KRAS基因突变高分辨率熔解曲线检测技术参数的优化

目的建立KRAS基因突变高分辨率熔解曲线分析法(high resolution melting,HRM)检测方法,确立最佳反应体系和反应条件,为将此技术用于临床基因突变的快速检测奠定基础。方法根据引物Tm值设立退火温度梯度,确定适宜的退火温度。利用正交实验,对影响实时荧光定量PCR(qPCR)-HRM反应体系的模板DNA、Mg2+、引物等3种主要因素进行优化。通过对SW480和MDA-MB-231细胞基因组DNA进行HRM分析检测突变,并直接测序验证,观察反应体系和反应条件的可行性。结果引物最适退火温度为60.8℃。KRAS基因突变qPCR-HRM最佳反应体系为:20μl qPCR-HRM反应体系中,引物浓度为0.5μmol/L,Mg2+为2.5mmol/L,模板DNA为40ng。优化的qPCR-HRM反应体系和反应条件能检测基因突变,和直接测序结果一致。结论采用正交实验优化的qPCR-HRM反应体系,操作简便,电泳条带清晰,熔解曲线峰型单一,特异性好,结果准确可靠,可为进一步建立HRM技术检测基因突变的临床应用提供借鉴。     

非小细胞肺癌EGFR和KRAS基因突变和临床病理特征分析

目的 探讨非小细胞肺癌(NSCLC)中表皮生长因子受体(EGFR)和鼠类肉瘤病毒癌基因(KRAS)突变状态及其与临床病理特征的关系.方法 回顾性收集2019年1月至2021年6月首都医科大学大兴教学医院183例病理确诊NSCLC患者临床病理资料,采用扩增阻滞突变系统(ARMS)法检测肿瘤组织中EGFR和KRAS基因的突...     

非小细胞肺癌患者KRAS基因突变与临床病理特征分析

目的 检测非小细胞肺癌(NSCLC)患者的KRAS基因突变,并分析不同突变状态NSCLC患者的临床病理特征.方法 选择2018年9月—2020年9月就诊于潍坊市第二人民医院且临床资料完整的453例经病理组织学确诊的原发性NSCLC患者作为研究对象.采用环形引物双扩增技术检测所有患者组织标本中KRAS基因的突变状态,分析...     

高分辨率熔解曲线分析法检测急性髓系白血病c-kit基因突变

目的 应用高分辨率熔解(HRM)曲线分析法检测急性髓系白血病(AML)患者c-kit基因常见突变N822K和D816V.方法 对21例t(8;21)阳性AML患者骨髓标本的c-kit基因第17号外显子进行PCR扩增、HRM突变检测及DNA测序验证.结果 HRM分析发现21例患者中6例(28.6%)的c-kit基因扩增产物出现异常熔解曲线,经测序验证1例为D816V杂合子突变,5例为N822K杂合子突变.结论 HRM分析是一种简便、快速、特异和高通量的c-kit基因突变筛查方法.

Abstract：

Objective To detect the common mutations (D816V and N822K) of c-kit gene in acute myeloid leukemia (AML) using high-resolution melting analysis (HRM). Methods HRM analysis was established to screen c-kit mutations in PCR products of c-kit exon 17 in 21 AML patients with t(8;21 ). PCR products were sequenced to confirm the mutation. Results HRM analysis identified an aberrant melting curve in 6 cases (28.6%), which were confirmed by direct DNA sequencing as one D816V mutation and five N822K mutation. Conclusion HRM analysis is a convenient, rapid, specific and high-throughput technique for scanning c-kit gene mutation in AML.     

KRAS基因在非小细胞肺癌患者外周血和肿瘤组织中的突变研究

目的 KRAS基因突变可能导致非小细胞肺癌(NSCLC)表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)治疗的耐药。本研究旨在探讨外周血检测KRAS基因的可行性,以及对EGFR-TKIs治疗的疗效预测价值。方法应用实时荧光定量PCR(FQ-PCR)法检测120例NSCLC患者外周血标本和97例肿瘤组织标本KRAS基因突变情况,其中70例外周血标本和肿瘤组织相匹配。全组共55例接受EGFR-TKIs治疗,52例可评价疗效。比较外周血和组织标本突变一致性,分析KRAS基因突变和患者临床特征相关性。结果外周血标本中检测出14例突变(11.7%),组织中11例(11.3%),两种标本一致性为75.0%。接受EGFR-TKIs治疗患者中,外周血标本35例,其中检测KRAS突变者2例,疗效评价均为疾病进展(PD);未突变者33例,客观缓解率(ORR)27.3%,疾病控制率(DCR)57.6%。组织标本45例,其中检测KRAS突变者4例,疗效评价均为PD;未突变者41例,ORR26.8%,DCR56.1%。结论 NSCLC患者外周血和肿瘤组织中KRAS基因突变一致性较高,组织中检测KRAS基因突变和EGFR-TKIs...     

原发性肝癌中KRAS及BRAF基因突变检测的临床意义

目的探讨分析KRAS及BRAF基因突变检测对原发性肝癌的意义。方法回顾性分析2017年1月-2019年10月我院收治的158例原发性肝癌患者,根据病理临床证实是否存在肝外转移,分为转移组86例、非转移组72例,采集所有患者的血液,检测KRAS、BRAF基因以及突变情况,分析两组KRAS、BRAF基因的突变率以及突变的情况。结果转移组KRAS基因突变率明显比非转移组高(P<0.05),两组BRAF基因均未出现突变(P>0.05)。结论有肝外转移的患者KRAS存在突变,且突变率较高,而转移和非转移均未出现BRAF基因突变,因此可根据KRAS基因突变来判断原发性肝癌是否存在转移,其对原发性肝癌是否存在转移中具有临床价值。     

非小细胞肺癌靶向治疗的相关分子检测

非小细胞肺癌(non-small cell lung cancer,NSCLC)是成年人常见的恶性肿瘤之一。随着NSCLC分子生物学和新药开发研究的不断深入,基于分子标志物的靶向治疗已从实验室走到了临床,并在晚期NSCLC患者的治疗中取得了显著的效果。表皮生长因子受体(epidermal growth factor receptor,EGFR)基因、间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)基因、KRAS基因、BRAF基因是目前NSCLC靶向治疗研究的重要靶点,针对EGFR和ALK基因的靶向药物已相继被用于临床。     

HRM法检测结直肠癌患者循环DNA中KRAS和EGFR基因突变

摘要:目的：探讨高分辨率熔解曲线(HRM) 法检测结直肠癌患者循环DNA中KRAS和EGFR基因突变的可行性。 方法：用HRM法检测70例结直肠癌患者KRAS基因2号和3号外显子以及EGFR基因19和21号外显子的突变情况,用直接测序法验证结果的准确性。 结果：70 例结直肠癌患者血液标本经HRM法、直接测序法分别检测到18例(25.7%)和17例(24.3%)KRAS基因2号外显子突变;均检测到3例EGFR基因19号外显子突变(4.3%);两法均未检测到3号外显子和21号外显子突变。HRM检测与直接测序法结果符合率为99.6% (279/280)。 结论：HRM法检测结直肠癌患者血样本KRAS和EGFR基因突变,具有准确度高、操作简单、检测成本低等优点,适合在临床推广应用。     

某地区结直肠癌患者KRAS基因及BRAF基因突变状态的检测分析

目的 检测某地区结直肠癌患者KRAS基因和BRAF基因的突变状态及其与年龄、性别的关系.方法 由某地区30例结直肠癌患者石蜡组织中提取DNA,通过直接测序法及荧光定量PCR法检测KRAS基因及BRAF基因突变状态.结果 KRAS基因突变率为43.3%,共发现6种突变类型,主要位于12、13密码子,其中以c.38G>A突变率最高(38.5%).单因素及多因素分析均提示KRAS突变与年龄或性别无相关性.BRAF基因突变率为0.结论 某地区结直肠癌患者KRAS基因突变率高,靶向治疗前进行突变状态检测具有重要临床价值.     

非小细胞肺癌中EGFR和K-ras基因突变检测分析与病理特征的关系

目的探讨不同分型非小细胞肺癌的EGFR与K-ras基因突变及其相关病理特征的关系。方法基因测序方法检测260例非小细胞肺肿瘤患者肿瘤组织中的EGFR(18,19,20,21外显子)和K-ras(12,13,61密码子)基因的突变。结果 260例样本中,EGFR基因突变检出率为48.8%(127/260),突变主要集中在19号外显子的缺失和21号外显子的点突变。K-ras基因突变检出率为10%(26/260),其中12、13和61密码子均发现突变。受检的所有样本中没有发现EGFR与K-ras基因同时出现突变的情况。腺癌的EGFR突变检出率明显高于鳞状细胞癌等其他组织类型。在分化程度上,高分化与中分化癌基因突变检出率和中分化与低分化突变检出率比较差异显著(P<0.01)。EGFR突变与有无淋巴结转移无关(P>0.05)。K-ras基因突变与组织类型、患者性别、分化程度、有无淋巴结转移间无相关性。结论非小细胞肺癌EGFR基因突变检出率较高,K-ras基因突变检出率较低,且两种突变并不同时出现。EGFR基因突变与性别、肺癌组织类型、分化程度有关,存在多种突变。     

Mutation patterns and prognostic analysis of BRAF/KRAS/PIK3CA in colorectal cancer

Background and objectiveAberrant gene expression and abnormal signaling pathways often occur in patients with colorectal cancer, in which mutations in B‐Raf Proto‐Oncogene (BRAF), KRAS Proto‐Oncogene (KRAS), and Phosphatidylinositol‐4,5‐Bisphosphate 3‐Kinase Catalytic Subunit Alpha (PIK3CA) are quite common. In this study, the relationship between BRAF, KRAS, and PIK3CA mutations and clinicopathologic features and prognosis of colorectal cancer patients was investigated.MethodsOne hundred and fifty patients with colorectal cancer admitted to Affiliated people''s Hospital (Fujian Provincial People''s Hospital), Fujian University of Traditional Chinese Medicine were collected and grouped according to the mutation patterns of BRAF, KRAS, and PIK3CA. The association between BRAF, KRAS, and PIK3CA mutations and pathological factors (age, sex, etc.) was analyzed using the Chi‐square test. Subsequently, survival analysis was performed to screen the impact factors of overall survival time by Kaplan–Meier (K‐M) curve, and Cox regression model was established for the selected factors.ResultsBRAF, KRAS, and PIK3CA mutations were not associated with age, sex, and alcoholism. K–M curve and log‐rank test results demonstrated that among the factors included in this study, overall survival rate of colorectal cancer patients was only associated with mutation factors. The prognosis of KRAS+/PIK3CA−/BRAF−mutant and KRAS−/PIK3CA−/BRAF+mutant patients was better than that of KRAS+/PIK3CA+/BRAF−mutant patients.ConclusionThe mutant patterns of BRAF, KRAS, and PIK3CA were not related to the general and clinicopathological features of patients. The mutant pattern could be used as an independent prognostic factor for colorectal cancer.     

Assessment of the clinical application of detecting EGFR,KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing

Background: Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent? PGM in mutation detection of actionable genes in lung cancer.

Methods: The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays.

Results: Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement.

Conclusions: The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.     

高分辨熔解曲线技术在筛查21羟化酶缺乏症患者热点基因突变中的临床应用价值

目的 探讨利用高分辨熔解曲线技术对21羟化酶缺乏症患者中致病基因热点突变筛查的可行性及临床应用价值.方法 针对CYP21A2基因上的4个热点突变c.293-13A/C>G(I2G)、c.518T>A(p.I173N)、c.955C>T(p.Q319X)及c.1069C>T(p.R357W)设计相应的特异性引物,应用高分...     

胰腺癌中EGFR、KRAS、BRAF基因突变状况分析及意义

目的:检测胰腺癌组织EGFR、KRAS、BRAF基因突变状况,为胰腺癌EGFR靶向治疗研究奠定基础.方法:提取胰腺癌及胰腺良性病变石蜡组织切片中基因组DNA,PCR扩增EGFR 18、19、21外显子片段,KRAS2、3外显子片段,BRAF15外显子片段,采用直接测序法检测其突变状况.结果:32例胰腺癌患者中有24例患者存在KRAS基因突变,10例良性病变组织均未发现突变,两者差异具有统计学意义(x2=14.57,P=1.35× 10-4),其中22例12密码子突变(G12D14例,G12V8例);2例61密码子突变(Q61L).所有检测样本中未见BRAF突变.共3例EGFR突变,其中包括1例19外显子突变(de1746-750),2例21外显子突变(L858R),10例良性病变组织未见突变.结论:在胰腺癌中KRAS基因突变可能为EGFR通路失调的主要原因,其次是EGFR突变,BRAF突变未见.     

血清TK1水平检测在非小细胞肺癌诊断中的应用

目的研究血清胸苷激酶1(TK1)水平变化对小细胞肺癌(NSCLC)患者临床诊断价值。方法采用免疫印迹-增强化学发光法检测129例非小细胞肺癌(NSCLC)住院患者及90例健康查体者血清TK1水平,并对76例NSCLC患者手术前后血清TK1水平变化状况进行比较。结果NSCLC患者血清TK1水平明显高于健康对照组(P0.05),差异有统计学意义;NSCLC血清TK1水平ⅢⅣⅠ+ⅡTis,两两比较差异有统计学意义(P0.05);手术后30dNSCLC患者血清TK1水平明显低于手术前,差异有统计学意义(P=0.001);血清TK1水平与NSCLC患者是否发生淋巴结转移相关(P0.01),与病理类型、年龄、性别、是否吸烟均无相关性(P0.05);NSCLC患者TK1检测具有较高敏感度与特异性。结论血清TK1水平检测对NSCLC患者病情诊断有重要临床意义。     

HRM法检测结直肠癌组织KRAS基因密码子12和13点突变

目的 建立HRM法检测大肠癌患者肿瘤组织KRAS(v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)基因突变的方法 .方法 采用HRM法对含不同比例KRAS基因突变型质粒的系列混合样本进行检测,以评价其灵敏度.应用HRM法检测60份大肠癌患者新鲜肿瘤组织KRAS基因密码子12和13的突变状况,并与直接测序法的结果 进行比较分析.结果 HRM法只需在PCR结束后直接运行高分辨熔解,即可获得检测结果 .HRM法可检出系列混合样本中突变型质粒比例为10%的突变,其检测灵敏度达10%.HRM法从60份大肠癌患者组织标本中,检出17份KRAS基因密码子12或13突变(28.3%);直接测序法检出15份(25.0%)突变,2份未检出KRAS基因突变.HRM法检测的敏感度为100%(15/15),特异度为96%(43/45).结论 HRM法在筛选大肠癌标本的KRAS基因突变类型时,具有操作简便、快速、灵敏,单管避免污染等优点,完全符合临床个体化治疗的要求,值得推广.     

KRAS/BRAF mutations in brain arteriovenous malformations: A systematic review and meta-analysis

IntroductionSomatic KRAS mutations have been identified in the majority of brain arteriovenous malformations (AVM) specimens. The aim of our study was to evaluate the prevalence of Kirsten rat sarcoma (KRAS)/murine sarcoma viral oncogene homolog B1 (BRAF) mutations in brain AVM.MethodsA systematic literature review was performed in November 2019. We reviewed MEDLINE/PubMed, Cochrane Library, and ClinicalTrials.gov for citation or ongoing trials from January 2010 to March 2020.Results6 studies were identified as meeting the inclusion criteria of this review. The total frequency of KRAS mutations in 1726 patients with AVM was 55%. The prevalence of BRAF mutation was 7.5%. The prevalence of AVMs with grade 2 was the most (39%). Frontal and parietal lobes were the commonest sites of AVMs (21%). the most prevalent presentation of patients with AVM was hemorrhage (62%).ConclusionOur findings support a high prevalence of somatic activating mutations in KRAS and less commonly, BRAF in the overwhelming majority of brain AVMs. Practically and importantly, this pathway homogeneity in CNS arteriovenous malformations also supports the development of targeted therapies with RAS/RAF pathway inhibitors. However, more studies are needed to confirm this hypothesis.