Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients

AIM:To identify and assess mutations in the K-ras and BRAF genes in a cohort of Chinese patients with colorectal cancer (CRC) for their association with various clinicopathological parameters and prognosis.METHODS:Genomic DNA was isolated from frozen tissues.Pyrosequencing analysis was conducted to detect mutations in the K-ras (codons 12,13,and 61) and BRAF genes (codon 600).Statistical analysis was carried out using SPSS-15.0 software.RESULTS:Among the 118 colorectal cancer patients,we detected 41 (34.7%) mutations in the K-ras gene.Mutation frequencies at codon 12 and codon 13 were 23.7% (28/118) and 10.2% (12/118),respectively.Only one patient harbored a point mutation at codon 61 (0.8%,1/118).Gender was the only factor that showed an obvious relationship with K-ras gene mutation (female 44.7% vs male 28.2%,P=0.037).Other clinicopathological features,such as age,location of the tumor,tumor differentiation,Tumor,Node and Metastases classification,and the Union for International Cancer Control staging,showed no positive relationship with K-ras gene mutations.No significant correlation was observed between the presence of K-ras mutations (codons 12,13,and 61) and the survival of the patients.BRAF mutations were rare,and only two patients (1.7%) harbored a detectable mutation at codon 600.CONCLUSION:K-ras gene mutation is a common event in our 118 Chinese CRC patients,with an obvious relationship with gender.However,it seems not to be an independent prognostic factor in CRC patients.The BRAF gene is rarely mutated in Chinese CRC patients.

Hypermethylation of CpG island in O^6-methylguanine-DNA methyltransferase gene was associated with K-rasG to A mutation in colorectal tumor

AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas,62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutantallele-specific amplification was used to detect K-rasG to A point mutation in codon 12.RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation,respectively (P = 0.027, P＜0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P = 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

Highly sensitive ECL-PCR method for detection of K-ras point mutation

A highly sensitive electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for K-ras point mutation detection is developed. Briefly, K-ras oncogene was amplified by a Ru(bpy)3(2+) (TBR)-labeled forward and a biotin-labeled reverse primer, and followed by digestion with MvaI restriction enzyme, which only cut the wild-type amplicon containing its cutting site. The digested product was then adsorbed to the streptavidin-coated microbead through the biotin label and detected by ECL assay. The experiment results showed that the different genotypes can be clearly discriminated by ECL-PCR method. It is useful in point mutation detection, due to its sensitivity, safety, and simplicity.

Study of mutations of p53, APC and K-ras genes in 47 cases of intestinalmetaplasia of gastric mucosa

Objective:To study the role of the mutations of p53, APC and K-ras genes in 47 cases of 3 types of intestinal metaplasia (IM) of gastric mucosa. Methods:In 47 cases of IM, exons 5- 8 of p53 and exons 15 of APC were examined with PCR-SSCP and codon 12 of K-ras with PCR-RFLP to detect the existence of any mutations of these structures. Results:Muta- tions of p53, APC and K-ras were found in 29.8% (14/47),6.4% (3/47) and 6.4% (3/47) respectively in our series of patients who consisted of 33 with types I and II and 14 with type III of IM. The mutation rate of p53 was far higher in patients with type III IM (57.1%,8/14) than in those with types I and II IM(18.2%,6/33)(P <0.05). Though the mutation rate of APC and K-ras was also higher in the patients with type III IM than in those with types I and II IM, it was of no statistical significance (P >0.05). In one case of type III IM, mutation of both p53 and K-ras was found. Conclusion: The molecular changes of 3 types of IM are different. The mutation of p53 may be closely related to carcinogenesis in cases of type III IM and it serve as a sign for the early diagnosis of gastric carcinoma.

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.

Unique GGT→GTT mutation at K-ras codon 12 in six human pancreatic cancer cell lines from Chinese patients

Objective To investigate the K-ras mutation pattern in six pancreatic cancer cell lines from Chinese patients. Methods All six cell lines were analyzed for mutations in exon 1 of the K-ras gene by polymerase chain reaction (PCR) and direct sequencing.Results All 6 pancreatic cancer cell lines had GGT→GTT mutations at K-ras codon 12 but no mutations at codon 13.Conclusion The unique GGT→GTT mutation at codon 12 plays a potential role in the carcinogenesis of pancreatic cancers in Chinese.

Evaluation of Intraductal Ultrasonography, Endoscopic Brush Cytology and K-ras, P53 Gene Mutation in the Early Diagnosis of Malignant Bile Duct Stricture

Background： In qualitative diagnosis of bile duct stenosis, single diagnostic measure is difficult to make a correct diagnosis, to combine several diagnostic techniques may be helpful to make an accurate diagnosis. The aim of this study was to evaluate the value of intraductal ultrasonography （IDUS）, endoscopic brush cytology and K-ras, P53 gene mutation in the early diagnosis of malignant biliary stricture. Methods： From February 2012 to February 2013, 84 patients with suspected malignant biliary stricture were performed I DUS firstly, then endoscopic brush cytology and finally K-ras, P53 gene mutation detection, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of all above ways were evaluated and compared. Results： Of 84 patients, 52 cases were ultimately diagnosed malignant biliary stenosis; of which, 9 cases had no recurrence or metastasis to other organs after radical operation during the follow-up period. IDUS combined with brush cytology and K-ras ＋ P53 gene mutation detection had obvious advantage in the sensitivity, accuracy and negative predictive value than any other joint detection and single detection （the advantage was more significant compared with IDUS ＋ brush cytology or any single detection P 〈 0.01）. There were obvious statistical significance in the sensitivity and accuracy between IDUS ＋ brush cytology ＋ P53 or IDUS ＋ brush cytology ＋ K-ras and IDUS ＋ brush cytology or IDUS （P 〈 0.05）. There was no statistical significance in the sensitivity, specificity, positive predictive value, negative predictive value and accuracy between IDUS ＋ brush cytology ＋ P53 and IDUS ＋ brush cytology ＋ K-ras （P 〉 0.05）. Conclusions： IDUS combined with brush cytology and K-ras, P53 gene mutation detection is better than the separate detection and contribute to the early diagnosis of malignant biliary stricture. Its more widespread use is recommended.

Performance of K-ras mutation analysis plus endoscopic ultrasound- guided fine-needle aspiration for differentiating diagnosis of pancreatic solid mass： a meta-analysis

Background Difficulties persist in differentiating pancreatic ductal adenocarcinomas （PDAC） from pancreatic inflammatory masses （PIM）. Auxiliary diagnostic techniques which enhance the endoscopic ultrasound-guided fine-needle aspiration （EUS-FNA） diagnostic yield have been attempted, for example, K-ras mutation analysis. We aimed to evaluate the accuracy of K-ras mutation analysis combined with EUS-FNA for the differential diagnosis of PDAC and PIM by pooling data of existing trials. Methods We systematically searched the Medline, PubMed, Web of Science, Embase, and Cochrane Central Trials databases for relevant published studies. Meta-analysis was performed. Pooling was conducted in fixed-effect model or random-effect model. Results In total eight studies, with 696 cases of PDAC and 138 cases of PIM, met our inclusion criteria. The pooled sensitivity, specificity, positive likely ratio and negative likely ratio of K-ras mutation analysis combined with cytopathology for diagnosis of PDAC versus PIM were 90%, 95%, 13.45, and 0.13, respectively. Especially, among total 123 patients whose EUS-FNA results were inconclusive or negative, fifty-nine had K-ras mutations and were finally diagnosed with PDAC （48%, 59/123）. Publication bias was not present. Conclusions Combining K-ras mutation analysis with routine cytology moderately improves the ability of EUS-FNA to differentially diagnose between PDAC and PIM, especially for patients with suspected PDAC yet inconclusive EUS-FNA findings, and may prove to be a valuable supplemental method to EUS-FNA.

T790M mutation in stage IV EGFR-mutated NSCLC patient with acquired resistance reverted to original 19Del mutation after administration of a series of precision treatments:a case report

Existing studies have yet to elucidate clearly the mechanisms of secondary resistance to third generation epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs),neither is there any established standard therapy for patients resistant to third generation EGFR-TKIs.This case report demonstrates a rare mutation pattern in a male patient with a pathologic diagnosis of non-small cell lung cancer(NSCLC)harboring an EGFR exon 19 deletion(19Del)mutation,who then acquired an EGFR-T790M mutation after developing resistance to the first generation EGFR-TKI(gefitinib).The mutation reverted to the original EGFR-19Del mutation after the patient developed secondary resistance against the third generation TKI(osimertinib).This patient eventually achieved partial response(PR)with second generation TKI(afatinib)as a fourth-line treatment.

一种改进人工免疫的飞行状态规则提取方法

针对基本人工免疫系统寻优性能的不足,提出映射突变机制,将抗体信息映射为多维空间中的坐标点,然后依据各抗体相应的亲和力和相对位置对其进行移动,从而扩大了搜索范围,提供了更为高效的抗体产生方法。通过测试函数验证,映射突变机制的引入提高了寻优的精度和收敛速度。将改进的人工免疫系统用于飞行数据规则提取,仿真实验结果表明,基于改进人工免疫系统所提取的规则简洁、正确、可靠。

新型非拟肽磺胺类HIV蛋白酶抑制剂Darunavir的研究进展

综述新型非拟肽磺胺类人类免疫缺陷病毒(HIV)蛋白酶抑制剂Darunavir(TMC-114)的发现,抗病毒的活性,分子模拟以及结构优化的研究进展.Darunavir不但对野生型的HIV蛋白酶有很好的抑制活性,而且对多种抗性突变的HIV蛋白酶也有良好的活性,是针对耐药性HIV所开发的新药.

扶正解毒方药调控T790M及c-Met改善吉非替尼获得性耐药

目的:探讨扶正解毒方药改善吉非替尼获得性耐药的作用机制。方法:建立肺癌干细胞荷瘤小鼠模型,随机分为生理盐水组、吉非替尼组、扶正解毒方药联合吉非替尼组、扶正中药联合吉非替尼组、解毒中药联合吉非替尼组,测定荷瘤小鼠抑瘤率,应用ARMS法检测肿瘤组织T790M突变,RT-PCR法检测肿瘤组织c-Met基因扩增。结果:吉非替尼单药对肺癌干细胞荷瘤小鼠抑瘤率为31.02%;解毒中药联合吉非替尼组为33.43%;扶正中药联合吉非替尼组为37.51%;扶正解毒方药联合吉非替尼组为45.11%。上述各组与生理盐水组比较差异有统计学意义(P<0.05或P<0.01);扶正解毒方联合吉非替尼组与吉非替尼单药组比较差异有统计学意义(P<0.05);吉非替尼单药组T790M基因突变和c-Met基因扩增显著增加,与生理盐水组比较差异有统计学意义(P<0.01),扶正解毒方药联合吉非替尼组能够降低T790M基因突变和c-Met基因扩增,与吉非替尼单药组比较差异有统计学意义(P<0.05)。结论:扶正解毒方药能够改善吉非替尼获得性耐药,其作用机制与降低T790M突变及抑制c-Met扩增有关。

反思达尔文

Ｃａｉｒｎｓ 提出，Ｌｕｒｉａ 和Ｄｅｌｂｒｕｃｋ 的波动试验只是证实了随机突变———选择留存机制，并不能否定有定向突变存在． 天然遗传工程的存在则表明，生物在进化过程中并不总是被动地承受选择，有时也会主动地改变自己的遗传结构以适应环境． 这些新成果表明，自然选择不是驱动生物进化的唯一动力． 再者，无论是定向突变还是自然选择，它们解释的都是适应性进化，而适应性进化则只是生物进化中的一种非本质形式．事实上，解释适应性进化的达尔文学说，还不能回答生物如何从低级向高级进化的问题．

贵州省艾滋病患者耐药基因突变研究

目的研究艾滋病(AIDS)患者耐药基因突变情况,为防止AIDS耐药传播及流行提供科学依据。方法采用NueliSens EasyQ荧光实时定量PCR系统对治疗1年以上的AIDS患者进行病毒载量检测,病毒载量>1 000 U/ml者采用Abbott公司ViroSeqTMHIV-1 Genotyping System V2.0系统进行扩增、测序和基因突变分析。结果 1 088例AIDS患者中病毒载量>1 000 U/ml者45例,获得POL区基因序列30例,且均有不同程度基因突变,仅7例产生耐药,总耐药率为0.64%(7/1 088);发现交叉耐药2例。7例耐药的AIDS患者均出现至少1种耐药相关基因突变位点,突变率为23.3%(7/30)。7例AIDS患者均对非核苷类逆转录酶抑制剂产生耐药基因突变,其耐药突变位点为K103N 4例、G190A 2例、V179D 1例、Y181C 1例、K101E 1例、M230L 1例,临床主要表现为对地拉韦定、依非韦仑及奈韦拉平高度耐药,其中有2例对核苷类逆转录酶抑制剂产生耐药基因突变,突变位点为M184V,临床上表现为对拉米夫定和恩曲他滨高度耐药。未见蛋白酶抑制剂耐药基因突变。结论贵州省AIDS患者的耐药情况目前仍处于较低水平且耐药形式较为单一;核苷类逆转录酶抑制剂及非核苷类逆转录酶抑制剂对贵州省AIDS患者的广泛治疗和使用仍然有效;及时监控和掌握AIDS患者的耐药基因突变情况,对于指导临床用药尤为重要。

T790M的起源、遗传易感性和体内自然选择

3背景·研究一：酪氨酸激酶抑制剂（tyrosine kinase inhibitors．TKI）在部分非小细胞肺癌non-small cell lung cancer．NSCLC）患者，尤其是非吸烟者、妇女和患腺癌伴有支气管肺泡分化者中取得了较好的疗效。以往研究认为，表皮生长因子受体（epidermal growth factor receptor。EGFR）T790M突变是在应用TKI治疗后出现的继发突变。然而在另一些研究中发现，从未给予TKI治疗的NSCLC患者中，也可发现T790M突变。

获得性重型再生障碍性贫血患者穿孔素基因突变检测

本研究旨在探讨穿孔素(perforin)基因PRF1突变是否为获得性重型再生障碍性贫血(SAA)发病的遗传易感性基础。用聚合酶链反应(PCR)方法扩增31例SAA患者及15名正常对照者外周血单个核细胞基因组DNAPRF1外显子2(exon2)及外显子3(exon3);用双脱氧终止法测序,与GenBank报道序列核对寻找突变位点;发现突变序列克隆入M13噬菌体载体中,对所得2条染色体相应序列分别测序,以确定不同突变在染色体上的分布。结果表明:在SAA患者,发现了2处基因突变即822位C>T纯合子突变(无义突变)及907位G>A杂合子突变(Met303Val);rs885822单核苷酸多态性(SNP)位点分布病例组与对照组比较有统计学差异(p<0.05)。结论:穿孔素基因突变可能是部分SAA患者的遗传易感因素。

基于改进遗传算法的接口测试数据集的生成方法

针对用电信息采集统一接口平台需要大量包含足够测试用例占比的测试数据这一难题,提出基于改进遗传算法的用电信息采集系统统一接口平台测试数据集的生成方法;在对原始数据预处理的基础上,采用基于相似度的交叉算子对原始群体扩充,使用提出基于群体趋势不变的染色体变异算法,在保持群体数据集特性的情况下增大测试用例占比,从而形成测试数据的自动生成方法;应用该测试数据集生成方法,基于某省级电力公司2016年7—9月300万个典型用电客户用电数据进行数据集生成实验,利用熵原理比较无变异因子、插值法变异和改进遗传算法分别生成的测试数据与原始数据的重合度。结果表明,改进遗传算法生成的测试数据集,具有同用电信息采集系统采集数据相同的属性和属性值分布以及类似的属性关联关系,能够满足测试用例需求。

第三代EGFR-TKI获得性耐药相关机制研究

随着第一代和第二代靶向表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)的药物广泛应用于表皮生长因子受体突变的晚期非小细胞肺癌患者,极大地提高了患者的总生存期和无进展生存期,但EGFR-TKI也存在耐药。Osimertinib克服了第一代靶向药物吉非替尼、厄洛替尼的常见耐药突变(T790M),成为第三代靶向药,但随着临床应用其也出现了耐药。目前针对以Osimertinib为代表的第三代靶向药耐药机制研究已成为热点,了解目前最常见的第三代EGFR-TKI的耐药机制及克服耐药的新型药物或方案有助于提高非小细胞肺癌的治疗效果,改善患者的生活质量。

获得性再生障碍性贫血体细胞突变及其意义

再生障碍性贫血(AA)与骨髓增生异常综合征(MDS)都是造血干细胞(HSC)异常的克隆性疾病,两者有时难以区分。随着分子检测技术的进步,人们对两者发病机制的认识也在不断深入。本文将综述AA体细胞突变(SM)与细胞遗传学改变特点,分析其与MDS的分子联系以及在疾病转化中的作用。AA中最常见的体细胞性改变是PIGA和人类白细胞抗原(HLA)等位基因的缺失,它们及8三体、del(13q)与AA免疫发病机制相关。AA最常见的5个突变中,PIGA和BCOR/BCORL1突变预后更好,而DNMT3A和ASXL1突变与克隆演变和预后不良相关。AA继发MDS的危险因素包括预后不良的SM和细胞遗传学改变[如del(7q)]、疾病持续时间较长、AA发病年龄、端粒磨损等。因SM动态变化及意义未确定,其在疾病进展中的意义仍不明,但监测SM并结合临床评估预后仍可能指导治疗。

Comethylation of p16 and MGMT genes in colorectal carcinoma:Correlation with clinicopathological features and prognostic value

AIM: To investigate the significance of p16 and O6- methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. METHODS: p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. RESULTS: p16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCs, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation. Only 27% of patients with simultaneous p 16 and MGMT methylation showed the detectible occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P < 0.05, χ2-test). In addition, p16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 ± 6.0 mo vs 23.1 ± 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P < 0.001, χ2-test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 ± 1.9 mo vs 37.0 ± 1.8 mo, P < 0.001, Log-rank test). The comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. CONCLUSION: Our data suggest that comethylation of promoters of p 16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.