BRAFV600E: implications for carcinogenesis and molecular therapy

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is frequently mutated in human cancer. This pathway consists of a small GTP protein of the RAS family that is activated in response to extracellular signaling to recruit a member of the RAF kinase family to the cell membrane. Active RAF signals through MAP/ERK kinase to activate ERK and its downstream effectors to regulate a wide range of biological activities including cell differentiation, proliferation, senescence, and survival. Mutations in the v-raf murine sarcoma viral oncogenes homolog B1 (BRAF) isoform of the RAF kinase or KRAS isoform of the RAS protein are found as activating mutations in approximately 30% of all human cancers. The BRAF pathway has become a target of interest for molecular therapy, with promising results emerging from clinical trials. Here, the role of the most common BRAF mutation BRAF(V600E) in human carcinogenesis is investigated through a review of the literature, with specific focus on its role in melanoma, colorectal, and thyroid cancers and its potential as a therapeutic target.     

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.     

超声引导下细针抽吸细胞学检查联合BRAFV600E基因检测诊断甲状腺微小乳头状癌的价值

目的 探讨超声引导下细针抽吸细胞学检查(FNAC)联合BRAFV600E基因检测对甲状腺微小乳头状癌(PTMC)的术前诊断价值.方法 选取我院经手术病理确诊的PTMC患者126例,共计126个结节,按结节二维超声所测最大径分为≤5 mm组31个,5～10 mm组95个,比较两组FNAC、BRAFV600E基因检测诊断结果,分析二者联合诊断价值.结果 5～10 mm组FNAC的诊断准确率(86.32％)明显高于≤5 mm组(58.06％),两组比较差异有统计学意义(P<0.05);5～10 mm组与≤5 mm组BRAFV600E基因突变率分别为92.63％、90.32％,两组比较差异无统计学意义;FNAC联合BRAFV600E基因检测诊断≤5 mm组、5～10 mm组的准确率分别为93.54％、97.78％,均明显高于单独FNAC诊断,差异均有统计学意义(均P<0.05).结论 FNAC联合BRAFV600E基因检测可提高PTMC术前诊断准确率,且对5～10 mm PTMC诊断准确率更高.     

超声引导下FNAC联合BRAFV600E基因突变检测在甲状腺结节诊断中的意义

目的探讨超声引导下细针穿刺细胞学(UG-FNAC)联合BRAFV600E基因突变检测在甲状腺结节术前诊断中的作用。方法回顾性分析300例甲状腺结节UG-FNAC检查结果及BRAFV600E基因突变检测结果,同时对其中120例手术切除病例的组织病理学进行分析。结果300例UG-FNAC结果按照Bethesda报告系统(2017版)进行分类,Ⅰ类:25例,占8.3%;Ⅱ类:53例,占17.7%;Ⅲ类:14例,占4.7%;Ⅳ类:7例,占2.3%;Ⅴ类:20例,占6.7%;Ⅵ类:181例,占60.3%。300例FNAC样本中,BRAF V600E检测173例(57.7%)为突变型,127例(42.3%)为野生型。120例手术切除病例的组织病理学结果,良性病变6例,占5%;恶性肿瘤114例,占95%;BRAFV600E在甲状腺乳头状癌(PTC)中的突变率为87.5%。结论UG-FNAC联合BRAFV600E基因突变检测是诊断甲状腺结节良、恶性准确有效的方法,具有重要的临床指导作用。     

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.     

Differentiated thyroid gland carcinoma in a scintigraphically hot thyroid nodule: diagnosis and interdisciplinary therapeutic management]

A hyperfunctioning differentiated thyroid carcinoma is a rare occurrence. Nevertheless, this diagnosis must be considered in a scintigraphically hot nodule if there is a clinical or sonographic suggestion of malignancy. The case of a 57-year old patient with hyperthyreosis and a scintigraphically hot thyroid nodule is presented. Further evaluation led to the diagnosis of a differentiated thyroid carcinoma with extensive lymph node and pulmonary metastases (pT2b, pN1b, pM1). The scintigraphically hot nodule corresponded to the primary tumor, whereas scintigraphic detection of the lymph node metastases was only possible postoperatively. Extensive resection of the lymph node metastases was achieved by the intraoperative application of a gamma probe (2nd operation). This allowed sufficient uptake of radioiodine in the pulmonary metastases for their detection and subsequent devitalisation by radioiodine therapy. Complete elimination of all tumour tissue was documented at a control follow-up after six months. Gamma probe-guided surgery may allow for additional removement of non-palpable lymph node metastases. In selected cases this may optimize the surgical results and thereby facilitate the subsequent radioiodine elimination of advanced differentiated thyroid carcinomas.     

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.     

甲状腺细针穿刺残留物标本BRAF V600E突变检测联合细胞学诊断在甲状腺乳头状癌中的诊断价值

目的 探讨使用甲状腺细针穿刺(FNA)残留物标本检测BRAF V600E突变的可行性及其联合细胞学诊断在甲状腺乳头状癌中的诊断价值.方法 回顾性收集2017年3月至2018年12月的1674例进行甲状腺FNA、具有完整细胞学诊断及FNA残留物标本BRAF V600E突变检测结果的患者信息,其中876例患者进行了手术切除...     

The role of BRAF(V600E) mutation as poor prognostic factor for the outcome of patients with intrathyroid papillary thyroid carcinoma

Background

BRAF(V600E) mutation, which represents the most frequent genetic mutation in papillary thyroid carcinoma (PTC), is widely considered to have an adverse outcome on PTC outcome, however its real predictive value is not still well stated. The aim of the present study was to evaluate if BRAF(V600E) mutation could be useful to identify within patients with intrathyroid ultrasound-N0 PTC those who require more aggressive treatment, by central neck node dissection (CLND) or subsequent postoperative ¹³¹I treatment.

Methods

Among the whole series of 931 consecutive PTC patients operated on at 2nd Clinical Surgery of University of Padova and at General Surgery Department of University of Trieste during a period from January 2007 to December 2012, we selected 226 patients with an intrathyroid tumor and no metastases (preoperative staging T1–T2, N0, M0). BRAF(V600E) mutation was evaluated by PCR-single-strand conformation polymorphism analysis and direct genomic sequencing. We analyzed the correlation between the presence/absence of the BRAF(V600E) mutation in the fine-needle aspiration (FNA) and the clinical-pathological features: age, gender, extension of surgery, node dissection, rate of cervical lymph node involvement, tumor size, TNM stage, variant of histotype, mono/plurifocality, association with lymphocitary chronic thyroiditis, radioactive iodine ablation doses, and outcome.

Results

The BRAF(V600E) mutation was present in 104 of 226 PTC patients (47.8%). BRAF(V600E) mutation correlated with multifocality, more aggressive variants, infiltration of the tumoral capsule, and greater tumor's diameter. BRAF(V600E) mutation was the only poor prognostic factor in these patients.

Discussion

In our series, BRAF(V600E) mutation demonstrated to be an adverse prognostic factor indicating aggressiveness of disease and it could be useful in the management of low-risk PTC patients, as supplementary prognostic factor to assess the preoperative risk stratification with the aim to avoid unnecessary central neck node dissection (BRAF pos.) or to perform complementary ¹³¹I-therapy (BFAF neg.).     

Dedifferentiated thyroid cancer: A therapeutic challenge

Human papillary dedifferentiated thyroid cancer (HPDTC) represents a therapeutic dilemma. Targeted therapy (RET proto-oncogene or BRAF-targeting drugs) are promising treatments for HPDTC.     

Anemia in cancer: therapeutic implications and interventions

Significant progress has been made in the prevention and management of many symptoms associated with cancer and its therapy. Anemia in cancer may be secondary to blood loss, displacement of normal bone marrow cells by malignant cells, myelotoxic therapy, or the tumor itself. Practitioners may not always adequately assess anemia unless it represents a source of significant symptoms or patient distress. Risk factors include platinum-based treatment regimens, specific tumor types, and low baseline hemoglobin levels. Anemia may have an impact on patient performance status, quality of life, clinical symptoms, and possibly therapeutic efficacy and survival. Treatment interventions, directed toward the underlying etiology of the anemia, involve iron supplementation, blood transfusion, and administration of recombinant human erythropoietin. Future advances may include new tools to assess physical or functional symptoms and predict therapeutic response more accurately, and more cost-effective, convenient agents to prevent or treat anemia in cancer. Novel approaches that may add to the armamentarium of strategies designed to address anemia in patients with cancer currently are being developed.     

Kwak TI-RADS分类联合BRAF^V600E基因突变检测对不确定意义细胞学诊断结果的甲状腺结节的诊断价值

目的探讨Kwak TI-RADS分类联合BRAF^V600E基因突变检测诊断不确定意义细胞学诊断结果的甲状腺结节良恶性的应用价值。方法选取于我院行细针穿刺细胞学诊断且结果为不确定意义的76例甲状腺结节患者,均行常规超声和BRAF^V600E基因突变检测,以手术病理结果或临床随访为标准,绘制受试者工作特征(ROC)曲线,计算并比较Kwak TI-RADS分类与BRAF^V600E基因突变检测鉴别不确定意义细胞学诊断结果的甲状腺结节良恶性的诊断效能。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,包括恶性结节50例,良性结节26例。Kwak TI-RADS分类诊断甲状腺恶性结节的敏感性78.0%,特异性76.9%,准确率77.6%,曲线下面积0.775;BRAF^V600E基因突变检测的诊断敏感性62.0%,特异性100%,准确率75.0%,曲线下面积0.810;二者联合诊断的敏感性88.0%,特异性84.6%,准确率86.8%,曲线下面积0.873,与两者单独应用比较,差异均无统计学意义。结论Kwak TI-RADS分类联合BRAF^V600E基因突变检测可以一定程度上提高对不确定意义细胞学诊断结果的甲状腺结节良恶性的诊断效能,有一定的临床价值。     

Metabolic syndrome: definition and therapeutic implications

The collection of impaired glucose metabolism, central obesity, elevated blood pressure, and dyslipidemia is identified as metabolic syndrome (MetS). It is estimated that approximately 25% of the world's population has MetS. In the United States, MetS is more common in men and Hispanics, and its incidence increases with age. Metabolic syndrome increases the risk of developing cardiovascular disease and type 2 diabetes mellitus. The underlying risk factors include insulin resistance and abdominal obesity. Confusion about MetS exists in part due to the lack of a consensus definition and treatment protocol. Treatment of MetS begins with therapeutic lifestyle changes and then pharmacologic treatment of the syndrome's individual components. Effective interventions include diet modification, exercise, and use of pharmacologic agents to treat risk factors. Weight loss and increasing physical activity significantly improve all aspects of MetS. A diet that includes more fruits, vegetables, whole grains, monounsaturated fats, and low-fat dairy products will benefit most patients with MetS. Physicians can be most effective in advising patients by customizing specific lifestyle recommendations after assessing patients for the presence of risk factors.     

Regulation of c-Raf-1: therapeutic implications

The mitogen activated protein kinase (MAPK) or Ras/Raf/MAPK kinase (MEK)/ERK signaling cascade is a ubiquitously expressed intracellular signaling pathway that transmits mitogenic stimuli to the nucleus through a series of sequential phosphorylation events and controls such cellular functions as proliferation, differentiation, and apoptosis. Components of this pathway such as ras and raf are oncogenes and as such aberrancy in their encoded proteins results in malignant transformation. The MAPK pathway is dysregulated in approximately 30% of all human tumors and therefore targeting specific components of this pathway that regulate pleiotropic cellular processes using such strategies as isoprenylation inhibitors, antisense oligodeoxyribonucleotides, and inhibitors of the kinase function represent attractive therapeutic options. raf kinase, a downstream effector of ras, has been known to be functionally aberrant in various human tumors. The 3 isoforms of raf have been studied extensively, and agents targeting c-raf are presently undergoing early-phase clinical testing. The outcomes of these trials have wide-ranging clinical implications in the management of cancers. This review addresses the rationale for targeting raf, the diverse cellular functions regulated by c-raf, and the current status of various pharmacological approaches targeting c-raf.     

C-peptide: new findings and therapeutic implications in diabetes

In contrast to earlier views, new data indicate that proinsulin C-peptide exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. C-peptide in nanomolar concentrations binds specifically to cell membranes, probably to a G-protein coupled receptor. Ca(2+)- and MAP-kinase dependent signalling pathways are activated, resulting in stimulation of Na(+), K(+)-ATPase and endothelial nitric oxide (NO) synthase, two enzyme systems known to be deficient in diabetes. C-peptide may also interact synergistically with insulin signal transduction. Studies in intact animals and in patients with type 1 diabetes have demonstrated multifaceted effects. Thus, C-peptide administration in streptozotocin-diabetic animals results in normalization of diabetes-induced glomerular hyperfiltration, reduction of urinary albumin excretion and diminished glomerular expansion. The former two effects have also been observed in type 1 diabetes patients given C-peptide in replacement dose for up to 3 months. Peripheral nerve function and structure are likewise influenced by C-peptide administration; sensory and motor nerve conduction velocities increase and nerve structural changes are diminished or reversed in diabetic rats. In patients with type 1 diabetes, beneficial effects have been demonstrated on sensory nerve conduction velocity, vibration perception and autonomic nerve function. C-peptide also augments blood flow in several tissues in type 1 diabetes via its stimulation of endothelial NO release, emphasizing a role for C-peptide in maintaining vascular homeostasis. Continued research is needed to establish whether, among the hormones from the islets of Langerhans, C-peptide is the ugly duckling that--nearly 40 years after its discovery--may prove to be an endogenous peptide hormone of importance in the treatment of diabetic long-term complications.