基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

ARMS法检测非小细胞肺癌表皮生长因子受体基因突变及临床意义

目的：探讨应用突变特异性扩增系统法（ARMS）检测非小细胞肺癌（NSCLC）表皮生长因子受体（EGFR）基因突变的特点。方法：收集220例浙江南部地区NSCLC患者肿瘤样本,分别采用ARMS法和测序法检测EGFR基因18、19、20、21号外显子突变情况,并分析EGFR突变与病理类型、突变种类、患者年龄和性别的关系。结果：两方法比较,相符率为86．81％（158/182,Kappa=0．732,P〈0．01）。总突变率为47．27％（104/220）,其中腺癌的突变率明显高于鳞癌（52．46％zJs17．14％;P〈0．01）。肺腺癌中,女性患者EGFR突变率明显高于男性（65．56％∞39．78％;P〈0．01）。突变样本中,21外显子错义突变（L858R）所占比例最高（62．5％,65/104）,19外显子缺失（19Del）其次（43．27％,45/104）,其中两者同时突变占5．77％（6/104）;但腺癌女性与男性患者L858R突变率（64．41％∞56．76％）不存在显著性差别。结论：采用ARMS法检测EGFR基因突变较测序法敏感,突变主要发生在女性肺腺癌患者,以L858R突变为主。     

应用高分辨率熔解曲线分析石蜡标本中KRAS基因突变

目的:KRAS基因在结直肠癌患者突变率为30％～50％,并且KRAS基因状态与抗EGFR抗体疗效的密切相关.常用的直接测序法其检测灵敏度低、容易出现假阴性结果.采用高分辨率溶解曲线分析法检测结直肠癌中KRAS基因突变,探索其应用于临床检测的可行性.方法:本文收集20例结直肠癌患者石蜡包埋组织标本,应用高分辨率熔解曲线方法检测KRAS基因突变(2,3号外显子).将KRAS基因突变型DNA(p.G12D)和野生型DNA梯度混合稀释,突变型DNA浓度分别为40％、20％、10％、2％,进行高分辨率熔解曲线方法进行检测,并将扩增产物进行直接测序,比较两种方法的灵敏度.结果:在20例结直肠癌患者中检出5例2号外显子突变,p.G12D 2例,p.G12V 2例,p.G13D 1例,3号外显子未检出突变.HRM方法检测基因突变的检出限可达2％,直接测序法检测基因突变的检出限为20％.结论:HRM法比直接测序法灵敏度高,能精确检测出结直肠癌石蜡标本中低浓度的KRAS突变,有望应用于临床基因突变检测.     

高分辨率熔解曲线分析法检测结直肠癌KRAS基因突变

目的:采用高分辨率熔解曲线分析法检测结直肠癌中KRAS基因突变,探讨其用于临床检测的可行性。方法:首先用高分辨率熔解曲线分析法检测64例结直肠癌患者KRAS基因第2外显子的突变情况,再用直接测序法对结果进行验证。结果:通过高分辨率熔解曲线分析法检测,发现有23例KRAS基因突变(35.9%),经直接测序法验证,证实所有患者的突变情况与高分辨率熔解曲线法的结果完全一致;共检测出6种KRAS突变类型,G12D(GGT>GAT)的突变率最高(47.8%),G12D、G12V和G13D等3种常见突变型占总突变数的78.3%。结论:与直接测序法相比,应用高分辨率熔解曲线分析法检测KRAS基因突变具有操作简单快捷、结果准确、成本低的优点,适合应用于临床检测。     

EML4-ALK突变阳性的肺癌患者EGFR突变检测及其临床特征

目的:检测表皮生长因子受体(epidermal growth factor receptor,EGFR)在间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)融合基因突变的原发性肺癌(primary lung cancer)人群中的突变率,并分析其与病人临床病理特征间的关系。方法:入选的106例病例均为中国西北五省人群,且经ALK融合基因检测为阳性。将106例患者的组织标本采用ARMS方法检测EGFR基因18-21外显子的突变情况,统计分析双突变患者的临床病理特征。结果:106例ALK融合基因突变阳性的原发性肺癌患者的组织标本,有7例(6.6%)同时存在EGFR突变,其中19外显子缺失突变(19-del)的3例(42.9%),L858R突变的2例(28.5%),L861Q和G719X突变的各1例(14.3%);7例ALK和EGFR双突变的患者中ALK融合基因的突变均为EML4-ALK突变亚型1(variant 1,V1)。7例双阳性的患者中,6例患者的年龄小于总体患者的中位年龄(53岁),占85.7%;男性患者4例,占57.1%;不吸烟患者7例,占100%;腺癌患者4例,占57.1%,其中女性3例;肉瘤样癌2例,占28.6%;粘液表皮样癌1例,占14.3%。结论:EML4-ALK融合基因和EGFR突变能够共存,在EML4-ALK阳性的肺癌患者中,EGFR的突变率为6.6%,双突变的患者大多年轻且均无吸烟史,且双突变的女性患者均为腺癌。     

湖南地区人群中线粒体12S rRNA基因A1555G突变筛查

为建立快速、简便、准确筛查线粒体DNA 12S rRNA基因A1555G突变的基因检测技术平台,收集1 758例（女性808例,男性950例）正常人群样本,利用Bsm AⅠ酶切法筛查线粒体DNA A1555G突变以及通过实时荧光定量Taqman探针法和直接测序法对筛查结果进行验证,结果检测到2例A1555G阳性突变样本,其中1例为男性,1例为女性。实时荧光定量Taqman探针法与Bsm AⅠ酶切法、直接测序法检测结果完全相符,未发现假阳性和假阴性,该方法具有结果准确直观、简单省时,特异性强,敏感性高的优点,适用于对母系遗传性耳聋线粒体DNA A1555G突变的大规模筛查或氨基糖甙类抗生素应用前的预防性检测。     

非小细胞肺癌患者K-ras基因突变与预后的相关性分析

目的:探讨非小细胞肺癌(NSCLC)患者K-ras基因突变与预后的相关性。方法:回顾分析2006年3月~2008年7月江西省肿瘤医院收治的92例NSCLC患者的临床资料,所有患者均采用蝎形探针扩增阻遏突变系统(ARMS)法行K-ras基因突变检测。结果:92例NSCLC患者中,11例K-ras基因突变阳性,阳性率为11.96%,包括Gly12Cys(GGTTGT)突变3例、Gly12Val(GGTGTT)突变2例、Gly12Ala(GGTGCT)突变2例、Gly12Arg(GGTCGT)突变2例、Gly12Cys(GGTTGT)突变与Gly12Val(GGTGTT)突变并存1例、Gly12Cys(GGTTGT)突变与Gly12Ala(GGTGCT)突变并存1例;K-ras基因突变阳性组与阴性组之间的患者性别、年龄、病理类型、吸烟史相比,差异无统计学意义(P0.05);K-ras基因突变阳性组Ⅲa~Ⅳ期患者中位生存期显著低于K-ras基因突变阴性组Ⅲa~Ⅳ期患者,差异有统计学意义(P0.05)。结论:NSCLC患者K-ras基因突变阳性率较低,但它可以对NSCLC患者预后产生负性影响。     

应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变

目的:建立简便、快速、灵敏的锁核酸(locked nucleic acid,LNA)探针实时荧光聚合酶链反应(PCR)检测方法,检测乙型肝炎病毒(hepatitis B virus,HBV)阿德福韦酯(Adefovir dipivoxil,ADV)耐药相关位点(rtA181V、rtN236T)突变。方法:通过基因测序筛选阳性样本,进而构建ADV rt181和rt236位点野生株和突变株重组质粒,设计包含扩增阿德福韦酯rtA181V和rtN236T耐药位点在内的特异性引物和LNA荧光探针,以构建的重组质粒为标准品建立实时荧光PCR反应体系,并通过与基因测序平行检测血清样本以判断检测方法的可行性与准确性。结果:所建立的LNA-PCR法能够检测102copies/ml的HBV中ADV基因突变,同时具备较高的特异性。通过对89例ADV治疗一年后HBV阳性临床样本进行检测,有8例(8.98%)rtA181V突变、5例(5.61%)rtN236T突变、2例(2.24%)rtA181V和rtN236T混合突变,检测结果与测序结果一致。结论:所建立的LNA-PCR法是一种简便、快速、灵敏的基因突变检测方法,能有效的区分单碱基突变,对慢性乙型肝炎患者德福韦治疗过程中耐药突变的监控和抗病毒药物的调整具有指导意义。     

电化学发光法检测梅毒螺旋体特异性抗体的应用研究

目的对罗氏电化学发光免疫法(ECLIA)检测梅毒螺旋体特异性抗体的临床价值进行评估。方法收集梅毒疑似病例血清标本132份,分别用ECLIA、梅毒螺旋体明胶凝集试验(TPPA)和免疫印迹法(WB)进行检测,以WB为金标准,计算并比较ECLIA和TPPA的灵敏度和特异性,进而比较化学发光免疫法检测低S/CO值和高S/CO值的灵敏度和特异性差异。结果针对132份血清标本,ECLIA敏感性为100.00%,特异性为83.33%,阳性预测值为96.43%,阴性预测值为100.00%,总符合率为96.97%。TPPA敏感性为93.52%,特异性为87.50%,阳性预测值为97.12%,阴性预测值为75.00%,总符合率为92.42%。ECLIA检测1≤S/CO3组与S/CO≥3组的敏感性均为100.00%,特异性分别为86.96%和95.24%,结论 ECLIA检测具有较高的敏感性,适合临床大样本筛查,对S/CO值低的标本应结合TPPA、WB及临床资料确诊。     

实时荧光核酸恒温扩增技术 在检测分泌物中MRSA的应用

目的对实时荧光核酸恒温扩增技术(simultaneous amplification and testing method,SAT)检测创面分泌物中耐甲氧西林金黄色葡萄球菌(MRSA)核酸试剂盒(RNA恒温扩增)应用进行评价。方法收集我院临床各科室于2016年12月至2017年1月送至检验科微生物室347份分泌物标本,分别用实时恒温扩增技术和ChromID MRSA产色平板筛选MRSA。当SAT法和MRSA培养结果不相符时,进行冻存的备用标本PCR扩增、第三方测序,以MRSA培养结果加PCR测序结果作为本次试验"扩大金标准",计算SAT的灵敏度、特异性、阳性预测值、阴性预测值,并进行相应的统计学分析。结果以ChromID MRSA产色平板筛选加PCR测序作为"扩大金标准",SAT法检测MRSA的敏感度为90.91%、特异度为99.40%、阳性预测值为83.33%、阴性预测值为99.40%,对MRSA的最低检出下线为102拷贝/mL,Kappa系数为0.85。结论 SAT技术在检测分泌物中MRSA具有很高的灵敏度、特异性,而且准确、可靠,与传统的细菌培养相比耗时短,为MRSA的实验室诊断提供新的检测方法。     

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.     

Predictive Efficacy of Low Burden EGFR Mutation Detected by Next-Generation Sequencing on Response to EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Carcinoma

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.     

Investigation of sensitivity,specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR,based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

Purpose

Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients.

Methods

Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing.

Results

All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results.

Conclusion

Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.     

A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry

The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.     

Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

Background

Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods.

Results

All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations.

Conclusions

Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS.     

Epidermal Growth Factor Receptor Exon 20 Mutation Increased inPost-Chemotherapy Patients with Non-Small Cell Lung Cancer Detected with Patients' Blood Samples

PURPOSE: Patients with non-small cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR)-mutations have excellent response to EGFR tyrosine kinase inhibitors (TKIs), and exon 20 mutation accounts for most of TKI drug resistance. Nested polymerase chain reaction (PCR) was used to detect EGFR exon 20 mutations of patients with NSCLC after chemotherapy. The same is being analyzed with patients' characteristics. METHODS: Peripheral blood samples were collected from 273 patients with NSCLC, including 143 with adenocarcinoma (ADC) and 130 with squamous cell carcinoma (SCC), after chemotherapy. DNA was extracted from whole blood for nested PCR amplification and purification. Sequencing was carried out in an automated 3730 sequencer, followed by analysis of EGFR exon 20 mutations from nested PCR products. RESULTS: The mutations of EGFR exon 20 were mainly point mutations in rs1050171 (c.2361A>G) and rs56183713 (c.2457G>A). The point mutation was 28.21%, 28.46%, and 27.97% in patients with NSCLC, ADC and SCC, respectively. Men had an equivalent mutation (27.18%) to women (30.77%). The mutation in smokers and nonsmokers was 27.68% and 29.17%, respectively. In unselected patients, there was no correlation between EGFR exon 20 mutations and patients' characteristics of age, gender, smoking history, histologic type, or tumor-node-metastasis (TNM) staging system. In subgroup analyses, the EGFR mutation of patients with SCC was correlated with TNM stage [P = .013; odds ratio = 1.758; 95% confidence interval (CI) = 1.125-2.747]. CONCLUSIONS: The data indicate that the chemotherapy may induce EGFR-TKI-resistant mutation in NSCLC cells and EGFR-TKI should be used in the early stage of NSCLC but not after chemotherapy.     

The Relationship between TTF-1 Expression and EGFR Mutations in Lung Adenocarcinomas

Objective

To explore the relationship between TTF-1 and EGFR mutations in lung adenocarcinoma tissues to guide clinical treatment timely and effectively.

Materials and Methods

we collected 664 tissue samples from patients with histologically confirmed lung adenocarcinoma from May 2010 to April 2013. All tumor tissues were collected prior to administering therapy. TTF-1 was detected byimmunohistochemistry and EGFR mutations by DNA direct sequencing. Finally, the correlation between TTF-1 expression and the presence of EGFR mutations was analyzed using χ² test or Fisher’s exact test with SPSS software version 18.0.

Results

Of the 664 lung adenocarcinoma tissue samples, 18 were partially positive for TTF-1 (+−), and 636 were positive for TTF-1 (+) resulting in a total positive rate of 98.49% (+,+−)(including partial positive). In only 10 cases was the TTF-1 negative (−); the negative rate was 1.51%. There were 402 cases without an EGFR mutation and 262 cases with EGFR mutations; the rate of mutations was 39.46%. The location of the EGFR mutation was exon 19 for 121 cases resulting in a mutation rate in exon 19 of 18.22%. The location of the EGFR mutation was exon 21 for 141 cases resulting in a mutation rate in exon 21 of 21.23%. Exon 18 and 20 detected by DNA direct sequencing no mutations.A Fisher’s exact test was used to determine the correlation between EGFR mutations and TTF-1 expression.for the whole, TTF-1 positive expression(including partial positive) has correlation with EGFR mutations (p<0.001),especially for Exon 21 expression,the correlation is significant (p = 0.008).

Conclusion

In lung adenocarcinomas, positive and partial positive TTF-1 expression has a significant positive correlation with EGFR mutations(exon 19 and 21). In clinical practice, TTF-1 expression combine with EGFR mutations, especially exon 21 mutation can guide clinical treatment timely for lung adenocarcinomas.     

High sensitivity of both sequencing and real‐time PCR analysis of KRAS mutations in colorectal cancer tissue

The KRAS mutation status predicts the outcome of treatment with epidermal growth factor receptor targeted agents, and therefore the testing for KRAS mutations has become an important diagnostic procedure. To optimize the quality of this test, we compared the results of the two most commonly used KRAS mutation tests, cycle sequencing and a real‐time PCR‐based assay, in DNA extracted from formalin‐fixed paraffin‐embedded (FFPE) colorectal cancer samples of 511 patients. The results were interpreted in the context of the tumour cell percentage and the assay parameters. In 510 samples KRAS mutation status assessment was successful. A KRAS mutation was detected in 201 tumours (39.4%). Sequencing and the real‐time PCR‐based assay generated the same result in 486 samples (95.3%). The sequencing result was considered false positive in one (0.2%) and false negative in nine samples (1.8%). The assay result was considered false positive in six (1.2%) and false negative in seven samples (1.4%). Explanations for discrepant test results were a higher sensitivity of the assay in samples with a low tumour cell percentage, occurrence of mutations that are not covered by the assay and δ Ct values approximating the cut‐off value of the assay. In conclusion, both sequencing and the real‐time PCR‐based assay are reliable tests for KRAS mutation analysis in FFPE colorectal cancer samples, with a sensitivity of 95.5% (95% confidence interval [CI] 91.7–97.9%) and 96.5% (95% CI 93.0–98.6%), respectively. The real‐time PCR based assay is the method of choice in samples with a tumour cell percentage below 30%.     

Quantification and Dynamic Monitoring of EGFR T790M in Plasma Cell-Free DNA by Digital PCR for Prognosis of EGFR-TKI Treatment in Advanced NSCLC

Background

Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.

Materials and Methods

We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.

Results

Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively).

Conclusion

Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.     

Transmission of synovial sarcoma from a single multi-organ donor to three transplant recipients: case report

Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination. Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant. Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.