BRAF基因V600E突变与Ⅰ～Ⅱ期乳头状甲状腺癌临床病理特征及预后的相关性分析

目的 探讨Ⅰ～Ⅱ期乳头状甲状腺癌（PTC）患者肿瘤组织中BRAF基因V600E突变情况,并分析其与临床病理特征及预后的关系。方法 采用RT-PCR法检测374例Ⅰ～Ⅱ期PTC患者组织样本中BRAF基因V600E突变,并分析其与临床病理特征及预后的关系。 结果 374例PTC患者标本中BRAF基因V600E的突变率为40.37％（151／374）。BRAF基因V600E突变与包膜侵犯、多灶、多次131I治疗相关（P＜0.05）。374例 PTC患者中31例复发,复发患者中有BRAF基因V600E突变的22例。31例复发病例中,BRAF基因V600E突变患者的中位生存期为5.3年（95％CI：3.9～6.7年）,未突变患者为7.0年（95％CI：5.1～8.9年）,差异无统计学意义（P＞0.05）。Logistic多因素回归分析显示,BRAF基因V600E突变及包膜侵犯是Ⅰ～Ⅱ期PTC患者复发的独立危险因素（P＜0.05）。 结论 BRAF基因V600E突变可能是Ⅰ～Ⅱ期PTC患者预后的一项重要预测指标。     

结直肠癌Ras、BRAF和PIK3CA基因突变分析及与临床病理特征的关系

目的 分析结直肠癌患者中Ras（K-Ras／N-Ras）、BRAF和PIK3CA基因突变情况及其与临床病理特征的关系。方法 回顾性分析2013年12月至2014年10月于北京大学肿瘤医院消化肿瘤内科接受诊治的200例结直肠癌患者的肿瘤组织标本,采用PCR扩增-直接测序法检测Ras,包括K-Ras（第2、3、4外显子）、N-Ras（第2、3、4外显子）、BRAF（第15外显子）及PIK3CA（第9、20外显子）基因的突变状态,分析其与结直肠癌临床病理特征的关系。结果 200例患者中存在Ras基因突变92例（46.0％）,其中K-Ras基因突变87例（43.5％）,主要发生在外显子2,N-Ras基因突变5例（2.5％）;其中1例患者存在K-Ras、N-Ras基因同时突变。存在BRAF基因突变15例（7.5％）,突变类型均为V600E,且与K-Ras突变存在排他性。存在PIK3CA基因突变9例（4.5％）,可与Ras或BRAF基因突变共存。Ras（K-Ras／N-Ras）基因在年龄≥65岁患者中的突变率明显高于＜65岁者（P＜0.05）,其表达与性别、原发部位、组织学类型、分化程度、TNM分期、区域淋巴结转移、远处转移、术后复发转移均无关（P均＞0.05）。BRAF、PIK3CA基因在原发部位为右半结肠患者中的突变率明显升高（P＜0.05）,但与年龄、性别、组织学类型、分化程度、TNM分期、区域淋巴结转移、远处转移、术后复发转移均无关（P＞0.05）。结论 N-Ras、PIK3CA基因在中国结直肠癌患者中的突变率较低。K-Ras、N-Ras基因突变与年龄相关,BRAF、PIK3CA基因与肿瘤原发部位相关;对结直肠癌患者进行Ras（K-Ras／N-Ras）、BRAF及PIK3CA基因的联合检测将会为提高临床靶向治疗的疗效提供更加可靠的依据。     

DEK 的表达与结直肠癌患者预后的相关性研究

目的：采用免疫组化技术,分析DEK基因在结直肠癌患者中的表达情况及其与预后的相关性。方法：运用免疫组化SP法,检测DEK基因在169例结直肠癌患者肿瘤组织及癌旁组织中的表达情况,并运用统计分析软件分析DEK表达水平与结直肠癌患者预后的相关性。结果：DEK的表达率在结直肠癌组织（85／169,50．30％）明显高于癌旁正常组织（28／169,16．57％）,差异具有统计学意义（P〈0．01）。DEK在肿瘤组织中的表达情况与病人年龄、性别、肿瘤分化程度、TNM分期及肿瘤大小等因素无相关性,与总生存率（OS）显著相关（P〈0．05）。结论：DEK表达水平与结直肠癌预后密切相关,DEK高表达的结直肠癌患者预后较差。     

结直肠癌组织EGFR基因表达及其与临床意义的研究

目的：通过对结直肠癌上皮生长因子受体（epidermal growth factor receptor，EGFR）基因的表达及其在体细胞突变的研究，探讨EGFR基因的表达与临床病理特征的关系。方法：收集解放军总医院病理科2004～2006年手术治疗的675例结直肠癌患者的临床及病理资料，采用免疫组织化学法检测结直肠癌组织中EGFR基因的表达并探讨其与患者临床病理特征的关系。在上述病例中随机选取25例，其中5例经免疫组织化学染色显示EGFR表达呈强阳性，提取这25例石蜡组织DNA，进行PCR扩增并测序从而分析在结直肠癌中EGFR基因的突变情况。结果：免疫组化结果显示EGFR基因的阳性率为21．33％（144／675）。EGFR基因的表达与结直肠癌的肿瘤直径（Χ^2=0．0204，P=0。8836）、肠壁浸润深度（Χ^2=1．1286，P=0．2881）、淋巴结转移情况（Χ^2=0．0708，P=0．7902）及肿瘤的病理分期（Χ^2=5．3691，P=0．1467）无关，但与肿瘤的分化程度（Χ^2=5．7793，P=0．0162）、腹腔及远处转移情况（Χ^2=4．3983，P=0．0360）密切相关。25例结直肠癌中有1例显示EGFR基因存在错义突变，有5例显示同义突变。结论：EGFR基因的表达与结直肠癌的分化程度及远处转移有关，可辅助判断患者的预后和转移情况以及指导临床治疗。EGFR基因在结直肠癌的突变可能为低频率事件。     

结直肠癌患者KRAS和BRAF基因突变检测及其临床意义

目的探讨结直肠癌患者组织中KRAS和BRAF基因突变情况,分析突变与临床病理特征的关系。方法应用荧光PCR-优化寡核苷酸探针法检测304例结直肠癌石蜡包埋标本中KRAS基因2号外显子的12和13密码子、BRAF基因的15号外显子的突变情况,分析KRAS和BRAF基因突变与临床病理特征的关系。结果 KRAS和BRAF基因在结直肠癌的突变率分别为38.8%(118/304)和4.3%(13/304)。KRAS基因突变阳性标本中12密码子的突变率为78.0%,其中p.G12D发生率最高,占总突变的45.3%;13密码子的突变率为22.0%。高年龄组(≥60岁)患者的KRAS基因突变率为47.2%(58/123),高于低年龄组(<60岁)的33.1%(60/181),差异有统计学意义(P<0.05)。转移性结直肠癌患者19例,其KRAS基因突变率为36.8%(7/19),与285例原发性结直肠癌患者的突变率(38.9%,111/285)差异无统计学意义(P>0.05)。BRAF基因在结肠、低分化、黏液腺癌患者中的突变率明显高于直肠、中或高分化和管状腺癌的患者。结论结直肠癌患者中KRAS基因突变的发生率较高,与年龄相关,而与性别、部位、病理类型和分化程度不相关。BRAF基因突变与肿瘤部位、病理类型和分化程度有关。原发性与转移性结直肠癌患者KRAS基因突变率无明显差异。     

结直肠癌K-ras基因突变与临床病理特征的相关性分析

 【摘要】 目的：研究结直肠癌组织中K-ras基因的突变情况与临床病理特性之间的相关性。 目的　研究结直肠癌组织中K-ras基因的突变情况与临床病理特性之间的相关性。方法　采用直接测序法对90例结直肠癌石蜡标本进行K-ras基因突变检测,并对K-ras基因的突变情况及其与结直肠癌患者临床病理特征的关系进行统计学分析。结果　90例结直肠癌患者组织中,K-ras基因12、13密码子总突变者31例,总突变率为34.4 %。12密码子单突变21例,突变率为23.3 %;双突变1例。13密码子突变者9例,突变率为10.0 %。结直肠癌K-ras基因突变率与肿瘤部位有明显相关性（P＝0.042）。12密码子单突变和13密码子突变与临床病理参数均无明显相关性（P＞0.05）。结论　不同肿瘤部位患者的K-ras基因突变率存在明显不同。     

直肠癌患者术前MRI特征与鼠类肉瘤病毒癌基因同源物B基因状态的关系

目的 探讨直肠癌患者术前MRI特征与鼠类肉瘤病毒癌基因同源物B(BRAF)基因状态的关系。方法根据BRAF基因检测结果的不同将150例直肠癌患者分为突变组（n=43）和未突变组（n=107）。比较两组患者的术前MRI参数[表观扩散系数（ADC）、肿瘤最大轴向长度（ATL）、肿瘤最大纵向长度（LTL）、ATL/LTL],采用Spearman相关分析法分析ATL、ATL/LTL与BRAF基因突变的相关性。比较两组患者的术前MRI影像特征（肿瘤位置、肿瘤大体形态、累及肠周范围、环周切缘）,采用Logistic回归模型分析直肠癌患者BRAF基因突变的影响因素,采用受试者工作特征（ROC）曲线及曲线下面积（AUC）分析ATL、ATL/LTL对BRAF基因突变的诊断价值。结果 突变组患者的ATL、ATL/LTL均明显高于未突变组,差异均有统计学意义（P﹤0.01）;两组患者的ADC、LTL比较,差异均无统计学意义（P﹥0.05）。Spearman相关性分析结果显示,ATL、ATL/LTL与直肠癌患者BRAF基因突变均呈正相关（P﹤0.01）。两组患者的肿瘤大体形态比较,差异有统计学意义（P﹤0.0...     

血清TSH水平及BRAF基因V600E突变联合检测在甲状腺癌诊断中的价值

目的 探讨血清促甲状腺激素(TSH)水平及BRAF基因V600E位点突变联合检测在甲状腺癌诊断中的价值。方法 选取2019年6月至2021年3月在江苏省肿瘤医院住院手术治疗的甲状腺结节患者246例为研究对象,分为甲状腺癌组和良性病变组。采用全自动电化学发光免疫分析法检测其治疗前血清TSH水平,采用微滴式数字PCR法检测组织DNA BRAF基因V600E位点突变状态,并对其在良恶性组间差异,诊断效能等进行分析。结果 TSH表达阳性在良恶性组间差异无统计学意义(P=0.533),BRAF基因V600E位点突变在良恶性组间差异有统计学意义(P<0.001)。血清TSH阳性标本中BRAF基因V600E位点突变检出率为44.44%,略低于血清TSH阴性标本中BRAF基因V600E位点突变检出率(57.62%),差异无统计学意义(P=0.141)。二者联合检测时敏感度70.64%,特异性89.28%,阳性预测值98.09%,阴性预测值28.09%,准确度72.76%。结论 血清学标志物TSH和分子标志物BRAF基因V600E位点突变联合检测能够更好地区分甲状腺癌与甲状腺良性病变,尤其适用于经...     

云南地区结直肠癌患者RAS/BRAF/PIK3CA基因突变状态及与临床病理特征的关系

目的:本研究主要对结直肠癌患者的KRAS/NRAS/BRAF V600E/PIK3CA基因突变情况及与临床病理特征的关系进行探究。方法:我们对2018年01月至2020年01月在我院分子诊断中心进行基因检测的结直肠癌患者的KRAS/NRAS/BRAF V600E/PIK3CA突变情况进行回顾性分析,共纳入209例具有完整临床信息的患者,所有患者的基因检测都是采用高通量测序方法获得的,并对患者的人口统计学和临床信息进行分析。结果:209例结直肠癌患者中,KRAS、NRAS、BRAF V600E、PIK3CA突变例数分别为98例、7例、10例和25例,突变率分别为46.9%、3.3%、4.8%和12.0%。KRAS 2号外显子突变最常见,其中又以G12D亚型突变高发,肿瘤原发部位在直肠的患者和微卫星稳定的CRC患者KRAS突变率明显高于原发部位在左右半结肠和微卫星不稳定的患者(P<0.05);BRAF V600E在MSI-H的CRC患者中突变率显著高于MSS的患者(P<0.05);男性患者、既往没有吸烟史、有淋巴结转移、临床分期较早以及MSI-H的CRC患者有更高的PIK3CA...     

结直肠癌组织中KRAS和BRAF基因突变与临床病理特征及预后的关系

目的:探讨结直肠癌组织中KRAS和BRAF基因突变与临床病理和预后的关系。方法:采用扩增阻滞突变系统（ARMS）方法对134例结直肠癌组织进行回顾性分析。结果:134例CRC组织中KRAS基因突变率49.3%（66/134）,第2外显子突变率42.5%（57/134）,其中12及13密码子突变率分别为35.1%（47/134）和7.5%（10/134）,第3外显子61密码子突变率1.5%（2/134）,第4外显子117/146密码子突变率5.2%（7/134）,BRAF V600E突变率4.5%（6/134）。左半结肠KRAS基因突变率54.5%（54/99）高于右半结肠34.3%（12/35）,差异有统计学意义（P＜0.05）,右半结肠BRAF基因突变率14.3%（5/35）高于左半结肠1.0%（1/99）,差异有统计学意义（P＜0.05）,而KRAS和BRAF基因突变与性别、年龄、民族、肿块大小、分化程度、淋巴结转移及病理分期等临床病理特征,差异均无统计学意义（P＞0.05）。KRAS基因突变型CRC患者的中位生存时间48个月（39.9%）,与野生型47个月（46.2%）相比较,差异无统计学意义（P＞0.05）。BRAF基因突变型CRC患者的中位生存时间21个月（26.7%）,与野生型48个月（44.7%）相比较,差异有统计学意义（P＜0.05）。多因素COX回归风险模型结果显示,BRAF基因突变型是CRC患者预后不良的独立危险因素（B=1.664,OR=5.278,95%CI:1.505~18.516, P＜0.05）,而性别、年龄、民族、肿块大小、分化程度、肿瘤部位、淋巴结转移及病理分期均不是CRC患者生存时间的独立危险因素（P＞0.05）。结论:CRC组织中KRAS基因突变率高,BRAF基因突变率低。KRAS和BRAF基因突变型与肿瘤部位有关。BRAF基因突变型是CRC预后不良的独立危险因素。     

甲状腺乳头状癌NIS蛋白表达对131^I疗效的预测及与BRAF基因突变的关系

目的：探讨甲状腺乳头状癌钠碘转运体（sodium／iodide symporter,NIS）蛋白表达对131^I疗效的预测及与BRAF基因突变的关系。方法：收集甲状腺乳头状癌根治术后行131^I治疗的患者60例,临床随访131^I疗效分为完全缓解和不完全缓解,不完全缓解组再分为摄碘和不摄碘。免疫组化法对患者肿瘤原发灶NIS蛋白表达进行分析,根据表达强弱分为0,1,2,3分。应用Pyrosequencing焦磷酸测序技术对肿瘤原发灶BRAF基因突变进行分析。对131^I疗效各组和BRAF突变两组的NIS蛋白表达水平进行统计分析。结果：甲状腺乳头状癌NIS蛋白染色阳性颗粒主要定位于细胞浆,阳性评分为1．45±0．77。131^I治疗完全缓解患者29例,其NIS蛋白水平（1．62±0．86）与不完全缓解组（1．29±0．64）无统计学差异（P=0．100）。但对不完全缓解组进一步研究发现,不摄碘组NIS蛋白水平（1．00±0．60）显著低于摄碘组（1．47±0．61）,P=0．043。伴BRAF基因突变患者检出47例（78．3％）,其NIS蛋白评分（1．34±0．73）与携带野生型基因患者（1．85±0．80）比较差异仃统计学意义,P：0．035。结论：131^I疗效与肿瘤原发灶NIS蛋白表达水平没有明显关系,但不摄碘的持续不缓解患者常伴有肿瘤NIS蛋白表达降低和缺失;伴BRAF基因突变患者其NIS蛋白表达降低,分化程度更差。     

胃癌Kai-1基因缺失与突变的研究

目的：探讨Kai-1基因的缺失与突变在胃癌演进与转移中的意义。方法：提取30例胃癌患者的肿瘤组织（16例无淋巴转移,14例有淋巴转移）,30例癌旁正常组织的RNA,RT—PCR扩增,电泳检测,测序验证Kai-1基因的缺失情况;并提取发生缺失的肿瘤组织的DNA,PCR扩增,测序探讨Kai-1基因的突变情况。结果：30例组织标本中12例出现Kai-1基因exon9缺失（10例杂合缺失,2例纯合缺失）,30例癌旁正常组织中有5例出现Kai-1基因exon9缺失（5例杂合缺失）;在胃癌组织中Kai-1基因缺失频率显著高于在癌旁正常组织（P〈0．05）;在有淋巴转移的胃癌组织中,缺失的频率明显高于无淋巴转移的胃癌组织（P〈0．05）,在胃癌中晚期（Ⅲ-Ⅳ期）明显高于早期（I-Ⅱ期）（P〈0．05）,而Kai-1基因的缺失频率与胃癌患者的年龄、性别、组织学类型以及分化程度无关（P〉0．05）。结论：Kai-1基因缺失与突变可能与胃癌演进、转移有关,检测其缺失或突变可作为判断胃癌的演进与转移的客观临床指标。     

Frequency and application of the hot spot BRAF gene mutation (p.V600E) in the diagnostic strategy for Hereditary Nonpolyposis Colorectal Cancer

BACKGROUND: BRAF somatic mutations were reported with high frequency in sporadic colorectal cancers (CRCs) with microsatellite instability (MSI). The hot spot c. 1799 T>A, p.V600E gene mutation is very rarely involved in the tumorigenesis of CRC linked to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). These data suggested that the screening of mismatch repair (MMR) genes could be avoided in cases positive for p.V600E. The aim of our study was to analyze the frequency of this hotspot mutation in a group of 140 CRC patients and the applicability of BRAF 15 exon mutation screening in the diagnosis of HNPCC. METHODS: Exon 15 of the BRAF gene was PCR amplified and subjected to single-strand conformation polymorphism (SSCP) analysis. Samples showing an altered mobility pattern were then subjected to direct sequencing. Associations between BRAF mutation and clinical, pathological or molecular features were evaluated using Fisher's exact chi-squared tests as appropriate. RESULTS: The mutation was detected in eight of 140 (5.7%) CRC samples with common characteristic features such as MSI, proximal tumor location, moderate differentiation, mucinous production and early Dukes' stage. CONCLUSIONS: We conclude that screening for this mutation is an efficient tool in the diagnostic strategy for HNPCC.     

Distribution of KRAS and BRAF Mutations in Metastatic Colorectal Cancers in Turkish Patients

The results of this study demonstrate the potential prognostic and predictive values of KRAS and BRAF gene mutations in patients with colorectal cancer (CRC). It has been proven that KRAS and BRAF mutations are predictive biomarkers for resistance to anti-EGFR monoclonal antibody treatment in patients with metastatic CRC (mCRC). We demonstrated the distribution of KRAS (codons 12, 13 and 61) and BRAF (codon 600) gene mutations in 50 mCRCs using direct sequencing and compared the results with clinicopathological data. KRAS and BRAF mutations were identified in 15 (30%) and 1 (2%) patients, respectively. We identified KRAS mutations in codon 12, 13 and 61 in 73.3% (11/15), 20% (3/15) and 6.67% (1/15) of the positive patients, respectively. The KRAS mutation frequency was significantly higher in tumors located in the ascending colon (p=0.043). Thus, we found that approximately 1/3 of the patients with mCRC had KRAS mutations and the only clinicopathological factor related to this mutation was tumor location. Future studies with larger patient groups should yield more accurate data regarding the molecular mechanism of CRC and the association between KRAS and BRAF mutations and clinicopathological features.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

Value of KRAS,BRAF, and PIK3CA Mutations and Survival Benefit from Systemic Chemotherapy in Colorectal Peritoneal Carcinomatosis

Background: It is well known that peritoneal carcinomatosis (PC) from colorectal cancer (CRC) is associated with a poor prognosis. However, data on the prognostic significance of modern chemotherapy containing bevacizumab, cetuximab or panitumumab are not available. Materials and Methods: This retrospective review concerned 526 patients with metastatic CRC who were classified into two groups according to the presence or absence of PC, and were treated with systemic chemotherapy, with or without bevacizumab or anti-EGFR antibodies. The genetic background, in particular KRAS, BRAF, and PIK3CA gene mutations, and overall survival (OS) were compared between the two groups. Results: The median OS values were 23.3 and 29.1 months for PC and non-PC patients, respectively (hazard ratio [HR]=1.20; p=0.17). Among all patients, tumor location, number of metastatic sites and BRAF mutation status were significant prognostic factors, whereas the presence of PC was not. In the PC group, chemotherapy with bevacizumab resulted in a significantly longer OS than forchemotherapy without bevacizumab (HR=0.38, p<0.01), but this was not the case in the non-PC group (HR=0.80, p=0.10). Furthermore, the incidence of the BRAF V600E mutation was significantly higher in PC than in non-PC patients (27.7% versus 7.3%, p<0.01). BRAF mutations displayed a strong correlation with shorter OS in non-PC (HR=2.26), but not PC patients (HR=1.04). Conclusions: Systemic chemotherapy, especially when combined with bevacizumab, improved survival in patients with PC from CRC as well as non-PC patients. While BRAF mutation demonstrated a high frequency in PC patients, but it was not associated with prognosis.     

BRAF V600E mutation in metastatic colorectal cancer: Methods of detection and correlation with clinical and pathologic features

The screening for BRAF V600E mutation is employed in clinical practice for its prognostic and potentially predictive role in patients with metastatic colorectal carcinoma (mCRC). Little information is available on the sensitivity and specificity of the testing methods to detect this mutation in CRC. By using serial dilution of BRAF mutant DNA with wild type DNA, we found that the sensitivity of allelic discrimination-Real Time PCR was higher than PCR-Sequencing (10% vs 20%). In agreement, the Real Time PCR assay displayed increased analytical sensitivity in detecting the BRAF V600E mutation as compared with PCR-Sequencing in a cohort of 510 consecutive CRCs (21 vs 16 cases). Targeted resequencing demonstrated that all cases negative by PCR-Sequencing had an allelic frequency of the BRAF mutation <20%, thus suggesting tumor heterogeneity. The association of BRAF mutations with clinical and pathological features was assessed next in a cohort of 840 KRAS exon 2 wild type CRC patients screened with the Real Time PCR assay. The BRAF V600E mutation frequency in this cohort was 7.8% that increased to 33.4% in females over 70 y of age with right-sided tumor location. BRAF mutations were also detected in 4.4% of male patients with left-sided tumors and aged <70 y. Fourteen of 61 (22.9%) BRAF V600E mutation bearing patients exhibited microsatellite instability (MSI) as assessed by T17 mononucleotide sequence within intron 8 of HSP110. Our study indicates that Real Time PCR-based assays are more sensitive than PCR-Sequencing to detect the BRAF V600E mutation in CRC and that BRAF mutations screening should not be restricted to selected patients on the basis of the clinical-pathological characteristics.