Renewable standard reference material for the detection of TP53 mutations.

BACKGROUND: Numerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0 kb region of the TP53 gene, including exons 5-9. Eleven of these clones contain a single mutation within the mutational hot spots of the TP53 gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-type TP53 from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms. METHODS AND RESULTS: The single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM. CONCLUSION: The generation of a standard TP53 reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.     

A locked nucleic acid clamp-mediated PCR assay for detection of a p53 codon 249 hotspot mutation in urine

Hepatocellular carcinoma (HCC) has a 5-year survival rate of <10% because it is difficult to diagnose early. Mutations in the TP53 gene are associated with approximately 50% of human cancers. A hotspot mutation, a G:C to T:A transversion at codon 249 (249T), may be a potential DNA marker for HCC screening because of its exclusive presence in HCC and its detection in the circulation of some patients with HCC. A locked nucleic acid clamp-mediated PCR assay, followed by melting curve analysis (using the SimpleProbe), was developed to detect the TP53 249T mutation. In this assay, the locked nucleic acid clamp suppressed 10(7) copies of wild-type templates and permitted detection of 249T-mutated template, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard within 2 hours. With an amplicon size of 41 bp, it detects target DNA sequences in short fragmented DNA templates. The detected mutations were validated by DNA sequencing analysis. We then tested DNA isolated from urine samples of patients with HCC for p53 mutations and identified positive TP53 mutations in 9 of 17 samples. The possibility of using this novel TP53 249T assay to develop a urine or blood test for HCC screening is discussed.     

Mutation scanning of the RET protooncogene using high-resolution melting analysis

BACKGROUND: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. METHODS: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen, and the high-resolution melter, HR-1, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. RESULTS: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. CONCLUSION: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.     

表皮生长因子受体-酪氨酸激酶抑制剂治疗后耐药的肺腺癌患者二次组织活检与外周血基因特征一致性分析

目的 对表皮生长因子受体(epidermal growth factor receptor,EGFR)-酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)治疗后耐药的肺腺癌患者行二次组织活检并采集外周血标本进行基因检测,分析二次组织活检与外周血标本基因特征的一致性.方法 EGFR-TKI...     

Multiplex amplification coupled with COLD-PCR and high resolution melting enables identification of low-abundance mutations in cancer samples with low DNA content

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification.     

Genetic variants of the NOTCH3 gene in migraine--a mutation analysis and association study

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Exons 3 and 4 are mutation hotspots. Migraine is a clinical hallmark of CADASIL. The objective of this study was to investigate whether genetic variants in exons 3 and 4 of the NOTCH3 gene are associated with migraine. Exons 3 and 4 of the NOTCH3 were analysed for mutations and polymorphisms by direct DNA sequencing in 97 migraineurs and the same number of control individuals. No mutations in exons 3 and 4 of the NOTCH3 gene were found in 97 patients with migraine. However, association analysis revealed significant association of the single nucleotide polymorphism (SNP) rs1043994 with migraine.     

Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model

Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.     

Towards fast and inexpensive molecular diagnostic: the case of TP53

BACKGROUND: Much research suggests that TP53 mutations have prognostic importance and sometimes are a significant factor in clinical oncology. A considerable effort has been made to develop fast and inexpensive methods for TP53 mutations detection. METHODS: On the basis of describing the role of TP53 as tumor suppressor gene and TP53 mutation spectrum, the authors discuss conventional methods and new technologies for TP53 mutations detection. This discussion is supported by more recent publications in the field of both molecular genetics and analysis technologies. RESULTS: Biosensors and gene chips are of considerable recent interest, due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional methods. CONCLUSIONS: New methods such as biosensors and gene chips appear promising as analytical methods of detecting mutations.     

中国肾移植患者中细胞色素P450 3A4的新突变位点

背景：环孢素A在人体内主要经过细胞色素P450 3A4 代谢。已有研究表明细胞色素 P450 3A4 基因多态性影响环孢素A的药代动力学,而且环孢素A慢性肾毒性主要是由于环孢素A在体内的长期蓄积。因此推测细胞色素 P450 3A4 基因多态性可能是肾移植移植后环孢素A慢性肾毒性的主要原因之一。目的：分析细胞色素 P450 3A4 基因多态性与肾移植移植后环孢素A慢性肾毒性的相关性。方法：纳入 200 例服用环孢素A的肾移植患者参加此项研究,其中105例经肾活检和（或）血肌酐值的变化诊断为环孢素 A慢性肾毒性,其他 95 例未发生肾毒性）。采集受试者外周静脉血并提取基因组DNA,采用聚合酶链式反应和直接测序法检测细胞色素P450 3A4基因外显子5,7,9 和 12 的点突变。结果与结论：中国肾移植患者中发现 3 个细胞色素 P450 3A4 新突变位点,即 336A〉G,837 G〉A 和 406 A〉C。其中 3 例肾移植患者可检测出 336 A〉G,8 例肾移植患者可检测出 837 G〉A。新突变点 406A〉C 仅发现于 3 例环孢素A慢性肾毒性患者,在非肾毒性患者中未发现新突变位点,且 406 A〉C 可导致细胞色素 P4503A4 保守区 136 位苏氨酸（Thr）转变为苯丙氨酸（Phe）。文章未发现已报道的中国人群细胞色素P450 3A4基因在外显子5,7,9 和 12 的多态性。结果证实,文章在中国肾移植患者中发现了 3 个细胞色素P450 3A4 的新突变位点 336 A〉G,837G〉A 和 406 A〉C,其中 406 A〉C 可能引起细胞色素 P450 3A4 酶活性的改变。     

406例肺癌小活检标本中表皮生长因子受体基因的突变分析

目的探讨肺癌小活检标本中表皮生长因子受体（epidermal growth factor receptor,EGFR）基因的突变情况。方法采用蝎形探针扩增阻滞突变系统（Scorpions ARMS）检测406例肺癌小活检标本中EGFR基因第18、19、20和21外显子的突变情况。结果在406例肺癌小活检标本中,检测到190例存在EGFR突变（占46.8%）,其中80例为第19外显子框内多核苷酸缺失、88例为第21外显子L858R突变、4例为第21外显子L861Q突变、9例为第18外显子G719X突变、2例为第20外显子S768I突变、1例为第20外显子T790M突变;此外,发现6例存在双位点突变。肺腺癌EGFR突变率为55.5%（186/335）,鳞状细胞癌突变率为5.8%（3/52）,5例腺鳞癌中有1例检测到EGFR基因突变,其它类型肺癌均未检测到EGFR基因突变（0/14）。女性患者EGFR基因突变率（62.4%）明显高于男性患者（32.1%,P〈0.01）,非吸烟患者EGFR基因突变率（59.8%）明显高于吸烟患者（24.7%,P〈0.01）。结论 EGFR基因突变多见于女性、非吸烟和肺腺癌患者;Scorpions ARMS是检测活检小标本EGFR基因突变的可靠方法。     

ICC患者血浆ctDNA突变检测数字PCR平台的建立及临床应用价值

目的建立KRAS G12D、TP53 C242S、IDH1 R132C突变数字聚合酶链反应(dPCR)检测平台,并初步评估其检测性能及临床应用价值。方法选取行切除手术的肝内胆管细胞癌(ICC)患者22例,设计KRAS G12D、TP53 C242S、IDH1 R132C突变位点的引物及探针,建立dPCR突变检测平台。并采用不同浓度的自配标准品质粒验证该平台的准确性、精密度、空白限、功能灵敏度及线性范围。将外周血dPCR检测结果与外周血Oseq-ctDNA及组织Oseq-T靶向测序结果进行比较。ICC患者行切除术后,每6个月采集1次外周血并跟踪随访,评估该突变检测平台对ICC患者术后疗效监测的作用。结果 dPCR平台的准确性良好[3种突变(KRAS G12D、TP53 C242S和IDH1 R132C)3个丰度的检测结果与理论值的偏差均<±15%],批内和批间精密度[变异系数(CV)]均<20%,空白限为4拷贝,功能灵敏度为0.1%,且在0.1%~10.0%范围内线性良好。ROC曲线分析结果显示,ctDNA突变谱诊断ICC的曲线下面积(AUC)为0.659,敏感性为31.8%,特异性为100%;与糖类抗原19-9(CA19-9)联合检测诊断ICC的AUC为0.841,敏感性为68.2%,特异性为100%。dPCR平台的检测结果与Oseq-ctDNA测序结果有较高的一致性(kappa=0.792,P=0.007)。随访结果显示,有1例ICC患者术后18个月KRAS G12D突变升高,与影像学检查确认的复发时间一致。结论建立了检测KRAS G12D、TP53C242S、IDH1 R132C突变的dPCR平台,可用于ICC患者的辅助诊断、疗效评估及术后动态监测。     

Germline p53 mutations are frequently detected in young children with rhabdomyosarcoma.

We investigated the possibility that a proportion of children with sporadic rhabdomyosarcoma (RMS) carry constitutional mutations of the p53 tumor suppressor gene. 33 patients with sporadic RMS at two large outpatient pediatric oncology clinics submitted blood samples. Genomic DNA was extracted from peripheral blood leukocytes and PCR was used to amplify exons 2-11 of the p53 gene. Amplified genomic DNA was screened for the presence of germline p53 mutations using single-strand conformation polymorphism (SSCP) analysis. The DNA sequence of those samples that showed aberrant migration of bands on SSCP analysis was determined to identify the precise nature of the gene mutations. Patient records were reviewed to assess clinical correlates of the mutant p53 carrier state. Heterozygous constitutional mutations were detected in 3/33 patient samples screened. Two of these missense mutations are located in exon 7 and one in exon 8 of the p53 gene. The presence of mutations was not correlated with tumor histology, stage, or site. However, an association between young age at diagnosis and presence of a constitutional p53 mutation was noted: 3/13 children under the age of 3 yr at diagnosis carried mutations, whereas none of 20 children over 3 yr of age at diagnosis harbored a detectable constitutional mutation. These results in children with RMS corroborates previous findings in other clinical settings suggesting that the mutant p53 carrier state may predispose individuals to malignancy at an early age. Although this study did not assess whether the mutations were preexisting or new germline alterations, assessment of close relatives of RMS patients for cancer risk and predictive genetic testing may be indicated.     

Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography

OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation. DESIGN AND METHODS: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency. RESULTS: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results. CONCLUSION: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.     

Rapid mutation screening for HRPT2 and MEN1 mutations associated with familial and sporadic primary hyperparathyroidism

Familial hyperparathyroidism, a disease of the parathyroid glands, may occur in conjunction with pituitary and pancreatic tumors (multiple endocrine neoplasia type I), kidney and bone tumors (hyperparathyroidism jaw tumor syndrome), or alone (familial isolated hyperparathyroidism). This study describes the development and validation of rapid scanning for mutations in two tumor suppressor genes linked to familial hyperparathyroidism-MEN1 and HRPT2. Denaturing high-performance liquid chromatography mutation scanning for MEN1 was performed using a set of 10 amplicons covering the nine coding exons and flanking intronic regions and for HRPT2 using a set of three amplicons for exons 1, 2, and 7 and flanking intronic regions, in which 80% of the mutations identified to date are located. All 52 MEN1 mutations or polymorphisms, 46 known and six unknown, were successfully detected. Mutation detection in exon 9 was not confounded by the presence of the common polymorphism D418D. In addition, all 10 HRPT2 mutations were successfully detected, and a two-step approach was able to distinguish IVS2 common polymorphisms from exon 2 mutations. The development of rapid denaturing high performance liquid chromatography mutation scanning of MEN1 and HRPT2 facilitates a molecular diagnosis of the associated familial syndromes for both clinically affected and at-risk family members.     

中国人群KEL等位基因2例新突变的检出

目的研究中国人群中KEL基因多态性。方法用聚合酶链反应-限制性酶切片段-单链构象多态性分析(PCR-RF-SSCP)合并异源二聚体分析,筛选找出具有不同格局的样本并对其测序,找出突变位点。结果在500份随机的中国人群样本中,发现2份样本格局与正常格局不同,测序显示其核苷酸发生了突变,分别是KEL基因外显子9上1086G>A和内含子7的第67位C>A点突变,外显子9上碱基突变是同义突变。结论在中国人群中检出2例新突变的KEL基因。     

New p53 mutations in hilar cholangiocarcinoma

BACKGROUND: Inactivation of the tumour suppressor gene, p53, is the commonest genetic abnormality in human cancer. The study of the type of p53 mutation in a given tumour may provide prognostic information, clues to aetiology and become useful for therapeutics. MATERIALS AND METHODS: The molecular characterisation of p53 was performed by restriction analysis, denaturing gradient gel electrophoresis, and gene sequencing for exons 5-9. RESULTS: We report, p53 mutational analysis in exons 5-9 in 29 European patients with hilar cholangiocarcinoma who underwent attempted resection. Four patients (14%) showed somatic single nucleotide substitutions with amino acid changes (146, 163, 175, 158, and 175) with one showing mutations in two different positions involving a loss of two CfoI sites. All the mutations occurred in exon 5. Three patients had a germline polymorphism (10%) with a silent substitution in codon 213 (exon 6). CONCLUSIONS: The systematic screening for p53 mutations in European patients with hilar cholangiocarcinoma has shown that the type of mutation (except 175) is different and its incidence is much lower when compared to the pattern previously reported for intrahepatic cholangiocarcinoma in East Asian patients. A probable explanation is that the presence and type of p53 mutation is dependent on geographic and environmental factors which vary in different populations.     

二代测序技术在肺癌组织相关基因突变检测中的临床应用价值

目的基于二代测序技术分析肺癌相关基因突变及位点分布状况,探讨突变基因与临床病理特征之间的关系及临床应用价值。 方法收集唐都医院呼吸与危重症医学科2017年12月至2018年6月行支气管镜活检和手术切除的肺癌组织样本53例,采用Illumina二代测序平台分别检测22个基因共103个突变热点区域,描述检测结果,统计分析高频突变基因与临床病理特征之间的关系。 结果22个肺癌相关基因中共检测到19个基因突变;103个肺癌相关突变位点中共检测到71个突变,其中肿瘤蛋白P53(TP53)基因的突变率为45.07%(32/71),表皮生长因子受体(EGFR)基因突变率为9.86%(7/71),细胞周期蛋白依赖性激酶抑制因子2A(CDKN2A)基因的突变率为8.45%(6/71),大肠癌K-鼠肉瘤病毒癌基因(KRAS)突变率为5.63%(4/71),肿瘤组织腺瘤样息肉病基因(APC)的突变率为4.23%(3/71),磷酸酰肌醇-3-激酶催化亚基(PIK3CA)、ERBB4、神经营养性酪氨酸激酶受体1(NTRK1)、SMO、KIT基因的突变率均为2.82%(2/71),GNAQ、CTNNB1、MAP2K1、ATM、成纤维细胞生长因子受体(FGFR3)、NOTCH1、上皮钙黏蛋白编码基因(CDH1)、成视网膜细胞瘤1(RB1)、HRAS基因的突变率均为1.41%(1/71)。TP53突变者共有29例,其中3例患者具有双位点突变,共有32个突变位点。EGFR突变者共有6例,其中1例患者具有双位点突变,共有7个突变位点。CDKN2A突变均为单位点突变。TP53的5号外显子Val173Glu错义突变、8号外显子Arg273Leu错义突变、9号外显子Lys320Ter无义突变的突变率均为6.25%(2/32),其他位点突变率均为3.13%(1/32)。EGFR的19号外显子Glu746Ala750del缺失突变、21号外显子Arg868Leu错义突变的突变率分别为42.86%(3/7)、28.57%(2/7),其他位点突变率均为14.29%(1/7)。CDKN2A所有突变位点的突变率均为16.67%(1/6)。TP53突变频率在低、中分化肺癌患者中较高,差异具有统计学意义(χ²＝7.58,P＝0.023)。EGFR突变在病理类型(χ²＝7.45,P＝0.024)和分化程度(χ²＝14.51,P＝0.001)方面的差异具有统计学意义;临床各分期间比较,差异无统计学意义(χ²＝6.37,P＝0.081)。 结论Illumina二代测序平台可作为分析肺癌多基因突变的检测手段,TP53、EGFR和CDKN2A突变频率较高,对肺癌的临床诊疗具有一定的指导意义。     

Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC

BACKGROUND: Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutations, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and further defined whether HC probands with an incomplete HFE genotype carry uncommon mutations. METHODS: Analytical conditions for each coding exon were determined by a combination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and optimize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA samples harboring the C282Y, H63D, or S65C mutations and 17 artificially created positive controls that carried either 1 of the 14 other known HFE mutations or 3 selected polymorphisms. RESULTS: Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more complex melting profile and required two analysis temperatures. DHPLC detected all known HFE mutations as well as the three selected polymorphisms. CONCLUSIONS: DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation.     

Identification of liquid biopsy-based mutations in colorectal cancer by targeted sequencing assays

Recently, liquid biopsy, as a promising approach was introduced for the analysis of different tumor-derived circulating markers including tumor DNA and cell free DNA (ct/cfDNA). Identification of mutations in cfDNA may allow the early detection of tumors, as well as predicting and monitoring treatment responses in a minimally invasive way. In the present study, we used commercially available gene panels to verify the mutation overlap between liquid biopsy and abnormalities detected in colorectal tumor tissue. The two panels (Archer®VariantPlex®Solid Tumor and LIQUIDPlexTM ctDNA) overlap in 23 genes, which enables a comprehensive view of tumor-plasma mutational status by next generation sequencing. We successfully analyzed 16 plasma and 16 tumor samples. We found that 87% of tumor tissues contained 44 mutations in 12 genes and 43.8% of cfDNA harbored 13 mutations in 5 genes. To verify whether the mutation pattern of the tumor DNA could be consistently detected in plasma cfDNA, we compared the alterations between cfDNA and matched tissue DNA in nine patients. Six of the 9 tumor tissues harbored mutations in TP53, KRAS or MET genes, those were not detectable by the ctDNA kit, even eventhough the exons of these genes overlap in both panels. Comparing the mutational patterns of the matched samples, we found that only one cfDNA had the same mutations (KRAS, SMAD4 and TP53) in the paired tissue. The results of the comparison between tumor tissue DNA and matched plasma cfDNA underline the importance of studying the paired solid tumor and plasma samples together.