 and thyroid peroxidase, and BRAF^V600E mutation was also evaluated. Univariate and multivariate analyses were performed to identify the risk factors for central CLNM, and a risk model was established. Pathologically, 44% (99/225) of the PTCs had central CLNMs. Multivariate analysis revealed that size ≤10mm, microcalcification, internal vascularity, capsule contact or involvement and BRAF^V600E mutation were independent risk factors for central CLNM. The risk score for central CLNM was calculated as follows: risk score?=?1.5?×?(if lesion size ≤10 mm)?+?1.9?×?(if microcalcification)?+?0.8?×?(if internal flow)?+?3.0?×?(if capsule contact or involvement)?+?1.5?×?(if BRAF^V600E mutation). The rating result was divided into six stages, and the relevant risk rates of central CLNM were 0% (0/1), 0% (0/22), 7.4% (4/54), 48.6% (34/70), 71.2% (42/59) and 100% (19/19), respectively. In conclusion, PTC ≤10mm, microcalcification, internal vascularity, capsule contact or involvement and BRAF^V600E mutation are risk factors for central CLNM. The risk model may be useful in treatment planning and management of patients with PTCs.     

Clinical and molecular genetic analysis of a Chinese family with hereditary elliptocytosis caused by a novel mutation in the EPB41 gene

BackgroundHereditary elliptocytosis (HE) is a heterogeneous red blood cell membrane disorder characterized by the presence of elliptocytes on a peripheral blood smear. Clinical manifestations of HE vary widely from asymptomatic carriers to patients with severe transfusion‐dependent anemia. Most patients are asymptomatic or have mild anemia, which hinders diagnosis. The proband in this case had mild anemia and jaundice over a period of 4 years, the etiology of which was unclear. Hence, he was admitted to our hospital for further diagnosis.MethodsPeripheral blood smears and routine blood tests were performed and biochemical parameters of the proband, and his family members were determined. To confirm the diagnosis, gene mutations were screened in the proband using next‐generation sequencing (NGS) and verified by Sanger sequencing in other family members.ResultsA novel mutation (c.1294delA, p.Ser432 fs) in exon 15 of the EPB41 gene was detected in the proband and his family members. This mutation results in a frameshift and a premature stop codon at position 455, encoding a truncated protein. The variant was likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. SWISS‐MODEL protein structure prediction indicated partial loss of the spectrin and actin binding and C‐terminal domains.ConclusionA heterozygous mutation 1294delA in exon 15 of the EPB41 gene was identified using NGS and Sanger sequencing in members of a Chinese family. This identification expands the spectrum of EPB41 mutations and contributes to the genetic diagnosis of families with HE.     

A case of CADASIL caused by NOTCH3 c.512_605delinsA heterozygous mutation

BackgroundAutosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebrovascular disease closely related to the NOTCH3 gene. More than 200 mutations in this gene have been reported to be associated with this disease.MethodsThe NOTCH3 gene from CADASIL patient was screened for mutations by whole‐exome sequencing (WES). PCR amplification and direct Sanger sequencing were used to verify the suspicious gene mutation sites detected by WES.ResultsWe performed second‐generation sequencing on a sample of the patient''s genome and found a heterozygous deletion‐insertion mutation c.512_605delinsA in exon 4 of NOTCH3, which resulted in amino acid changes p.G171_A202delinsE. This variation was confirmed by the direct Sanger sequencing. It may be rated as a CADASIL clinical variation.ConclusionDiscovery of this mutation site provides an important theoretical basis for specific gene‐based diagnosis and treatment of CADASIL.     

Dangerous liaisons: flirtations between oncogenic BRAF and GRP78 in drug-resistant melanomas

BRAF mutations in aggressive melanomas result in kinase activation. BRAF inhibitors reduce BRAF^V600E tumors, but rapid resistance follows. In this issue of the JCI, Ma and colleagues report that vemurafenib activates ER stress and autophagy in BRAF^V600E melanoma cells, through sequestration of the ER chaperone GRP78 by the mutant BRAF and subsequent PERK activation. In preclinical studies, treating vemurafenib-resistant melanoma with a combination of vemurafenib and an autophagy inhibitor reduced tumor load. Further work is needed to establish clinical relevance of this resistance mechanism and demonstrate efficacy of autophagy and kinase inhibitor combinations in melanoma treatment.     

Host immunity contributes to the anti-melanoma activity of BRAF inhibitors

The BRAF mutant, BRAF^V600E, is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF^V600E metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF^V600E-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf^V600E-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf^V600E mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8⁺ T/FoxP3⁺CD4⁺ T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.     

Prognostic value of BRAF/MIR‐17 signature and B‐Raf protein expression in patients with colorectal cancer: A pilot study

BackgroundDespite the recent improvement in colorectal cancer (CRC) treatment, it still has a poor prognosis with a low survival rate. Genetic and epigenetic mechanisms have proved to play a substantial role in CRC tumorigenesis and progression. According to Gene Ontology and TargetScan analyses, the B‐Raf proto‐oncogene (BRAF) gene is one of the microRNA‐17 (miR‐17) targets. We aimed to explore the prognostic value of B‐Raf protein and BRAF/microRNA‐17 (MIR‐17) gene expression signature in CRC archived samples.MethodsB‐Raf protein expression was identified by immunohistochemistry, while gene expression studies were quantified by real‐time qPCR in 53 paired archived CRC specimens.ResultsThe BRAF showed higher expressions in CRC specimens relative to non‐cancer tissues (p = 0.006). MIR17 expression was inversely and significantly correlated with both B‐Raf protein (r = −0.79, p < 0.001) and gene expression (r = −0.35, p = 0.010) and showed a significant direct correlation with a high rate of relapse (p = 0.020). BRAF/miR‐17 expression in CRC was associated inversely with tumor size, high grade of colonic carcinoma, lymph node metastasis, and carcinoma subtype. Spearman correlation and Kaplan‐Meier survival curve analyses revealed that disease‐free survival and overall survival were inversely and significantly correlated with positive B‐Raf protein expression (r = −0.31 and −0.35, p = 0.023 and 0.011, respectively) and directly correlated with log BRAF/MIR17 ratio (r = 0.50 and 0.41, p < 0.001 and = 0.003, respectively). Cox hazard regression analysis revealed the BRAF/MIR17 ratio could predict both types of patients'' survival, among other variables.Conclusion BRAF/MIR17 ratio could have prognostic utility in patients with CRC. Further larger‐scale studies are warranted to confirm this utility.