高分辨率熔解曲线分析检测大肠肿瘤粪便DNA突变性能评价
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摘要
研究背景和目的
全球结直肠癌(CRC)发病率位于恶性肿瘤的第3位,我国位于第4-6位,呈逐年上升的趋势。广东省CRC发病率的上升速度较快。CRC的预后与早期诊断密切相关。绝大多数CRC是由“正常-腺瘤-癌”逐步演变而来,从可辨认的腺瘤发展为侵袭性癌约需5-10年,这为预防及早期筛查提供了充裕的时间。大量研究已证实发生癌变的腺瘤主要为晚期腺瘤(AA,指直径≥1cm或组织学证实为绒毛状腺瘤或重度异型性增生的腺瘤),通过临床筛查并及时切除癌前病变如腺瘤,可降低CRC发病率及病死率。
结肠镜检查因准确性高且能治疗而被首选,但存在需要专业的内镜医生、风险较大、费用高、患者接受性差等不足,难以大规模应用于无症状人群的筛查；粪便隐血试验(FOBT)具有简便、无创、经济,接受性好等优点而被优选,但存在假阳性率及假阴性率较高、需要多次送检等不足,由于腺瘤检出率敏感度低,因而对癌症发病率的影响小；粪便DNA检测具有只需一次送检、易接受、依从性高、不需服药或控制饮食、不受肿瘤部位影响等优点而被重视。大量研究表明粪便DNA检测的敏感性和特异性比FOBT好,尤其腺瘤检出率高引人注目。已列为美国指南候选方法。
但现有的粪便DNA检测方案尚需完善,主要是敏感性与特异性有待提高,费用需降低、操作需简单化等。除了尚需进一步优化/更替现有的标志物组合外,升级现有的检测方法也是关键环节。现有的高敏感性和特异性的粪便DNA检测方法,普遍存在费用高、操作复杂、过于专业化、受制于待检目标基因位点的等问题。因此寻求一种便捷易用、经济高效的检测方法是提升粪便DNA检测实用价值的必要条件。
高分辨率熔解曲线分析(HRMA)是犹他州大学Wittwer实验室与美国Idaho公司联合研制的一种PCR后闭管操作技术：通过高密度荧光数据采集,直接用熔解曲线检测PCR扩增片段中微小序列差别。具有低成本、高通量、无污染、快捷、操作简单、不受变异位置影响、敏感性及特异性好等优势。自2003年被引入检测基因突变以来,HRMA的应用与发展如“雨后春笋”,在医学分子诊断中越来越受到关注。经荟萃分析发现其灵敏度达97.5%(95%置信区间(CI),96.8-98.5)、特异度达95.8%(95%CI：95.3-96.3),DNA样本的来源及饱合染料类型均对HRMA的灵敏度无影响。我们前期的研究已经初步展示HRMA用于粪便DNA突变检测,获得较高的检出率,具有潜在临床应用价值。目前国内尚无有关评价HRMA方法用于粪便DNA检测筛查CRC的文献报道。
因此,本研究在前期研究基础上,以DNA测序结果作为金标准,扩大样本量、更新试验设计方案、优化实验条件及操作流程等方式对HRMA的准确性展开较为全面的评价性研究。首先在组织样本中建立与优化HRMA操作流程与实验参数；然后,在2个独立的样本集(包括“学习集”与“验证集”阶段)中对HRMA应用于粪便DNA KRAS与TP53突变的检测的敏感性和特异性进行全面评价；同时,在DNA系列稀释试验中评估HRMA技术的敏感性；最后,探讨检出的粪便KRAS、TP53突变与大肠肿瘤性病变的临床病理特征参数的关系及诊断价值。
方法
研究对象：2010.1-2010.7间在惠州市中心医院及南方医院接受肠镜检查者和/或普外科住院的术前CRC与AA患者中,及肠镜检查未发现明显异常的病人中。按下列纳入与排除标准,连续收集这些患者的粪便标本与临床病理特征参数资料,在“学习集”部分收集相应的肿瘤的FFPE组织标本。
研究组纳入标准：(1)年龄大于40岁、且病理组织学检查确诊为结直肠AA与CRC患者；(2)能同时收集病变组织与合格粪便样本(重量不低于5克)的患者。
研究组排除标准：(1)家族性或遗传性结直肠腺瘤或肿瘤；其他系统或器官肿瘤。(2)共患有炎症性肠病；(3)术前有放疗或化疗史；(4)妊娠或有严重肝肾功能不全者；(5)非原发癌灶者及不能确诊等其他情况；(6)既往有CRC或腺瘤病史；(7)肉眼见全血性大便者。
粪便样本采集：在内镜检查或肿瘤切除手术前、接受任何形式的结直肠侵入性操作或服用泻剂肠道准备的1周后,收集至少5克新鲜粪便量于48小时内尽快冻存于-80℃冰箱待用。
DNA提取与质控：按QIAamp粪便DNA、FFPE DNA的提取试剂盒的说明书方法提取DNA,在FFPE组织取样时,采用传统手工微切的方法,保留每—块样本中含有肿瘤细胞占的比例≥50%。提取DNA的浓度和纯度测定均用NanoDrop ND-1000分光光度计检测,达标要求：浓度≥50ng/μL,260/280nm的吸光度位于1.6-2.2且230/260nm位于2.0-2.5。达标的样本DNA均稀释到50ng/μL的终浓度,置入-20℃冰箱保存待用。
HRMA检测：基于NCBI的TP53外显子5-8、KRAS2-3的参考基因序列设计引物,KRAS Exon2(92bp) f-TTATAAGGCCTGCTGAAAATGACTGAA, r-TGAAT-TAGCTGTATCGTCAAGGCACT; Exon2(155bp) f-TTATAAGGCCTG CTGAAAAT-GACTGAA, r-TGAATTAGCTGTATCGTCAAGGCACT, Exon3f-GACTGTGTTTCT-CCCTTCTC, r-TGTACTGGTCCCTCATTGC; TP53Exon5f-GTGCAGCTGTGGGTT-GATT; r-AACCAGCCCTGTCGTCTCT, Exon6f-GAT TCCTCACTGATTGCTCTTA-G r-GGGCACCACCACACTATG; Exon7f-TTGGGCCTGTGTTATCTCCT r-TGGCA AG TGGCTCCTGAC,Exon8f-TTGCTTCTC TTTTCCTATCCTGAr-GCTTCTTGTCCTGCTTGCTT。其中, KRAS基因外显子2,设计2对引物,扩增的片段分别为92bp和155bp。KRAS基因外显子2(155bp)的检测是在LightCycler480PCR仪上操作；其他的KRAS基因外显子2(92bp)-3,TP53基因外显子5-8基因均在Rotor-GeneTM6000PCR仪上操作。反应体系：2μL(100ng)样本DNA,上下引物各为lμL(200nmol/L),镁离子2μL(2.5mmol/L), LightCycler480HRM Master/qPCR Master预混液12.5μL,加ddH2O至总体积25μL。反应条件参数：95℃预热5分钟,95℃×15秒→60℃-63℃×50秒→72℃×30秒→72℃×10分钟,共50个循环。PCR产物在95℃变性5分钟,冷却至40℃×1分钟(让异源双链形成)然后在65℃升至95℃间,荧光采集密度为25次/℃。HRMA分析：原始采集的荧光曲线经过正常化和温度调整处理后,野生型基因被用来作为阴性对照。曲线图形的差异表示扩增的样本基因序列存在变异。HRMA突变阳性样本记为异常熔解曲线。采集的数据经罗氏公司的基因分析软件(1.5版)及RotorGene的软件(V1.7.25版)分析得出结果。
DNA测序检测：首先,对PCR产物进行酶切纯化：1、将每个样本PCR产物使用1%的琼脂糖凝胶进行检测,确认目的条带是否单一；2、经上述琼脂糖凝胶检测条带单一的,使用SAP(虾碱性磷酸酶)和Exo I(外切核酸酶)进行酶切纯化；3、经上述琼脂糖凝胶检测有非特异性的条带,使用BioMIGA的琼脂糖凝胶回收试剂盒进行割胶回收。接着,在用在ABI3730DNA测序仪上进行单向测序,采用的是Sanger法。最后,使用3730XL软件、DNAMAN软件对测序数据进行分析,得出最终结果。所有样本DNA的测序均送上海硕士生物有限公司检测分析。
统计方法
以α=0.05为检验水准,各组间年龄,两样本t检验；所有的计数资料的比较,多样本间采用χ2检验,四格表采用Fisher确切概率法,若配对资料采用McNemar检验,符合率采用Kappa分析。统计分析均使用SPSS13.0软件包及EpiCalc2000统计软件。
结果
参与患者的临床病理特征参数
最初,有145例大肠肿瘤性病变患者,70例结肠镜检查阴性的病人(NC)作对照组。28例因粪便样本不符合纳入与排除标准而剔除。接下来,187提取DNA,12例因DNA质量不达标而排除。最终纳入研究共175例,63例CRC,52例AA,60例年龄相匹配的NC,所有参与者均是中国人。患者病情诊断的确定均是根据结肠镜检查结果和/或完整切除肿瘤的标本病理诊断。CRC组的平均年龄为61岁(41-87岁),AA组的平均年龄为58岁(40-80岁),对照组平均年龄为47岁(40-73岁)；病例组男女性比例69/57,其中CRC为32/31, AA为37/26,而对照组为24/36；CRC、AA原发部位位于近端结直肠分别为88.9%(56/63)、86.5%(45/52)；Duck's分期中,A+B期占60.3%；病例组KRAS外显子2-3突变率为26.1%(30/115),TP53外显子5-8突变率为21.7%(25/115),对照组突变率3.3%(2/60)。
“学习集”阶段研究
为了建立及优化HRMA技术的实验条件与参数设定。我们在CRC与AA的组织样本中,对HRMA检测突变基因的准确性进行评价。先对合格的40例病人的FFPE的样本进行测序证实KRAS外显子2-3与TP53外显子5-8基因突变情况,再用HRMA检测其基因突变的情况,我们检出22例病人有基因突变,其中17例来自29例CRC,5个来自AA,突变检出率55%(22/40)。两者检测结果完全一致,其敏感度与特异度均为100%。随后基于上述优化的HRMA的技术操作流程,我们就对其相应的粪便DNA的突变进行检测,结果发现,HRMA检出18例(18/40,45%)突变,其中,CRC14例(14/29,48.28%),AA4例(4/11,36.36%),这些检测结果均被随后的测序所证实,HRMA的敏感度与特异度均为100%。粪便DNA与相应组织的突变检出率的符合率高度一致,18/22(81.82%),其中CRC14/17(82.35%),AA4/5(80%),Kappa值为0.794,在CRC、AA中分别为0.794、0.814。
HRMA的敏感性分析
为了评价HRMA在结直肠癌筛查的环境中检测粪便DNA突变的可靠性,我们采用DNA的系列浓度稀释试验评估HRMA检测最低水平基因突变的灵敏度。选用已知KRAS基因外显子2及TP53基因外显子6-8突变具体情况的粪便样品DNA系列稀释实验,选用同样的粪便DNA中的野生型样本作为稀释实验的背景。我们发现,HRMA检出突变基因的最低稀释浓度限度为1%,但样本间存在的差异大；HRMA能够稳定可靠的检出浓度为≥5%。
“验证集”阶段研究
基于前二个部分研究得出的HRMA具有与测序一致的敏感性与特异性,且检出突变基因的最低浓度限度能达1%。促使我们在“验证集”中进一步评价其准确性。75例病例组,包括34例CRC和41例AA,60例对照组。在病例组的粪便DNA中,共检出含有KRAS和／或TP53基因突变频率为(49.3%,37/75)显著高于对照组(3.3%,2/60)(P<0.001)；在病例组的亚组分析中,基因突变率在CRC亚组(58.8%,20/34)与AA亚组(41.5%,17/41)之间的存在显着性差异(P=0.02)。HRMA检测的结果,均被随后的测序证实,其敏感度与特异度均为100%。
粪便基因突变与临床病理特征参数关系及临床诊断价值
1、病例组中,CRC、AA原发于近端结直肠(88.9%、86.5%)明显多于远端结直肠(13.5%、11.1%)；Duck's分期中,A+B期(60.3%)明显多于C+D期(39.7%)；在病例组与对照组间,CRC亚组与AA亚组间,均存在显著的性别构成比差异(P=0.004,P=0.035),前者男性所占比例均明显多于后者；对KRAS基因外显子2突变,我们同时检测二个不同长度的片段(155bp和92bp),发现两者的突变检出率完全一致。
2、在CRC组中,检出19例(19/63,30.1%)KRAS突变,15例(15/63,23.8%)TP53突变。发现各基因突变频率与性别、部位、分化度、组织类型、大小、Duck's分期、年龄均无统计学上差异。
3、在AA组中,检出11例(11/52,21.2%)KRAS突变,10例(11/52,19.2%)TP53突变。发现TP53突变与年龄分组存在差异(P=0.036),发现年龄≥60岁组突变检出率32.0%(8/25)高于<60岁组7.4%(2/27)。但发现各基因突变频率与性别、部位、异型增生度、组织类型、大小均无统计学差异。
4、两基因联合突变发生率,CRC中为54.0%(34/63),AA中为40.4%(21/52)；联合检测粪便DNA KRAS与TP53基因突变筛查CRC与AA的性能欠佳,敏感度分别为54%[95%CI0.41,0.66]与40%[95%CI0.27,0.55],特异度均为97%[95%CI0.87,0.99],准确性分别为75%与71%。
结论
“学习集”阶段
1、在CRC与AA患者的组织样本中,HRMA检测KRAS外显子2-3与TP53外显子5-8基因突变准确性与DNA测序结果完全一致,其敏感性与特异度均达100%。
2、在其相应的粪便DNA样本中,HRMA检测KRAS外显子2-3与TP53外显子5-8基因突变准确性与DNA测序结果完全一致,其敏感性与特异度均达100%。
3、粪便DNA突变检出率与组织DNA突变检测出率的符合率高度一致,Kappa值为0.794,在CRC、AA中分别为0.794、0.814。
HRMA的敏感性分析
在野生型基因稀释的背景下,这可粗略估计出HRMA检测突变的敏感性能达1%稀释浓度,并且发现待检样本的序列变异直接影响HRMA检测的灵敏度,因此HRMA能稳定可靠地检出突变的敏感性是≥5%稀释浓度。
“验证集”阶段
1、病例组基因突变检出率(49.3%,37/75)显著高于对照组(3.3%,2/60)。表明KRAS和/或TP53基因突变与大肠肿瘤性病变相关。
2、CRC患者基因突变检出率的显著高于AA患者,表明KRAS和/或TP53基因突变与CRC关系更密切。
3、HRMA检测基因突变的结果均能被随后的测序所证实,其检测粪便DNA突变的敏感度与特异度均达100%。
粪便基因突变与临床病理特征参数关系及临床诊断价值
1、在所有病例患者的粪便DNA样本中,KRAS突变检出率为26.1%(30/115),TP53突变检出率为21.7%(25/115)；CRC、AA原发于近端结直肠多于远端结直肠；Duck's分期中,较早期病变(A-B期)明显多于较晚期(C+D期)；在病例组与对照组间、CRC亚组与AA亚组间,均存在显著的性别差异,前者男性所占比例均明显多于后者；同一基因的不同长度片段(155bp和92bp)对HRMA的突变检出率无影响。
2、病例组中,均未发现KRAS、TP53各基因突变频率与性别、部位、分化度/异型增生度、组织类型、大小、Duke's分期、年龄存在相关性。
3、在AA组中,发现TP53突变与年龄相关,高龄组(年龄≥60岁)检出率高于低龄组(年龄<60岁)
4、联合检测粪便DNA KRAS与TP53基因突变(CRC54.0%(34/63)AA40.4%(21/52))诊断CRC与AA的敏感度欠佳,分别为54%与40%,特异度均为97%。
Background and Objection
Incidence of colorectal cancer (CRC) is listed3in global malignant tumor, and our country is4-6, which is continuously increasing trend. In Guangdong, the incidence of CRC in increase is very fast. The prognosis of CRC is related closely with early diagnosis. The cancer evolution mode of most of CRC is "normal-adenoma-cancer". The evolution from recognizable adenoma to invasive carcinoma need about5-10years, which provided a plenty of time to screen and prevent. The cancerous adenoma mainly resulted from the late adenoma has been confirmed by a lot of studies (refers to:>1cm in diameter or histologically confirmed for villous adenomas or severe atypia hyperplasia of adenomas). Pre-cancerous lesions such as adenomas are removed in time by clinical screening and can reduce mortality and morbidity of CRC.
Colonoscopy is recommended because of the high accuracy and treatment, but colonoscopy requires specialized endoscopic doctor, risky, high cost, difficult to accept for patients, and difficult to used in the screening of asymptomatic people. Fecal occult blood test (FOBT) is to be preferred because it's simple, noninvasive, economic, and good tolerability, but there are high false positive and false negative rate, and need to repeatedly inspect sample. Although FOBT can reduce cancer mortality, the effect on incidence of cancer is small owing to the poor detection rate of adenoma; stool DNA testing has many advantages, such as only-one submission, easy to accept, high compliance, without effect from medication or dietary control. A large number of studies have shown that the sensitivity and specificity of fecal DNA testing is better than FOBT, especially adenoma detection rate is striking. Stool DNA testing has be listed in The United States guide as candidate program.
Current stool DNA testing programs still need to improve the sensitivity and specificity, reduce the costs, and simplify operations. In addition, the further optimization/replacement of current markers, the upgrade of detection methods are also the key. With increasing sensitivity and specificity of stool DNA testing methods. There are some prevalent deficiencies, such as high costs, difficult to operate, too specialized, free to affect on the tested target gene loci and others. Therefore, seeking to a convenient and easy-to-use, cost-effective detection methods is a necessary condition to enhance the practical value of stool DNA testing.
The high-resolution melting curve analysis (HRMA) is the closed tube operation was jointly developed by the Wittwer laboratory of the University of Utah and the United States Idaho PCR technology:high-density fluorescence data collection, the melting curve is directly used to detect the small sequence difference in PCR amplified fragment. There are some advantages of HRMA:low cost, high-throughput, non-polluting, efficient, simple to operate, free from the impact of the location variance, good sensitivity and specificity. In2003, HRMA was introduced to detect mutations. The application and development of HRMA have sprung up, and become more and more attention in medical molecular diagnostics. Meta-analysis study showed the sensitivity of HRMA was97.5%(95%confidence interval (CI),96.8-98.5), and specificity was95.8%(95%CI:95.3-96.3). The source of the DNA samples and saturation dye type had no effect on the sensitivity of HRMA. Our previous studies have shown HRMA obtained a higher detection rate of stool DNA mutation detection, and potential clinical value. There are still no relevant studies to evaluate HRMA method as stool DNA testing screening for CRC.
Therefore, based on our previous study, DNA sequencing as the gold standard, this research was planned to expand the sample size, to update test design, and to optimize the experimental conditions and operational processes in order to more comprehensively evaluate the accuracy of HRMA. First, for the establishment and optimization of the HRMA operating processes and the experimental parameters, the HRMA was used to detect KRAS and TP53mutation in tissue samples; next, for a comprehensive evaluation of the sensitivity and specificity, the HRMA was used to detect KRAS and TP53mutation in fecal DNA in two independent sample sets (including the stage of "learning set" and "validation set"); then, the sensitivity of HRMA was assessed in DNA serial dilutions of test; finally, the relationship between fecal KRAS, TP53mutations detected by HRMA and the parameters of clinical pathological features of the colon neoplastic lesions and diagnostics value were investigated.
Materials and Methods
Experimental subjects
Consecutive subjects over40years of age, from Huizhou Municipal Central Hospital and Nanfang Hospital (China) were enrolled in this study from January2009to June2010. All subjects were identified with CRCs, AAs or normal control (NC) by colonoscopy. Subjects were matched for age and chosen to represent a mixed distribution of neoplasms from both proximal and distal colorectal sites (Table2). Individuals were excluded who had any of the following conditions, including contraindication to colonoscopy, personal history of or coexistent with cancer, active therapy with chemotherapy or radiation therapy for a concurrent cancer, personal history of colorectal adenomas or CRC, familial adenomatous polyposis, hereditary non-polyposis CRC, any other high-risk conditions (inflammatory bowel disease, strong family history of CRC (two or more first degree relatives with CRC, or one or more first-degree relatives with CRC younger than age50), prior colorectal resection for any reason, current pregnancy or lactation) and the weight of stool samples less than5g. At each site, the preparation for, and performance of colonoscopy, was done according to standard operating procedures. CRC and AA initially identified by colonoscopy were further verified by histological diagnosis. Cancers were staged in accordance to Dukes'classification system. This investigation was approved by each clinic institutional review board, and comprised two clinical pilot studies. All patients or their legal representatives signed informed consent.
Preparation of fecal samples
Within48hrs after collection, stool samples were sent to our laboratory and stored in-80℃. Patients were instructed to collect an aliquot of feces (at least5g) using a fecal collection tube, and to store the samples in a hermetically sealed plastic container. More than1week after any colorectal diagnostic procedure or cathartic preparation, and before either endoscopic or surgical neoplasm resection, stools were collected.
DNA extraction and dilution
Tissue DNA was isolated from formalin-fixed-paraffin-embedded (FFPE) neoplasm tissue using the QIAamp DNA FFPE tissue kit.40neoplasm areas were marked on the haematoxylin and eosin (H&E)-stained slides, and corresponding unstained slides were manually micro-dissected so that at least50%neoplasm cells were contained in the samples. Stool DNA was extracted with QIAamp DNA Stool Mini Kit after homogenization. Processes were operated according to the instructions of the manufacturer. The concentration and purity of extracted DNA were measured using the NanoDrop ND-1000Spectrophotometer and the samples were diluted to a final concentration of50ng/μl. Extracted DNA was excluded if concentration was less than50ng/μl. The absorbance at260/280nm was1.8-2.0, and at260/230nm was2.0-2.5.
HRMA assay
Primers for TP53/KRAS were designed from the NCBI RefSeq NG_017013.1/NG_007524.1. The primers and the length of all amplicons are listed:KRA Sexon2(92bp) f-TTATAAGGCCTGCTGAAAATGACTGAA, r-TGAATTAGCT G TATCGTCAAGGCACT; exon2(155bp) f-TTATAAGGCCTGCTGAAAATGA C TGAA, r-TGAATTA-GCTGTATCGTCAAGGCACT. Exon3f-GACTGTGTTT CTCCC TTCTC, r-TGTACTGGTCCCTCATTGC; TP53Exon5f-GTGCAGCTGT GGGTTGATT; r-AACCAGCCCTGTCGTCTCT, Exon6f-GATTCCTCACTGAT TGCTCTTAG, r-GG GCACCACCACACTATG,Exon7f-TTGGGCCTGTGTTAT CTCCT, r-TGGCAAGT GGCTCCTGAC,Exon8f-TTGCTTCTCTTTTCCTATCC TGA, r-GCTTCTTGTCCTG CTTGCTT. Two fragments of KRAS exon2with92bp and155bp each, were amplified. PCR was carried out to amplify KR AS exon2(155bp) on a LightCycler480and KRAS exons2(92bp)-3, TP53exons5-8on Rotor-GeneTM6000. Each PCR reaction mixture contained2μl of DNA solution (100ng),200nmol/L of each primer,2.5mmol/L MgCl2,12.5μl of LightCycler480High Resolution Melting Master/qPCR Master Mix, and ddH2O to a final volume of25μl. PCR paremeters were95℃for5mi n, followed by50cycles of15s at95℃,63℃/60℃for50s to72℃for30s, and10min at72℃. After amplification, the PCR product was denatured at95℃for5min and cooled to40℃for1min to allow heteroduplex form ation. The final HRMA step was performed from65℃to95℃with an increa se in25acquisitions per degree. For sample analysis, after normalization and t emperature-adjustment steps, melting curve shapes were compared between the neoplasm samples and control samples. Wild-type genes were used as a negati ve control. All samples, including wild-type, were plotted according to their m elting profiles. In the different graphs, the melting profiles of each sample wer e compared with those of the wild-type (which were converted to a horizontal line). Significant deviations from the horizontal line were indicative of sequen ce changes within the amplicon. Samples with aberrant melting curves were re corded as HRMA mutation positive. Data were acquired and analyzed using th e Gene Scanning software1.5(Roche)/the associated RotorGene Series Software (V1.7.25). All analyses were done by two independent researchers. The final j udgment was concluded by consensus after joint viewing of the melting curves from both researchers. If the first PCR products were not available for the m utational analyses of the melting curves, the second PCR with the same primer s were performed. All samples were amplified and detected in duplicate.
DNA sequencing
Data from direct DNA sequencing was compared to results from the HRMA assay. Primers used in the HRMA assay were also used for DNA sequencing (Table1). All PCR products were analyzed by DNA sequencing in order to evaluate the accuracy of HRMA in stool DNA testing. First, the PCR products were purified by digest:1. PCR products of each sample was detected and confirmed the purpose of the strip single using1%agarose gel;2. PCR products with a single strip, the SAP (shrimp alkaline phosphatase) and Exo Ⅰ(exo-nuclease) were used to digest and purify; BioMIGA agarose gel extraction kit was used to recycle by tapping the non-specific bands after agarose gel detected. Then, on the ABI3730DNA sequencing instrument, the one-way Sangers sequencing the method was performed. The3730XL software DNAMAN software were applied to analyze the sequencing data to obtain the final results. DNA sequencing of all samples were sent to the Chaoshi-bio for detection analysis.
Statistical analysis
The sensitivity and specificity of the results acquired from HRMA were compared with those results acquired from sequencing. McNemar's test for matched pairs test was used to compare the sensitivity, specificity, negative predictive value, positive predictive value of the HRMA/mutation with direct sequencing/clinicopathological parameters. Comparison of proportions between various subgroups was based on the Chi-square and Fisher exact test. Comparison between the stool DNA test and tissue DNA test was performed using the kappa coefficient. P≤0.05were considered statistically significant. All data were analyzed using the SPSS version13.0statistical package (SPSS Inc, Chicago, Illinois, USA) and EpiCalc2000statistical software.
Results
Clinical and histological characteristics of eligible subjects
Initially,145patients with colorectal neoplasia, and70subjects in the NC group were enrolled. Of all the subjects involved,8were excluded because their stool samples did not meet the requirements (less than5g);14in the colorectal neoplasia group, and6in the NC group were excluded due to the strict criteria of subject selection. Next, of the187extracted DNA,12were excluded (5CRCs,3AAs,4 NCs). Eventually,175participants were retained in this study. Among them,63patients had primary CRCs,52patients had AAs, and60patients were age-matched controls. All patients were Chinese. Among the patients, a definitive diagnosis was established by the complete resection specimens of colonoscopy or/and surgical resection specimens, the average age of CRC group was61years (41-87years), AA group58years (40-80years), and control group47years (40-73years); the sex ratio of the case group was69/57, including CRC32/31, AA37/26, and control group24/36; the primary sites of CRC and AA were located in the proximal colorectal88.9%(56/63) and86.5%(45/52); Duck's staging of A+B were accounted for60.3%; in case group, KRAS exon2-3mutation rate was26.1%(30/115), the TP53exon5-8mutations21.7%(25/115), and3.3%(2/60) in control group.
The study in learning set stage
In order to establish and optimize the HRMA experimental conditions and parameters, we evaluated the HRMA detection accuracy of the mutant gene in tissue samples of CRC and AA. First, in40FFPE tissue samples of patients, KRAS exons2-3and TP53exons5-8mutation were confirmed by sequencing; then, the HRMA was applied to detected these gene mutations.22patients with gene mutations were detected, including17cases from29CRCs and5from AAs. The rate of mutation detection was55%(22/40). Both results from HRMA and sequencing were fully consistent, the sensitivity and specificity of HRMA were100%. Subsequently, based on the above optimized the HRMA methodology, the HRMA detection accuracy of the mutant gene were assessed in referred stool DNA samples, and results showed that18cases (18/40,45%) with mutations were detected by HRMA, including14CRCs (14/29,48.28%) and4(4/11,36.36%) AAs. These detection results are confirmed by sequencing. The sensitivity and specificity of HRMA was100%. The rates of mutation detection in fecal DNA and in tissue DNA samples were18/22(81.82%) with a high degree of consistency, including CRC14/17(82.35%), and AA4/5(80%). Pooled Kappa value is0.794, in which CRC and AA were0.794and0.814respectively.
The sensitivity analysis of the HRMA
In order to evaluate the HRMA reliability of mutations detection in stool DNA from the CRC screening settings, we assess the lowest level of HRMA detection mutation by DNA series dilution test. Fecal samples of known KRAS exon2and TP53exon6-8mutations were selected for DNA serial dilution experiments, and the choice of the wild-type samples used as the background of the dilution experiments. The lowest level of HRMA detection mutation was is1%. but the reliable detection concentration was not less than5%owing to huge variation of samples.
The study in validation set stage
In the above assessment study, The sensitivity and specificity of the HRMA were100%and detection limit1%. and encouraged us to further evaluate its accuracy in the validation set.75cases groups, including34cases of CRC,41cases of AA, and60patients in the control group. The frequency of KRAS/TP53mutation detection in the case group(49.3%,37/75) were significantly higher than that of the control group (3.3%,2/60)(P<0.001); the subgroup analysis in the case group, the rates of KRAS/TP53mutation detection in CRC subgroup (58.8%,20/34) were significantly different from AA subgroup (41.5%,17/41)(P=0.02).The results of the HRMA are subsequently confirmed by sequencing, its sensitivity and specificity were100%.
The relationship between fecal mutations with parameter of clinicopathological features&clinical diagnostic value
CRC/AA originated in the proximal colorectum (88.9%/86.5%) than the remote colorectum (13.5%/11.1%);Duck's staging of A+B (60.3%) significantly were more than C+D (39.7%); there were significant proportions of gender between the case group/CRC subgroup and control group/AA subgroup (P=0.004, P=0.035), males among the former are more than the latter; the fragments of different lengths (155bp and92bp) in KRAS exon2, mutation detection was is exactly the same.
In the CRC group, KRAS mutations were detected in9cases (19/63,30.1%), and TP53mutations in15cases (15/63,23.8%); there were no statistically differences between frequencies of mutation and gender, location, degree of differentiation, histological type, size, Duck's staging, age.
In the AA group, KRAS mutations were detected in11cases (11/52,21.2%), and TP53mutations in10cases (11/52,19.2%); there were statistically differences in rates of TP53mutation detection among age groups (P=0.036), the age≥60group32.0%(8/25) was higher than the<60age group7.4%(2/27); there were no statistically differences between frequencies of mutation and gender, location, degree of dysplasia, histological type, size, Duck's staging.
There were54.0%(34/63) mutation incidences in CRC patients, and40.4%(21/52) in AA patients; the performance of stool KRAS and TP53mutation detections for screening CRC and AA's was poor, sensitivities were54%[95%CI0.41,0.66] and40%[95%CI,0.27,0.55], the both specificity97%[95%CI0.87,0.99], the accuracy of diagnosis75%and71%.
Conclusion
The stage of learning set
1. Among tissue samples of CRC and AA patients, the accuracy of the HRMA is consistent with DNA sequencing in detecting KRAS exon2-3/TP53exon5-8mutations, its sensitivity and specificity were100%.
2. Among the corresponding stool DNA samples, there is the same accuracy in detecting KRAS exon2-3/TP53exon5-8mutations between HRMA and DNA sequencing, the sensitivity and specificity of HRMA were100%.
3. the rates of stool DNA mutation detection is high degree of consistency with tissue DNA, Kappa value is0.794, CRC0.794, and AA0.814.
The sensitivity analysis
In the context of the dilution of wild-type gene,1%mutation detection limit can be a rough estimate of the sensitivity of the HRMA, the HRMA detection sensitivity directly is affected by the sequence variation of the samples; the mutation detection limit of HRMA is reliably≥5%.
The stage of validation set
1. The rates of stool mutation detected by HRMA are49.3%(37/75) in case group, and3.3%(2/60) in control group, KRAS and/or TP53gene mutations were associated with colorectal neoplastic lesions.
2. Among case group, the rates of stool mutation detection in CRC group are significantly higher than in AA group, KRAS and/or TP53gene mutations was closer relations with CRC.
3. The results of mutation detection by the HRMA are confirmed by sequencing, the sensitivity and specificity of the HRMA reach100%.
The relationship between fecal mutations with parameter of clinicopathological features&clinical diagnostic value
1. among all cases of patients, the rates of stool KRAS mutation detection is26.1%, and TP53mutation detection21.7%; the primary sites of CRC, and AA locate in the proximal colorectal more than the distal colorectal; compared with later stage of Duck's staging(C+D staging), early lesions (A+B staging) is significantly more; proportions of males in the case group/CRC subgroup is significantly more than the control group/AA subgroup; different lengths of the same gene have no effect on the performance of the HRMA.
2. Among the case group, the frequency of KRAS/TP53mutation are correlated with gender, location, degree of differentiation/dysplasia degree, histological type, size, age, and Duke's staging.
3. Among the AA group, TP53mutations were positively related to age group.
4. The stool KRAS/TP53mutations are poorly sensitive for screening CRC and AA, the sensitivity is54.0%for CRC;40.0%for AA, and specificity is97%.
全球结直肠癌(CRC)发病率位于恶性肿瘤的第3位,我国位于第4-6位,呈逐年上升的趋势。广东省CRC发病率的上升速度较快。CRC的预后与早期诊断密切相关。绝大多数CRC是由“正常-腺瘤-癌”逐步演变而来,从可辨认的腺瘤发展为侵袭性癌约需5-10年,这为预防及早期筛查提供了充裕的时间。大量研究已证实发生癌变的腺瘤主要为晚期腺瘤(AA,指直径≥1cm或组织学证实为绒毛状腺瘤或重度异型性增生的腺瘤),通过临床筛查并及时切除癌前病变如腺瘤,可降低CRC发病率及病死率。
结肠镜检查因准确性高且能治疗而被首选,但存在需要专业的内镜医生、风险较大、费用高、患者接受性差等不足,难以大规模应用于无症状人群的筛查；粪便隐血试验(FOBT)具有简便、无创、经济,接受性好等优点而被优选,但存在假阳性率及假阴性率较高、需要多次送检等不足,由于腺瘤检出率敏感度低,因而对癌症发病率的影响小；粪便DNA检测具有只需一次送检、易接受、依从性高、不需服药或控制饮食、不受肿瘤部位影响等优点而被重视。大量研究表明粪便DNA检测的敏感性和特异性比FOBT好,尤其腺瘤检出率高引人注目。已列为美国指南候选方法。
但现有的粪便DNA检测方案尚需完善,主要是敏感性与特异性有待提高,费用需降低、操作需简单化等。除了尚需进一步优化/更替现有的标志物组合外,升级现有的检测方法也是关键环节。现有的高敏感性和特异性的粪便DNA检测方法,普遍存在费用高、操作复杂、过于专业化、受制于待检目标基因位点的等问题。因此寻求一种便捷易用、经济高效的检测方法是提升粪便DNA检测实用价值的必要条件。
高分辨率熔解曲线分析(HRMA)是犹他州大学Wittwer实验室与美国Idaho公司联合研制的一种PCR后闭管操作技术：通过高密度荧光数据采集,直接用熔解曲线检测PCR扩增片段中微小序列差别。具有低成本、高通量、无污染、快捷、操作简单、不受变异位置影响、敏感性及特异性好等优势。自2003年被引入检测基因突变以来,HRMA的应用与发展如“雨后春笋”,在医学分子诊断中越来越受到关注。经荟萃分析发现其灵敏度达97.5%(95%置信区间(CI),96.8-98.5)、特异度达95.8%(95%CI：95.3-96.3),DNA样本的来源及饱合染料类型均对HRMA的灵敏度无影响。我们前期的研究已经初步展示HRMA用于粪便DNA突变检测,获得较高的检出率,具有潜在临床应用价值。目前国内尚无有关评价HRMA方法用于粪便DNA检测筛查CRC的文献报道。
因此,本研究在前期研究基础上,以DNA测序结果作为金标准,扩大样本量、更新试验设计方案、优化实验条件及操作流程等方式对HRMA的准确性展开较为全面的评价性研究。首先在组织样本中建立与优化HRMA操作流程与实验参数；然后,在2个独立的样本集(包括“学习集”与“验证集”阶段)中对HRMA应用于粪便DNA KRAS与TP53突变的检测的敏感性和特异性进行全面评价；同时,在DNA系列稀释试验中评估HRMA技术的敏感性；最后,探讨检出的粪便KRAS、TP53突变与大肠肿瘤性病变的临床病理特征参数的关系及诊断价值。
方法
研究对象：2010.1-2010.7间在惠州市中心医院及南方医院接受肠镜检查者和/或普外科住院的术前CRC与AA患者中,及肠镜检查未发现明显异常的病人中。按下列纳入与排除标准,连续收集这些患者的粪便标本与临床病理特征参数资料,在“学习集”部分收集相应的肿瘤的FFPE组织标本。
研究组纳入标准：(1)年龄大于40岁、且病理组织学检查确诊为结直肠AA与CRC患者；(2)能同时收集病变组织与合格粪便样本(重量不低于5克)的患者。
研究组排除标准：(1)家族性或遗传性结直肠腺瘤或肿瘤；其他系统或器官肿瘤。(2)共患有炎症性肠病；(3)术前有放疗或化疗史；(4)妊娠或有严重肝肾功能不全者；(5)非原发癌灶者及不能确诊等其他情况；(6)既往有CRC或腺瘤病史；(7)肉眼见全血性大便者。
粪便样本采集：在内镜检查或肿瘤切除手术前、接受任何形式的结直肠侵入性操作或服用泻剂肠道准备的1周后,收集至少5克新鲜粪便量于48小时内尽快冻存于-80℃冰箱待用。
DNA提取与质控：按QIAamp粪便DNA、FFPE DNA的提取试剂盒的说明书方法提取DNA,在FFPE组织取样时,采用传统手工微切的方法,保留每—块样本中含有肿瘤细胞占的比例≥50%。提取DNA的浓度和纯度测定均用NanoDrop ND-1000分光光度计检测,达标要求：浓度≥50ng/μL,260/280nm的吸光度位于1.6-2.2且230/260nm位于2.0-2.5。达标的样本DNA均稀释到50ng/μL的终浓度,置入-20℃冰箱保存待用。
HRMA检测：基于NCBI的TP53外显子5-8、KRAS2-3的参考基因序列设计引物,KRAS Exon2(92bp) f-TTATAAGGCCTGCTGAAAATGACTGAA, r-TGAAT-TAGCTGTATCGTCAAGGCACT; Exon2(155bp) f-TTATAAGGCCTG CTGAAAAT-GACTGAA, r-TGAATTAGCTGTATCGTCAAGGCACT, Exon3f-GACTGTGTTTCT-CCCTTCTC, r-TGTACTGGTCCCTCATTGC; TP53Exon5f-GTGCAGCTGTGGGTT-GATT; r-AACCAGCCCTGTCGTCTCT, Exon6f-GAT TCCTCACTGATTGCTCTTA-G r-GGGCACCACCACACTATG; Exon7f-TTGGGCCTGTGTTATCTCCT r-TGGCA AG TGGCTCCTGAC,Exon8f-TTGCTTCTC TTTTCCTATCCTGAr-GCTTCTTGTCCTGCTTGCTT。其中, KRAS基因外显子2,设计2对引物,扩增的片段分别为92bp和155bp。KRAS基因外显子2(155bp)的检测是在LightCycler480PCR仪上操作；其他的KRAS基因外显子2(92bp)-3,TP53基因外显子5-8基因均在Rotor-GeneTM6000PCR仪上操作。反应体系：2μL(100ng)样本DNA,上下引物各为lμL(200nmol/L),镁离子2μL(2.5mmol/L), LightCycler480HRM Master/qPCR Master预混液12.5μL,加ddH2O至总体积25μL。反应条件参数：95℃预热5分钟,95℃×15秒→60℃-63℃×50秒→72℃×30秒→72℃×10分钟,共50个循环。PCR产物在95℃变性5分钟,冷却至40℃×1分钟(让异源双链形成)然后在65℃升至95℃间,荧光采集密度为25次/℃。HRMA分析：原始采集的荧光曲线经过正常化和温度调整处理后,野生型基因被用来作为阴性对照。曲线图形的差异表示扩增的样本基因序列存在变异。HRMA突变阳性样本记为异常熔解曲线。采集的数据经罗氏公司的基因分析软件(1.5版)及RotorGene的软件(V1.7.25版)分析得出结果。
DNA测序检测：首先,对PCR产物进行酶切纯化：1、将每个样本PCR产物使用1%的琼脂糖凝胶进行检测,确认目的条带是否单一；2、经上述琼脂糖凝胶检测条带单一的,使用SAP(虾碱性磷酸酶)和Exo I(外切核酸酶)进行酶切纯化；3、经上述琼脂糖凝胶检测有非特异性的条带,使用BioMIGA的琼脂糖凝胶回收试剂盒进行割胶回收。接着,在用在ABI3730DNA测序仪上进行单向测序,采用的是Sanger法。最后,使用3730XL软件、DNAMAN软件对测序数据进行分析,得出最终结果。所有样本DNA的测序均送上海硕士生物有限公司检测分析。
统计方法
以α=0.05为检验水准,各组间年龄,两样本t检验；所有的计数资料的比较,多样本间采用χ2检验,四格表采用Fisher确切概率法,若配对资料采用McNemar检验,符合率采用Kappa分析。统计分析均使用SPSS13.0软件包及EpiCalc2000统计软件。
结果
参与患者的临床病理特征参数
最初,有145例大肠肿瘤性病变患者,70例结肠镜检查阴性的病人(NC)作对照组。28例因粪便样本不符合纳入与排除标准而剔除。接下来,187提取DNA,12例因DNA质量不达标而排除。最终纳入研究共175例,63例CRC,52例AA,60例年龄相匹配的NC,所有参与者均是中国人。患者病情诊断的确定均是根据结肠镜检查结果和/或完整切除肿瘤的标本病理诊断。CRC组的平均年龄为61岁(41-87岁),AA组的平均年龄为58岁(40-80岁),对照组平均年龄为47岁(40-73岁)；病例组男女性比例69/57,其中CRC为32/31, AA为37/26,而对照组为24/36；CRC、AA原发部位位于近端结直肠分别为88.9%(56/63)、86.5%(45/52)；Duck's分期中,A+B期占60.3%；病例组KRAS外显子2-3突变率为26.1%(30/115),TP53外显子5-8突变率为21.7%(25/115),对照组突变率3.3%(2/60)。
“学习集”阶段研究
为了建立及优化HRMA技术的实验条件与参数设定。我们在CRC与AA的组织样本中,对HRMA检测突变基因的准确性进行评价。先对合格的40例病人的FFPE的样本进行测序证实KRAS外显子2-3与TP53外显子5-8基因突变情况,再用HRMA检测其基因突变的情况,我们检出22例病人有基因突变,其中17例来自29例CRC,5个来自AA,突变检出率55%(22/40)。两者检测结果完全一致,其敏感度与特异度均为100%。随后基于上述优化的HRMA的技术操作流程,我们就对其相应的粪便DNA的突变进行检测,结果发现,HRMA检出18例(18/40,45%)突变,其中,CRC14例(14/29,48.28%),AA4例(4/11,36.36%),这些检测结果均被随后的测序所证实,HRMA的敏感度与特异度均为100%。粪便DNA与相应组织的突变检出率的符合率高度一致,18/22(81.82%),其中CRC14/17(82.35%),AA4/5(80%),Kappa值为0.794,在CRC、AA中分别为0.794、0.814。
HRMA的敏感性分析
为了评价HRMA在结直肠癌筛查的环境中检测粪便DNA突变的可靠性,我们采用DNA的系列浓度稀释试验评估HRMA检测最低水平基因突变的灵敏度。选用已知KRAS基因外显子2及TP53基因外显子6-8突变具体情况的粪便样品DNA系列稀释实验,选用同样的粪便DNA中的野生型样本作为稀释实验的背景。我们发现,HRMA检出突变基因的最低稀释浓度限度为1%,但样本间存在的差异大；HRMA能够稳定可靠的检出浓度为≥5%。
“验证集”阶段研究
基于前二个部分研究得出的HRMA具有与测序一致的敏感性与特异性,且检出突变基因的最低浓度限度能达1%。促使我们在“验证集”中进一步评价其准确性。75例病例组,包括34例CRC和41例AA,60例对照组。在病例组的粪便DNA中,共检出含有KRAS和／或TP53基因突变频率为(49.3%,37/75)显著高于对照组(3.3%,2/60)(P<0.001)；在病例组的亚组分析中,基因突变率在CRC亚组(58.8%,20/34)与AA亚组(41.5%,17/41)之间的存在显着性差异(P=0.02)。HRMA检测的结果,均被随后的测序证实,其敏感度与特异度均为100%。
粪便基因突变与临床病理特征参数关系及临床诊断价值
1、病例组中,CRC、AA原发于近端结直肠(88.9%、86.5%)明显多于远端结直肠(13.5%、11.1%)；Duck's分期中,A+B期(60.3%)明显多于C+D期(39.7%)；在病例组与对照组间,CRC亚组与AA亚组间,均存在显著的性别构成比差异(P=0.004,P=0.035),前者男性所占比例均明显多于后者；对KRAS基因外显子2突变,我们同时检测二个不同长度的片段(155bp和92bp),发现两者的突变检出率完全一致。
2、在CRC组中,检出19例(19/63,30.1%)KRAS突变,15例(15/63,23.8%)TP53突变。发现各基因突变频率与性别、部位、分化度、组织类型、大小、Duck's分期、年龄均无统计学上差异。
3、在AA组中,检出11例(11/52,21.2%)KRAS突变,10例(11/52,19.2%)TP53突变。发现TP53突变与年龄分组存在差异(P=0.036),发现年龄≥60岁组突变检出率32.0%(8/25)高于<60岁组7.4%(2/27)。但发现各基因突变频率与性别、部位、异型增生度、组织类型、大小均无统计学差异。
4、两基因联合突变发生率,CRC中为54.0%(34/63),AA中为40.4%(21/52)；联合检测粪便DNA KRAS与TP53基因突变筛查CRC与AA的性能欠佳,敏感度分别为54%[95%CI0.41,0.66]与40%[95%CI0.27,0.55],特异度均为97%[95%CI0.87,0.99],准确性分别为75%与71%。
结论
“学习集”阶段
1、在CRC与AA患者的组织样本中,HRMA检测KRAS外显子2-3与TP53外显子5-8基因突变准确性与DNA测序结果完全一致,其敏感性与特异度均达100%。
2、在其相应的粪便DNA样本中,HRMA检测KRAS外显子2-3与TP53外显子5-8基因突变准确性与DNA测序结果完全一致,其敏感性与特异度均达100%。
3、粪便DNA突变检出率与组织DNA突变检测出率的符合率高度一致,Kappa值为0.794,在CRC、AA中分别为0.794、0.814。
HRMA的敏感性分析
在野生型基因稀释的背景下,这可粗略估计出HRMA检测突变的敏感性能达1%稀释浓度,并且发现待检样本的序列变异直接影响HRMA检测的灵敏度,因此HRMA能稳定可靠地检出突变的敏感性是≥5%稀释浓度。
“验证集”阶段
1、病例组基因突变检出率(49.3%,37/75)显著高于对照组(3.3%,2/60)。表明KRAS和/或TP53基因突变与大肠肿瘤性病变相关。
2、CRC患者基因突变检出率的显著高于AA患者,表明KRAS和/或TP53基因突变与CRC关系更密切。
3、HRMA检测基因突变的结果均能被随后的测序所证实,其检测粪便DNA突变的敏感度与特异度均达100%。
粪便基因突变与临床病理特征参数关系及临床诊断价值
1、在所有病例患者的粪便DNA样本中,KRAS突变检出率为26.1%(30/115),TP53突变检出率为21.7%(25/115)；CRC、AA原发于近端结直肠多于远端结直肠；Duck's分期中,较早期病变(A-B期)明显多于较晚期(C+D期)；在病例组与对照组间、CRC亚组与AA亚组间,均存在显著的性别差异,前者男性所占比例均明显多于后者；同一基因的不同长度片段(155bp和92bp)对HRMA的突变检出率无影响。
2、病例组中,均未发现KRAS、TP53各基因突变频率与性别、部位、分化度/异型增生度、组织类型、大小、Duke's分期、年龄存在相关性。
3、在AA组中,发现TP53突变与年龄相关,高龄组(年龄≥60岁)检出率高于低龄组(年龄<60岁)
4、联合检测粪便DNA KRAS与TP53基因突变(CRC54.0%(34/63)AA40.4%(21/52))诊断CRC与AA的敏感度欠佳,分别为54%与40%,特异度均为97%。
Background and Objection
Incidence of colorectal cancer (CRC) is listed3in global malignant tumor, and our country is4-6, which is continuously increasing trend. In Guangdong, the incidence of CRC in increase is very fast. The prognosis of CRC is related closely with early diagnosis. The cancer evolution mode of most of CRC is "normal-adenoma-cancer". The evolution from recognizable adenoma to invasive carcinoma need about5-10years, which provided a plenty of time to screen and prevent. The cancerous adenoma mainly resulted from the late adenoma has been confirmed by a lot of studies (refers to:>1cm in diameter or histologically confirmed for villous adenomas or severe atypia hyperplasia of adenomas). Pre-cancerous lesions such as adenomas are removed in time by clinical screening and can reduce mortality and morbidity of CRC.
Colonoscopy is recommended because of the high accuracy and treatment, but colonoscopy requires specialized endoscopic doctor, risky, high cost, difficult to accept for patients, and difficult to used in the screening of asymptomatic people. Fecal occult blood test (FOBT) is to be preferred because it's simple, noninvasive, economic, and good tolerability, but there are high false positive and false negative rate, and need to repeatedly inspect sample. Although FOBT can reduce cancer mortality, the effect on incidence of cancer is small owing to the poor detection rate of adenoma; stool DNA testing has many advantages, such as only-one submission, easy to accept, high compliance, without effect from medication or dietary control. A large number of studies have shown that the sensitivity and specificity of fecal DNA testing is better than FOBT, especially adenoma detection rate is striking. Stool DNA testing has be listed in The United States guide as candidate program.
Current stool DNA testing programs still need to improve the sensitivity and specificity, reduce the costs, and simplify operations. In addition, the further optimization/replacement of current markers, the upgrade of detection methods are also the key. With increasing sensitivity and specificity of stool DNA testing methods. There are some prevalent deficiencies, such as high costs, difficult to operate, too specialized, free to affect on the tested target gene loci and others. Therefore, seeking to a convenient and easy-to-use, cost-effective detection methods is a necessary condition to enhance the practical value of stool DNA testing.
The high-resolution melting curve analysis (HRMA) is the closed tube operation was jointly developed by the Wittwer laboratory of the University of Utah and the United States Idaho PCR technology:high-density fluorescence data collection, the melting curve is directly used to detect the small sequence difference in PCR amplified fragment. There are some advantages of HRMA:low cost, high-throughput, non-polluting, efficient, simple to operate, free from the impact of the location variance, good sensitivity and specificity. In2003, HRMA was introduced to detect mutations. The application and development of HRMA have sprung up, and become more and more attention in medical molecular diagnostics. Meta-analysis study showed the sensitivity of HRMA was97.5%(95%confidence interval (CI),96.8-98.5), and specificity was95.8%(95%CI:95.3-96.3). The source of the DNA samples and saturation dye type had no effect on the sensitivity of HRMA. Our previous studies have shown HRMA obtained a higher detection rate of stool DNA mutation detection, and potential clinical value. There are still no relevant studies to evaluate HRMA method as stool DNA testing screening for CRC.
Therefore, based on our previous study, DNA sequencing as the gold standard, this research was planned to expand the sample size, to update test design, and to optimize the experimental conditions and operational processes in order to more comprehensively evaluate the accuracy of HRMA. First, for the establishment and optimization of the HRMA operating processes and the experimental parameters, the HRMA was used to detect KRAS and TP53mutation in tissue samples; next, for a comprehensive evaluation of the sensitivity and specificity, the HRMA was used to detect KRAS and TP53mutation in fecal DNA in two independent sample sets (including the stage of "learning set" and "validation set"); then, the sensitivity of HRMA was assessed in DNA serial dilutions of test; finally, the relationship between fecal KRAS, TP53mutations detected by HRMA and the parameters of clinical pathological features of the colon neoplastic lesions and diagnostics value were investigated.
Materials and Methods
Experimental subjects
Consecutive subjects over40years of age, from Huizhou Municipal Central Hospital and Nanfang Hospital (China) were enrolled in this study from January2009to June2010. All subjects were identified with CRCs, AAs or normal control (NC) by colonoscopy. Subjects were matched for age and chosen to represent a mixed distribution of neoplasms from both proximal and distal colorectal sites (Table2). Individuals were excluded who had any of the following conditions, including contraindication to colonoscopy, personal history of or coexistent with cancer, active therapy with chemotherapy or radiation therapy for a concurrent cancer, personal history of colorectal adenomas or CRC, familial adenomatous polyposis, hereditary non-polyposis CRC, any other high-risk conditions (inflammatory bowel disease, strong family history of CRC (two or more first degree relatives with CRC, or one or more first-degree relatives with CRC younger than age50), prior colorectal resection for any reason, current pregnancy or lactation) and the weight of stool samples less than5g. At each site, the preparation for, and performance of colonoscopy, was done according to standard operating procedures. CRC and AA initially identified by colonoscopy were further verified by histological diagnosis. Cancers were staged in accordance to Dukes'classification system. This investigation was approved by each clinic institutional review board, and comprised two clinical pilot studies. All patients or their legal representatives signed informed consent.
Preparation of fecal samples
Within48hrs after collection, stool samples were sent to our laboratory and stored in-80℃. Patients were instructed to collect an aliquot of feces (at least5g) using a fecal collection tube, and to store the samples in a hermetically sealed plastic container. More than1week after any colorectal diagnostic procedure or cathartic preparation, and before either endoscopic or surgical neoplasm resection, stools were collected.
DNA extraction and dilution
Tissue DNA was isolated from formalin-fixed-paraffin-embedded (FFPE) neoplasm tissue using the QIAamp DNA FFPE tissue kit.40neoplasm areas were marked on the haematoxylin and eosin (H&E)-stained slides, and corresponding unstained slides were manually micro-dissected so that at least50%neoplasm cells were contained in the samples. Stool DNA was extracted with QIAamp DNA Stool Mini Kit after homogenization. Processes were operated according to the instructions of the manufacturer. The concentration and purity of extracted DNA were measured using the NanoDrop ND-1000Spectrophotometer and the samples were diluted to a final concentration of50ng/μl. Extracted DNA was excluded if concentration was less than50ng/μl. The absorbance at260/280nm was1.8-2.0, and at260/230nm was2.0-2.5.
HRMA assay
Primers for TP53/KRAS were designed from the NCBI RefSeq NG_017013.1/NG_007524.1. The primers and the length of all amplicons are listed:KRA Sexon2(92bp) f-TTATAAGGCCTGCTGAAAATGACTGAA, r-TGAATTAGCT G TATCGTCAAGGCACT; exon2(155bp) f-TTATAAGGCCTGCTGAAAATGA C TGAA, r-TGAATTA-GCTGTATCGTCAAGGCACT. Exon3f-GACTGTGTTT CTCCC TTCTC, r-TGTACTGGTCCCTCATTGC; TP53Exon5f-GTGCAGCTGT GGGTTGATT; r-AACCAGCCCTGTCGTCTCT, Exon6f-GATTCCTCACTGAT TGCTCTTAG, r-GG GCACCACCACACTATG,Exon7f-TTGGGCCTGTGTTAT CTCCT, r-TGGCAAGT GGCTCCTGAC,Exon8f-TTGCTTCTCTTTTCCTATCC TGA, r-GCTTCTTGTCCTG CTTGCTT. Two fragments of KRAS exon2with92bp and155bp each, were amplified. PCR was carried out to amplify KR AS exon2(155bp) on a LightCycler480and KRAS exons2(92bp)-3, TP53exons5-8on Rotor-GeneTM6000. Each PCR reaction mixture contained2μl of DNA solution (100ng),200nmol/L of each primer,2.5mmol/L MgCl2,12.5μl of LightCycler480High Resolution Melting Master/qPCR Master Mix, and ddH2O to a final volume of25μl. PCR paremeters were95℃for5mi n, followed by50cycles of15s at95℃,63℃/60℃for50s to72℃for30s, and10min at72℃. After amplification, the PCR product was denatured at95℃for5min and cooled to40℃for1min to allow heteroduplex form ation. The final HRMA step was performed from65℃to95℃with an increa se in25acquisitions per degree. For sample analysis, after normalization and t emperature-adjustment steps, melting curve shapes were compared between the neoplasm samples and control samples. Wild-type genes were used as a negati ve control. All samples, including wild-type, were plotted according to their m elting profiles. In the different graphs, the melting profiles of each sample wer e compared with those of the wild-type (which were converted to a horizontal line). Significant deviations from the horizontal line were indicative of sequen ce changes within the amplicon. Samples with aberrant melting curves were re corded as HRMA mutation positive. Data were acquired and analyzed using th e Gene Scanning software1.5(Roche)/the associated RotorGene Series Software (V1.7.25). All analyses were done by two independent researchers. The final j udgment was concluded by consensus after joint viewing of the melting curves from both researchers. If the first PCR products were not available for the m utational analyses of the melting curves, the second PCR with the same primer s were performed. All samples were amplified and detected in duplicate.
DNA sequencing
Data from direct DNA sequencing was compared to results from the HRMA assay. Primers used in the HRMA assay were also used for DNA sequencing (Table1). All PCR products were analyzed by DNA sequencing in order to evaluate the accuracy of HRMA in stool DNA testing. First, the PCR products were purified by digest:1. PCR products of each sample was detected and confirmed the purpose of the strip single using1%agarose gel;2. PCR products with a single strip, the SAP (shrimp alkaline phosphatase) and Exo Ⅰ(exo-nuclease) were used to digest and purify; BioMIGA agarose gel extraction kit was used to recycle by tapping the non-specific bands after agarose gel detected. Then, on the ABI3730DNA sequencing instrument, the one-way Sangers sequencing the method was performed. The3730XL software DNAMAN software were applied to analyze the sequencing data to obtain the final results. DNA sequencing of all samples were sent to the Chaoshi-bio for detection analysis.
Statistical analysis
The sensitivity and specificity of the results acquired from HRMA were compared with those results acquired from sequencing. McNemar's test for matched pairs test was used to compare the sensitivity, specificity, negative predictive value, positive predictive value of the HRMA/mutation with direct sequencing/clinicopathological parameters. Comparison of proportions between various subgroups was based on the Chi-square and Fisher exact test. Comparison between the stool DNA test and tissue DNA test was performed using the kappa coefficient. P≤0.05were considered statistically significant. All data were analyzed using the SPSS version13.0statistical package (SPSS Inc, Chicago, Illinois, USA) and EpiCalc2000statistical software.
Results
Clinical and histological characteristics of eligible subjects
Initially,145patients with colorectal neoplasia, and70subjects in the NC group were enrolled. Of all the subjects involved,8were excluded because their stool samples did not meet the requirements (less than5g);14in the colorectal neoplasia group, and6in the NC group were excluded due to the strict criteria of subject selection. Next, of the187extracted DNA,12were excluded (5CRCs,3AAs,4 NCs). Eventually,175participants were retained in this study. Among them,63patients had primary CRCs,52patients had AAs, and60patients were age-matched controls. All patients were Chinese. Among the patients, a definitive diagnosis was established by the complete resection specimens of colonoscopy or/and surgical resection specimens, the average age of CRC group was61years (41-87years), AA group58years (40-80years), and control group47years (40-73years); the sex ratio of the case group was69/57, including CRC32/31, AA37/26, and control group24/36; the primary sites of CRC and AA were located in the proximal colorectal88.9%(56/63) and86.5%(45/52); Duck's staging of A+B were accounted for60.3%; in case group, KRAS exon2-3mutation rate was26.1%(30/115), the TP53exon5-8mutations21.7%(25/115), and3.3%(2/60) in control group.
The study in learning set stage
In order to establish and optimize the HRMA experimental conditions and parameters, we evaluated the HRMA detection accuracy of the mutant gene in tissue samples of CRC and AA. First, in40FFPE tissue samples of patients, KRAS exons2-3and TP53exons5-8mutation were confirmed by sequencing; then, the HRMA was applied to detected these gene mutations.22patients with gene mutations were detected, including17cases from29CRCs and5from AAs. The rate of mutation detection was55%(22/40). Both results from HRMA and sequencing were fully consistent, the sensitivity and specificity of HRMA were100%. Subsequently, based on the above optimized the HRMA methodology, the HRMA detection accuracy of the mutant gene were assessed in referred stool DNA samples, and results showed that18cases (18/40,45%) with mutations were detected by HRMA, including14CRCs (14/29,48.28%) and4(4/11,36.36%) AAs. These detection results are confirmed by sequencing. The sensitivity and specificity of HRMA was100%. The rates of mutation detection in fecal DNA and in tissue DNA samples were18/22(81.82%) with a high degree of consistency, including CRC14/17(82.35%), and AA4/5(80%). Pooled Kappa value is0.794, in which CRC and AA were0.794and0.814respectively.
The sensitivity analysis of the HRMA
In order to evaluate the HRMA reliability of mutations detection in stool DNA from the CRC screening settings, we assess the lowest level of HRMA detection mutation by DNA series dilution test. Fecal samples of known KRAS exon2and TP53exon6-8mutations were selected for DNA serial dilution experiments, and the choice of the wild-type samples used as the background of the dilution experiments. The lowest level of HRMA detection mutation was is1%. but the reliable detection concentration was not less than5%owing to huge variation of samples.
The study in validation set stage
In the above assessment study, The sensitivity and specificity of the HRMA were100%and detection limit1%. and encouraged us to further evaluate its accuracy in the validation set.75cases groups, including34cases of CRC,41cases of AA, and60patients in the control group. The frequency of KRAS/TP53mutation detection in the case group(49.3%,37/75) were significantly higher than that of the control group (3.3%,2/60)(P<0.001); the subgroup analysis in the case group, the rates of KRAS/TP53mutation detection in CRC subgroup (58.8%,20/34) were significantly different from AA subgroup (41.5%,17/41)(P=0.02).The results of the HRMA are subsequently confirmed by sequencing, its sensitivity and specificity were100%.
The relationship between fecal mutations with parameter of clinicopathological features&clinical diagnostic value
CRC/AA originated in the proximal colorectum (88.9%/86.5%) than the remote colorectum (13.5%/11.1%);Duck's staging of A+B (60.3%) significantly were more than C+D (39.7%); there were significant proportions of gender between the case group/CRC subgroup and control group/AA subgroup (P=0.004, P=0.035), males among the former are more than the latter; the fragments of different lengths (155bp and92bp) in KRAS exon2, mutation detection was is exactly the same.
In the CRC group, KRAS mutations were detected in9cases (19/63,30.1%), and TP53mutations in15cases (15/63,23.8%); there were no statistically differences between frequencies of mutation and gender, location, degree of differentiation, histological type, size, Duck's staging, age.
In the AA group, KRAS mutations were detected in11cases (11/52,21.2%), and TP53mutations in10cases (11/52,19.2%); there were statistically differences in rates of TP53mutation detection among age groups (P=0.036), the age≥60group32.0%(8/25) was higher than the<60age group7.4%(2/27); there were no statistically differences between frequencies of mutation and gender, location, degree of dysplasia, histological type, size, Duck's staging.
There were54.0%(34/63) mutation incidences in CRC patients, and40.4%(21/52) in AA patients; the performance of stool KRAS and TP53mutation detections for screening CRC and AA's was poor, sensitivities were54%[95%CI0.41,0.66] and40%[95%CI,0.27,0.55], the both specificity97%[95%CI0.87,0.99], the accuracy of diagnosis75%and71%.
Conclusion
The stage of learning set
1. Among tissue samples of CRC and AA patients, the accuracy of the HRMA is consistent with DNA sequencing in detecting KRAS exon2-3/TP53exon5-8mutations, its sensitivity and specificity were100%.
2. Among the corresponding stool DNA samples, there is the same accuracy in detecting KRAS exon2-3/TP53exon5-8mutations between HRMA and DNA sequencing, the sensitivity and specificity of HRMA were100%.
3. the rates of stool DNA mutation detection is high degree of consistency with tissue DNA, Kappa value is0.794, CRC0.794, and AA0.814.
The sensitivity analysis
In the context of the dilution of wild-type gene,1%mutation detection limit can be a rough estimate of the sensitivity of the HRMA, the HRMA detection sensitivity directly is affected by the sequence variation of the samples; the mutation detection limit of HRMA is reliably≥5%.
The stage of validation set
1. The rates of stool mutation detected by HRMA are49.3%(37/75) in case group, and3.3%(2/60) in control group, KRAS and/or TP53gene mutations were associated with colorectal neoplastic lesions.
2. Among case group, the rates of stool mutation detection in CRC group are significantly higher than in AA group, KRAS and/or TP53gene mutations was closer relations with CRC.
3. The results of mutation detection by the HRMA are confirmed by sequencing, the sensitivity and specificity of the HRMA reach100%.
The relationship between fecal mutations with parameter of clinicopathological features&clinical diagnostic value
1. among all cases of patients, the rates of stool KRAS mutation detection is26.1%, and TP53mutation detection21.7%; the primary sites of CRC, and AA locate in the proximal colorectal more than the distal colorectal; compared with later stage of Duck's staging(C+D staging), early lesions (A+B staging) is significantly more; proportions of males in the case group/CRC subgroup is significantly more than the control group/AA subgroup; different lengths of the same gene have no effect on the performance of the HRMA.
2. Among the case group, the frequency of KRAS/TP53mutation are correlated with gender, location, degree of differentiation/dysplasia degree, histological type, size, age, and Duke's staging.
3. Among the AA group, TP53mutations were positively related to age group.
4. The stool KRAS/TP53mutations are poorly sensitive for screening CRC and AA, the sensitivity is54.0%for CRC;40.0%for AA, and specificity is97%.
引文
[1]Sung JJ, Lau JY, Goh KL, et al. Increasing incidence of colorectal cancer in Asia:implications for screening[J]. Lancet Oncol,2005,6(11):871-6.
[2]许岸高,姜泊,钟旭辉,等.广东地区近20年大肠癌临床特征的变化趋势[J].中华医学杂志,2006,,(04)：272-5.
[3]许岸高,姜泊,余志金,等.广东地区大肠癌临床特征研究[J].中华消化杂志,2007,(07)：450-3.
[4]许岸高,姜泊,钟旭辉,等.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志,2006,(01)：9-12.
[5]曲利园,王亚东,王贵齐,等.国内外大肠癌筛查现状分析及对我国大肠癌筛查的建议[J].中国全科医学,2007,,(19)：1584-6.
[6]Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol, 2010,24(12):705-14.
[7]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
[8]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
[9]3rd SPC, Lal S, Glick JT, et al. Patient preferences for colorectal cancer screening:how does stool DNA testing fare?[J]. Am J Manag Care, 2007,13(7):393-400.
[10]Hardcastle JD, Chamberlain JO, Robinson MH, et al. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer[J]. Lancet, 1996,348(9040):1472-7.
[11]Kronborg O, Fenger C, Olsen J, et al. Randomised study of screening for colorectal cancer with faecal-occult-blood test[J]. Lancet, 1996,348(9040):1467-71.
[12]Mandel JS, Church TR, Bond JH, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer[J]. N Engl J Med, 2000,343(22):1603-7.
[13]Allison JE, Tekawa IS, Ransom LJ, et al. A comparison of fecal occult-blood tests for colorectal-cancer screening[J]. N Engl J Med,1996,334(3):155-9.
[14]Allison JE, Sakoda LC, Levin TR, et al. Screening for colorectal neoplasms with new fecal occult blood tests:update on performance characteristics[J]. J Natl Cancer Inst,2007,99(19):1462-70.
[15]Cole SR, Young GP. Effect of dietary restriction on participation in faecal occult blood test screening for colorectal cancer[J]. Med J Aust, 2001,175(4):195-8.
[16]van RLG, van RAF, Laheij RJ, et al. Random comparison of guaiac and immunochemical fecal occult blood tests for colorectal cancer in a screening population [J]. Gastroenterology,2008,135(1):82-90.
[17]Saito H, Soma Y, Koeda J, et al. Reduction in risk of mortality from colorectal cancer by fecal occult blood screening with immunochemical hemagglutination test. A case-control study [J]. Int J Cancer, 1995,61 (4):465-9.
[18]Lee KJ. Inoue M. Otani T, et al. Colorectal cancer screening using fecal occult blood test and subsequent risk of colorectal cancer:a prospective cohort study in Japan[J]. Cancer Detect Prev,2007,31(1):3-11.
[19]Parekh M, Fendrick AM, Ladabaum U. As tests evolve and costs of cancer care rise:reappraising stool-based screening for colorectal neoplasia[J]. Aliment Pharmacol Ther,2008,27(8):697-712.
[20]Ko CW, Dominitz JA, Nguyen TD. Fecal occult blood testing in a general medical clinic:comparison between guaiac-based and immunochemical-based tests[J]. Am J Med,2003,115(2):111-4.
[21]刘集鸿,许岸高,余志金,等.免疫胶体金便隐血试验在大肠癌普查中应用[J].中国肿瘤,2007,16(4)：219-20.
[22]许岸高,余志金,钟旭辉,等.自然人群大肠癌筛查方案的比较研究[J].中华健康管理学杂志,2009,3(3)：155-8.
[23]Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population[J]. N Engl J Med,2004,351(26):2704-14.
[24]Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia[J]. Ann Intern Med, 2008,149(7):441-50, W81.
[25]Scholefield JH, Moss S, Sufi F, et al. Effect of faecal occult blood screening on mortality from colorectal cancer:results from a randomised controlled trial[J]. Gut,2002,50(6):840-4.
[26]Myers RE, Balshem AM, Wolf TA, et al. Adherence to continuous screening for colorectal neoplasia[J]. Med Care,199331(6):508-19.
[27]Davies RJ, Miller R, Coleman N. Colorectal cancer screening:prospects for molecular stool analysis[J]. Nat Rev Cancer,2005,5(3):199-209.
[28]Albaugh GP, Iyengar V. Lohani A, et al. Isolation of exfoliated colonic epithelial cells, a novel, non-invasive approach to the study of cellular markers[J]. Int J Cancer,1992,52(3):347-50.
[29]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors [J]. Science (80-),1992,256(5053):102-5.
[30]Ratto C, Flamini G, Sofo L, et al. Detection of oncogene mutation from neoplastic colonic cells exfoliated in feces[J]. Dis Colon Rectum, 1996,39(11):1238-44.
[31]Osborn NK, Ahlquist DA. Stool screening for colorectal cancer:molecular approaches. [J]. Gastroenterology,2005,128(1):192-206.
[32]Diehl F, Schmidt K, Durkee KH, et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients[J]. Gastroenterology, 2008,135(2):489-98.
[33]Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
[34]Berger BM,3rd SPC, Rosenberg JL, et al. Colorectal cancer screening using stool DNA analysis in clinical practice:early clinical experience with respect to patient acceptance and colonoscopic follow-up of abnormal tests [J]. Clin Colorectal Cancer,2006,5(5):338-43.
[35]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer[J]. J Natl Compr Canc Netw,2010,8(1):81-92.
[36]Itzkowitz SH, Jandorf L, Brand R, et al. Improved fecal DNA test for colorectal cancer screening[J]. Clin Gastroenterol Hepatol,2007,5(1):111-7.
[37]Ahlquist DA. Next-generation stool DNA testing:expanding the scope [J]. Gastroenterology,2009,136(7):2068-73.
[38]Lansdorp-Vogelaar I, Kuntz KM, Knudsen AB, et al. Stool DNA testing to screen for colorectal cancer in the medicare population:a cost-effectiveness analysis[J]. Ann Intern Med,2010,153(6):368-77.
[39]刘志贞,李佩珍,韩晓立,等.大肠癌病人粪便和组织中APC基因突变的研究[J].现代预防医学,2007,(03)：446-7+451.
[40]Rengucci C, Maiolo P, Saragoni L, et al. Multiple detection of genetic alterations in tumors and stool[J]. Clin Cancer Res,2001,7(3):590-3.
[41]Hasegawa Y, Takeda S, Ichii S, et al. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA)[J]. Oncogene, 1995,10(7):1441-5.
[42]Traverso G, Shuber A, Levin B, et al. Detection of APC mutations in fecal DNA from patients with colorectal tumors [J]. N Engl J Med, 2002,346(5):311-20.
[43]Krypuy M, Ahmed AA, Etemadmoghadam D, et al. High resolution melting for mutation scanning of TP53 exons 5-8[J]. BMC Cancer,2007,7:168.
[44]Gundry CN. Vandersteen JG, Reed GH. et al. Amplicon melting analysis with labeled primers:a closed-tube method for differentiating homozygotes and heterozygotes[J]. Clin Chem,2003,49(3):396-406.
[45]Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus[J]. BMC Genet,2009,10:5.
[46]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling[J]. Hum Mutat, 2008,29(5):757-64.
[47]Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[48]Ma ES, Wong CL, Law FB, et al. Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis[J]. J Clin Pathol, 2009,62(10):886-91.
[49]De Leeneer K, Coene I, Poppe B, et al. Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting:comparison of two high-resolution melting platforms[J]. Clin Chem,2008,54(6):982-9.
[50]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
[51]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn,2009.
[52]Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics [J]. Pharmacogenomics, 2007,8(6):597-608.
[53]Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine[J]. Exp Mol Pathol,2008,85(1):50-8.
[54]Taylor CF. Mutation scanning using high-resolution melting[J]. Biochem Soc Trans,2009,37(Pt 2):433-7.
[55]Vossen RH, Aten E, Roos A, et al. High-resolution melting analysis (HRMA): more than just sequence variant screening[J]. Hum Mutat,2009,30(6):860-6.
[56]Wittwer CT. High-resolution DNA melting analysis:advancements and limitations[J]. Hum Mutat,2009,30(6):857-9.
[57]Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics [J]. Expert Rev Mol Diagn, 2010,10(2):219-40.
[58]Li BS, Wang XY, Ma FL, et al. Is high resolution melting analysis (HRMA) accurate for detection of human disease-associated mutations? A meta analysis[J]. PLOS ONE,2011,6(12):e28078.
[59]李琛,王新颖,李丙生,等.高分辨率熔解曲线分析在大肠癌筛查中的应用[J].广东医学,2011,32(1)：10-3.
1 Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol,2010,24(12):705-14.
2 Levin B, Lieberman DA, McFarland B. et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
3 Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
4 Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
5 Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol,2006,126(4):608-15.
6 Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
7 Fuster O, Barragan E, Bolufer P, et al. Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis[J]. J Mol Diagn,2009,11(5):458-63.
8 Heideman DA, Thunnissen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes[J]. Cell Oncol, 2009,31(5):329-33.
9 Dagar V, Chow CW, Ashley DM, et al. Rapid detection of SMARCB1 sequence variation using high resolution melting[J]. BMC Cancer,2009,9:437.
10 Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[1]Fu X, Deng Y, Fan X, et al. Detecting KRAS mutations in colorectal carcinoma patients with high resolution melting curve analysis[J]. Chin Arch Gen Surg (Electronic Edition),2009,3(5):376-8.
[2]Fukui T, Ohe Y, Tsuta K, et al. Prospective study of the accuracy of EGFR mutational analysis by high-resolution melting analysis in small samples obtained from patients with non-small cell lung cancer[J]. Clin Cancer Res, 2008,14(15):4751-7.
[1]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
[2]许岸高,姜泊,钟旭辉,等.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志,2006,45(1)：9-12.
[3]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
[4]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer[J]. J Natl Compr Canc Netw,2010,8(1):81-92.
[5]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors [J]. Science (80-),1992,256(5053):102-5.
[6]Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer:the multicenter "RASCAL" study[J]. J Natl Cancer Inst,1998,90(9):675-84.
[7]Russo A, Bazan V, Agnese V, et al. Prognostic and predictive factors in colorectal cancer:Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies[J]. Ann Oncol,2005,16 Suppl 4:iv44-9.
[8]Iacopetta B. TP53 mutation in colorectal cancer[J]. Hum Mutat, 2003,21(3):271-6.
[9]Iacopetta B, Russo A, Bazan V, et al. Functional categories of TP53 mutation in colorectal cancer:results of an International Collaborative Study [J]. Ann Oncol,2006,17(5):842-7.
[10]刘伟,王丽,余英豪,等k-ras基因在中国结直肠癌患者中的突变状态[J].世界华人消化杂志,2011,(13)：1367-74.
[1]曲利园,王亚东,王贵齐等.国内外大肠癌筛查现状分析及对我国大肠癌筛查的建议[J].中国全科医学.2007.(19)：1584-1586.
[2]李明.中国结直肠癌20年来发病模式的变化趋势[J].中华胃肠外科杂志.2004.(03)：214-217.
[3]许岸高,姜泊,钟旭辉,刘集鸿.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志.2006.45(1)：9-12.
[4]Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics,2009[J]. CA Cancer J Clin.2009.59(4):225-49.
[5]Zisman AL, Nickolov A, Brand RE, Gorchow A, Roy HK. Associations between the age at diagnosis and location of colorectal cancer and the use of alcohol and tobacco:implications for screening[J]. Arch Intern Med.2006. 166(6):629-34.
[6]Forde KA. Colonoscopic screening for colon cancer[J]. Surg Endosc.2006.20 Suppl2:S471-4.
[7]Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol.2010.24(12):705-14.
[8]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology [J]. CA Cancer J Clin.2008.58(3):130-60.
[9]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening [J]. Gut.2008.57(8):1166-76.
[10]3rd SPC, Lal S, Glick JT, Robinson PA, Zamor P, Heeren TC. Patient preferences for colorectal cancer screening:how does stool DNA testing fare[J]. Am J Manag Care.2007.13(7):393-400.
[11]许岸高,余志金,钟旭辉等.自然人群大肠癌筛查方案的比较研究[J].中华健康管理学杂志.2009.3(3)：155-158.
[12]Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population[J]. N Engl J Med.2004.351(26):2704-14.
[13]Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia[J]. Ann Intern Med.2008. 149(7):441-50, W81.
[14]Scholefield JH. Moss S, Sufi F, Mangham CM, Hardcastle JD. Effect of faecal occult blood screening on mortality from colorectal cancer:results from a randomised controlled trial [J]. Gut.2002.50(6):840-4.
[15]Hardcastle JD, Chamberlain JO, Robinson MH, et al. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer[J]. Lancet.1996. 348(9040):1472-7.
[16]Parekh M, Fendrick AM, Ladabaum U. As tests evolve and costs of cancer care rise:reappraising stool-based screening for colorectal neoplasia[J]. Aliment Pharmacol Ther.2008.27(8):697-712.
[17]Davies RJ, Miller R, Coleman N. Colorectal cancer screening:prospects for molecular stool analysis[J]. Nat Rev Cancer.2005.5(3):199-209.
[18]Markowitz SD, Bertagnolli MM. Molecular origins of cancer:Molecular basis of colorectal cancer[J]. N Engl J Med.2009.361(25):2449-60.
[19]Osborn NK, Ahlquist DA. Stool screening for colorectal cancer:molecular approaches [J]. Gastroenterology.2005.128(1):192-206.
[20]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors[J]. Science (80-).1992.256(5053):102-5.
[21]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer. J Natl Compr Canc Netw.2010.8(1):81-92.
[22]Ahlquist DA, Skoletsky JE. Boynton KA, et al. Colorectal cancer screening by detection of altered human DNA in stool:feasibility of a multitarget assay panel[J]. Gastroenterology.2000.119(5):1219-27.
[23]Diehl F, Schmidt K, Durkee KH, et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients[J]. Gastroenterology.2008. 135(2):489-98.
[24]Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay[J]. Gastroenterology.2009.136(2):459-70.
[25]Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics[J]. Expert Rev Mol Diagn.2010. 10(2):219-40.
[26]李丙生,许岸.高分辨率熔解曲线分析用于大肠肿瘤分子检测[J].广东医学.2011.(01)：6-7.
[27]Li BS, Wang XY, Ma FL, Jiang B, Song XX, Xu AG. Is high resolution melting analysis (HRMA) accurate for detection of human disease-associated mutations? A meta analysis[J]. PLOS ONE.2011.6(12):e28078.
[28]李琛,王新颖,李丙生等.高分辨率熔解曲线分析在大肠癌筛查中的应用[J].广东医学.2011.32(1)：10-13.
[29]Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer.2008.8:142.
[30]Fassina A, Gazziero A, Zardo D, Corradin M, Aldighieri E, Rossi GP. Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis[J]. J Clin Pathol.2009.62(12): 1096-102.
[31]Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples:KRAS codon 12 and 13 mutations in non-small cell lung cancer[J]. BMC Cancer.2006.6:295.
[32]傅新晖,邓艳红,范新娟等.利用高分辨率熔解曲线分析法检测结直肠癌中KRAS基因突变[J].中华普通外科学文献(电子版).2009.(05)：376-378.
[33]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation call ing [J]. Hum Mutat.2008.29(5):757-64.
[34]van ER, van PM, Chhatta AR, et al. Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues[J]. J Mol Diagn.2010.12(1):27-34.
[35]Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol.2006.126(4):608-15.
[36]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol.2009.31(3):161-7.
[37]Fuster O, Barragan E. Bolufer P, et al. Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis[J]. J Mol Diagn.2009.11(5):458-63.
[38]Heideman DA, Thunnissen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes[J]. Cell Oncol.2009. 31(5):329-33.
[39]Dagar V, Chow CW, Ashley DM, Algar EM. Rapid detection of SMARCB1 sequence variation using high resolution melting[J]. BMC Cancer.2009.9:437.
[40]Pichler M, Balic M, Stadelmeyer E, et al. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors[J]. J Mol Diagn.2009.11(2):140-7.
[41]Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing[J]. J Mol Diagn.2005.7(3): 413-21.
[42]Rapado I, Grande S, Albizua E, et al. High resolution melting analysis for JAK2 Exon 14 and Exon 12 mutations:a diagnostic tool for myeloproliferative neoplasms[J]. J Mol Diagn.2009.11(2):155-61.
[43]Grand RJ, Owen D. The biochemistry of ras p21[J]. Biochem J.1991.279 (Pt 3):609-31.
[44]Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer:the multicenter "RASCAL" study [J]. J Natl Cancer Inst.1998.90(9):675-84.
[45]Puig P, Urgell E, Capella G. et al. A highly sensitive method for K-ras mutation detection is useful in diagnosis of gastrointestinal cancer [J]. Int J Cancer.2000. 85(1):73-7.
[46]Hasegawa Y, Takeda S, Ichii S, et al. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA) [J]. Oncogene.1995.10(7):1441-5.
[47]范如英,李世荣,武子涛.结肠癌患者癌组织及其粪便K-ras基因突变的检测[J].中华消化杂志.2001.21(7)：445-446.
[48]Vogelstein B, Lane D, Levine AJ. Surfing the p53 network[J]. Nature.2000. 408(6810):307-10.
[49]Petitjean A, Mathe E, Kato S, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype:lessons from recent developments in the IARC TP53 database[J]. Hum Mutat.2007.28(6):622-9.
[50]Eguchi S, Kohara N, Komuta K, Kanematsu T. Mutations of the p53 gene in the stool of patients with resectable colorectal cancer [J]. Cancer.1996.77(8 Suppl): 1707-10.
[51]Rengucci C, Maiolo P, Saragoni L, Zoli W, Amadori D, Calistri D. Multiple detection of genetic alterations in tumors and stool [J]. Clin Cancer Res.2001. 7(3):590-3.
[52]占强,蒋志阳,陈涛,陶国清.粪便p53基因突变检测在结直肠癌诊断中的应用[J].中华内科杂志.2004.(07)：26-29.
[53]Russo A, Bazan V, Agnese V, Rodolico V, Gebbia N. Prognostic and predictive factors in colorectal cancer:Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies[J]. Ann Oncol.2005.16 Suppl 4:iv44-49.
[54]Iacopetta B. TP53 mutation in colorectal cancer[J]. Hum Mutat.2003.21(3): 271-6.
[55]Iacopetta B, Russo A, Bazan V, et al. Functional categories of TP53 mutation in colorectal cancer:results of an International Collaborative Study[J]. Ann Oncol. 2006.17(5):842-7.
[56]刘伟,王丽,余英豪等k-ras基因在中国结直肠癌患者中的突变状态[J].世界华人消化杂志.2011.(13)：1367-1374.
[57]占强,蒋志阳,陈涛,陶国清.粪便p53基因突变检测在结直肠癌诊断中的应用[J].中华内科杂志.2004.(07)：26-29.
[58]Al-Kuraya KS. KRAS and TP53 mutations in colorectal carcinoma[J]. Saudi J Gastroenterol.2009.15(4):217-9.
[59]Berg M, Danielsen SA, Ahlquist T, et al. DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset[J]. PLOS ONE.2010.5(11):e13978.
[60]Powell SM, Petersen GM, Krush AJ, et al. Molecular diagnosis of familial adenomatous polyposis[J]. N Engl J Med.1993.329(27):1982-7.
[61]Kronborg O, Fenger C, Olsen J, Jorgensen OD, Sondergaard O. Randomised study of screening for colorectal cancer with faecal-occult-blood test[J]. Lancet. 1996.348(9040):1467-71.
[62]Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping[J]. Clin Chem.2007.53(8):1544-8.
[63]Er TK, Chang YS, Yeh KT, Chang TJ, Chang JG. Comparison of two different screening methods for the KRAS mutation in colorectal cancer[J]. Clin Lab. 2010.56(5-6):175-86.
[64]Haug U, Hillebrand T, Bendzko P, et al. Mutant-enriched PCR and allele-specific hybridization reaction to detect K-ras mutations in stool DNA: high prevalence in a large sample of older adults[J]. Clin Chem.2007.53(4): 787-90.
[65]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn.2009.
[66]Takano EA, Mitchell G, Fox SB, Dobrovic A. Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis[J]. BMC Cancer.2008.8:59.
[67]der Stoep N v, van PCD, Janssens T, et al. Diagnostic guidelines for high-resolution melting curve (HRM) analysis:an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner[J]. Hum Mutat. 2009.30(6):899-909.
[68]Thomas RK, Nickerson E, Simons JF. et al. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing[J]. Nat Med.2006.12(7):852-5.
[1]Krypuy M, Ahmed AA, Etemadmoghadam D, et al. High resolution melting for mutation scanning of TP53 exons 5-8[J]. BMC Cancer,2007,7:168.
[2]Gundry CN, Vandersteen JG, Reed GH, et al. Amplicon melting analysis with labeled primers:a closed-tube method for differentiating homozygotes and heterozygotes[J]. Clin Chem,2003,49(3):396-406.
[3]Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen[J]. Clin Chem,2003,49(6 Pt 1):853-60.
[4]Liew M, Pryor R, Palais R, et al. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons[J]. Clin Chem, 2004,50(7):1156-64.
[5]Zhou L, Myers AN, Vandersteen JG, et al. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye[J]. Clin Chem, 2004,50(8):1328-35.
[6]Montgomery J, Wittwer CT, Palais R, et al. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis[J]. Nat Protoc, 2007.2(1):59-66.
[7]Zhou L, Wang L, Palais R, et al. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution[J]. Clin Chem, 2005,51(10):1770-7.
[8]Worm J, Aggerholm A, Guldberg P. In-tube DNA methylation profiling by fluorescence melting curve analysis[J]. Clin Chem,2001,47(7):1183-9.
[9]Malentacchi F, Forni G, Vinci S, et al. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol[J]. Nucleic Acids Res,2009,37(12):e86.
[10]Chateigner-Boutin AL, Small I. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons[J]. Nucleic Acids Res,2007,35(17):e114.
[11]Mader E, Lukas B, Novak J. A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM)[J]. BMC Genet, 2008,9:69.
[12]Zhou L, Vandersteen J, Wang L, et al. High-resolution DNA melting curve analysis to establish HLA genotypic identity [J]. Tissue Antigens, 2004,64(2):156-64.
[13]Takano T, Ohe Y, Tsuta K, et al. Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non small cell lung cancer treated with gefitinib[J]. Clin Cancer Res,2007,13(18 Pt 1):5385-90.
[14]Willmore-Payne C, Layfield LJ, Holden JA. c-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens[J]. Cancer,2005,105(3):165-70.
[15]Pichler M, Balic M, Stadelmeyer E, et al. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors[J]. J Mol Diagn,2009,11(2):140-7.
[16]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn,2009.
[17]Hollstein M. Sidransky D, Vogelstein B, et al. p53 mutations in human cancers[J]. Science (80-),1991,253(5015):49-53.
[18]Vogelstein B, Lane D, Levine AJ. Surfing the p53 network[J]. Nature, 2000,408(6810):307-10.
[19]Aylon Y, Oren M. Living with p53, dying of p53[J]. Cell, 2007,130(4):597-600.
[20]Vousden KH. Outcomes of p53 activation--spoilt for choice[J]. J Cell Sci, 2006,119(Pt 24):5015-20.
[21]Horn HF, Vousden KH. Coping with stress:multiple ways to activate p53[J]. Oncogene,2007,26(9):1306-16.
[22]Laframboise S, Chapman W, McLaughlin J, et al. p53 mutations in epithelial ovarian cancers:possible role in predicting chemoresistance[J]. Cancer J, 2000,6(5):302-8.
[23]Olivier M, Eeles R, Hollstein M, et al. The IARC TP53 database:new online mutation analysis and recommendations to users[J]. Hum Mutat, 2002,19(6):607-14.
[24]Petitjean A, Mathe E, Kato S, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype:lessons from recent developments in the IARC TP53 database[J]. Hum Mutat, 2007,28(6):622-9.
[25]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling[J]. Hum Mutat, 2008,29(5):757-64.
[26]Tindall EA, Petersen DC, Woodbridge P, et al. Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments [J]. Hum Mutat,2009,30(6):876-83.
[27]Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus[J]. BMC Genet,2009,10:5.
[28]Milbury CA, Li J, Makrigiorgos GM. COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations[J]. Clin Chem,2009.
[29]Zou H, Taylor WR, Harrington JJ. et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
[30]Adjei A A. Blocking oncogenic Ras signaling for cancer therapy [J]. J Natl Cancer Inst,2001,93(14):1062-74.
[31]Krypuy M, Newnham GM, Thomas DM, et al. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer[J]. BMC Cancer,2006,6:295.
[32]Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[33]Ma ES, Wong CL, Law FB, et al. Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis[J]. J Clin Pathol, 2009,62(10):886-91.
[34]Simi L, Pratesi N, Vignoli M, et al. High-resolution melting analysis for rapid detection of gene mutations in colorectal cancer [J]. Am J Clin Pathol, 2008,130(2):247-53.
[35]Liu ZM, Liu LN, Li M, et al. Mutation detection of KRAS by high-resolution melting analysis in Chinese with gastric cancer[J]. Oncol Rep, 2009,22(3):515-20.
[36]Seth R, Crook S, Ibrahem S, et al. Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer[J]. Gut, 2009.58(9):1234-41.
[37]Li M. Liu L, Liu Z, et al. The status of KRAS mutations in patients with non-small cell lung cancers from mainland China[J]. Oncol Rep, 2009,22(5):1013-20.
[38]Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers[J]. J Natl Cancer Inst,2005,97(5):339-46.
[39]Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib[J]. N Engl J Med,2004,350(21):2129-39.
[40]Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy [J]. Science (80-), 2004,304(5676):1497-500.
[41]Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol,2006,126(4):608-15.
[42]Fukui T, Tsuta K, Furuta K, et al. Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lung[J]. Cancer Sci,2007,98(11):1714-9.
[43]Takano T, Fukui T, Ohe Y, et al. EGFR mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma:a historical comparison of patients treated before and after gefitinib approval in Japan[J]. J Clin Oncol,2008,26(34):5589-95.
[44]Fukui T, Ohe Y, Tsuta K, et al. Prospective study of the accuracy of EGFR mutational analysis by high-resolution melting analysis in small samples obtained from patients with non-small cell lung cancer[J]. Clin Cancer Res, 2008,14(15):4751-7.
[45]Ikenoue T, Hikiba Y, Kanai F, et al. Functional analysis of mutations within the kinase activation segment of B-Raf inhuman colorectal tumors[J]. Cancer Res,2003,63(23):8132-7.
[46]Willmore-Payne C, Volmar KE, Huening MA, et al. Molecular diagnostic testing as an adjunct to morphologic evaluation of pancreatic ductal system brushings:potential augmentation for diagnostic sensitivity[J]. Diagn Cytopathol,2007,35(4):218-24.
[47]Friedenson B. BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian[J]. MedGenMed,2005,7(2):60.
[48]der Stoep N v, van PCD, Janssens T, et al. Diagnostic guidelines for high-resolution melting curve (HRM) analysis:an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner[J]. Hum Mutat, 2009,30(6):899-909.
[49]Takano EA, Mitchell G, Fox SB, et al. Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis[J]. BMC Cancer,2008,8:59.
[50]Jimenez IJ, Esteban CE, Palanca SS, et al. Advantages of the high resolution melting in the detection of BRCA1 or BRCA2 mutation carriers[J]. Clin Biochem,2009,42(15):1572-6.
[51]Vorkas PA, Christopoulos K, Kroupis C, et al. Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis[J]. Clin Biochem,2009.
[52]De Leeneer K, Coene I, Poppe B, et al. Genotyping of frequent BRCA1/2 SNPs with unlabeled probes:a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[J]. J Mol Diagn, 2009,11(5):415-9.
[53]Holden JA, Willmore-Payne C, Coppola D, et al. High-resolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors[J]. Am J Clin Pathol,2007,128(2):230-8.
[54]Er TK, Lin SF, Chang JG, et al. Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation[J]. Clin Chim Acta,2009,408(1-2):39-44.
[55]Do H, Solomon B, Mitchell PL, et al. Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting[J]. BMC Res Notes,2008,1:14.
[56]Rouleau E, Lefol C, Bourdon V, et al. Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements:application to MLH1 germline mutations in Lynch syndrome[J]. Hum Mutat,2009,30(6):867-75.
[57]Seth R, Keeley J, Abu-Ali G, et al. The putative tumour modifier gene ATP5A1 is not mutated in human colorectal cancer cell lines but expression levels correlate with TP53 mutations and chromosomal instability[J]. J Clin Pathol,2009,62(7):598-603.
[58]Nguyen-Dumont T, Calvez-Kelm FL, Forey N, et al. Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis[J]. Hum Mutat,2009,30(6):884-90.
[59]Sinthuwiwat T, Poowasanpetch P, Wongngamrungroj A, et al. High-resolution melting curve analysis for genotyping of common SNP in MTHFR gene using fixed-cell suspension [J]. Mol Cell Probes, 2008,22(5-6):329-32.
[60]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
[61]Kristensen LS, Dobrovic A. Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free high-resolution melting analysis[J]. Cancer Epidemiol Biomarkers Prev,2008,17(5):1240-7.
[62]Margraf RL, Mao R, Highsmith WE, et al. RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis[J]. J Mol Diagn,2007,9(2):184-96.
[63]Seipp MT, Durtschi JD, Liew MA, et al. Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting[J]. J Mol Diagn,2007,9(3):284-9.
[64]Illingworth R, Kerr A, Desousa D, et al. A novel CpG island set identifies tissue-specific methylation at developmentalgene loci[J]. PLoS Biol, 2008,6(1):e22.
[65]Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation[J]. N Engl J Med,2003,349(21):2042-54.
[66]Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM):a new approach for sensitive and high-throughput assessment of methylation[J]. Nucleic Acids Res,2007,35(6):e41.
[67]Snell C, Krypuy M, Wong EM, et al. BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype[J]. Breast Cancer Res,2008,10(1):R12.
[68]Balic M, Pichler M, Strutz J, et al. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis [J]. J Mol Diagn, 2009,11(2):102-8.
[69]Li Z, Zhang W, Shao Y, et al. High-resolution melting analysis of ADAMTS18 methylation levels in gastric, colorectal and pancreatic cancers[J]. Med Oncol,2009.[70] Wittwer CT. High-resolution DNA melting analysis:advancements and limitations[J]. Hum Mutat, 2009,30(6):857-9.
[71]Taylor CF. Mutation scanning using high-resolution melting[J]. Biochem Soc Trans,2009,37(Pt 2):433-7.
[72]Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine[J]. Exp Mol Pathol,2008,85(1):50-8.
[73]Do H, Dobrovic A. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies[J]. Mol Cancer,2009,8:82.
[2]许岸高,姜泊,钟旭辉,等.广东地区近20年大肠癌临床特征的变化趋势[J].中华医学杂志,2006,,(04)：272-5.
[3]许岸高,姜泊,余志金,等.广东地区大肠癌临床特征研究[J].中华消化杂志,2007,(07)：450-3.
[4]许岸高,姜泊,钟旭辉,等.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志,2006,(01)：9-12.
[5]曲利园,王亚东,王贵齐,等.国内外大肠癌筛查现状分析及对我国大肠癌筛查的建议[J].中国全科医学,2007,,(19)：1584-6.
[6]Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol, 2010,24(12):705-14.
[7]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
[8]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
[9]3rd SPC, Lal S, Glick JT, et al. Patient preferences for colorectal cancer screening:how does stool DNA testing fare?[J]. Am J Manag Care, 2007,13(7):393-400.
[10]Hardcastle JD, Chamberlain JO, Robinson MH, et al. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer[J]. Lancet, 1996,348(9040):1472-7.
[11]Kronborg O, Fenger C, Olsen J, et al. Randomised study of screening for colorectal cancer with faecal-occult-blood test[J]. Lancet, 1996,348(9040):1467-71.
[12]Mandel JS, Church TR, Bond JH, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer[J]. N Engl J Med, 2000,343(22):1603-7.
[13]Allison JE, Tekawa IS, Ransom LJ, et al. A comparison of fecal occult-blood tests for colorectal-cancer screening[J]. N Engl J Med,1996,334(3):155-9.
[14]Allison JE, Sakoda LC, Levin TR, et al. Screening for colorectal neoplasms with new fecal occult blood tests:update on performance characteristics[J]. J Natl Cancer Inst,2007,99(19):1462-70.
[15]Cole SR, Young GP. Effect of dietary restriction on participation in faecal occult blood test screening for colorectal cancer[J]. Med J Aust, 2001,175(4):195-8.
[16]van RLG, van RAF, Laheij RJ, et al. Random comparison of guaiac and immunochemical fecal occult blood tests for colorectal cancer in a screening population [J]. Gastroenterology,2008,135(1):82-90.
[17]Saito H, Soma Y, Koeda J, et al. Reduction in risk of mortality from colorectal cancer by fecal occult blood screening with immunochemical hemagglutination test. A case-control study [J]. Int J Cancer, 1995,61 (4):465-9.
[18]Lee KJ. Inoue M. Otani T, et al. Colorectal cancer screening using fecal occult blood test and subsequent risk of colorectal cancer:a prospective cohort study in Japan[J]. Cancer Detect Prev,2007,31(1):3-11.
[19]Parekh M, Fendrick AM, Ladabaum U. As tests evolve and costs of cancer care rise:reappraising stool-based screening for colorectal neoplasia[J]. Aliment Pharmacol Ther,2008,27(8):697-712.
[20]Ko CW, Dominitz JA, Nguyen TD. Fecal occult blood testing in a general medical clinic:comparison between guaiac-based and immunochemical-based tests[J]. Am J Med,2003,115(2):111-4.
[21]刘集鸿,许岸高,余志金,等.免疫胶体金便隐血试验在大肠癌普查中应用[J].中国肿瘤,2007,16(4)：219-20.
[22]许岸高,余志金,钟旭辉,等.自然人群大肠癌筛查方案的比较研究[J].中华健康管理学杂志,2009,3(3)：155-8.
[23]Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population[J]. N Engl J Med,2004,351(26):2704-14.
[24]Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia[J]. Ann Intern Med, 2008,149(7):441-50, W81.
[25]Scholefield JH, Moss S, Sufi F, et al. Effect of faecal occult blood screening on mortality from colorectal cancer:results from a randomised controlled trial[J]. Gut,2002,50(6):840-4.
[26]Myers RE, Balshem AM, Wolf TA, et al. Adherence to continuous screening for colorectal neoplasia[J]. Med Care,199331(6):508-19.
[27]Davies RJ, Miller R, Coleman N. Colorectal cancer screening:prospects for molecular stool analysis[J]. Nat Rev Cancer,2005,5(3):199-209.
[28]Albaugh GP, Iyengar V. Lohani A, et al. Isolation of exfoliated colonic epithelial cells, a novel, non-invasive approach to the study of cellular markers[J]. Int J Cancer,1992,52(3):347-50.
[29]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors [J]. Science (80-),1992,256(5053):102-5.
[30]Ratto C, Flamini G, Sofo L, et al. Detection of oncogene mutation from neoplastic colonic cells exfoliated in feces[J]. Dis Colon Rectum, 1996,39(11):1238-44.
[31]Osborn NK, Ahlquist DA. Stool screening for colorectal cancer:molecular approaches. [J]. Gastroenterology,2005,128(1):192-206.
[32]Diehl F, Schmidt K, Durkee KH, et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients[J]. Gastroenterology, 2008,135(2):489-98.
[33]Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
[34]Berger BM,3rd SPC, Rosenberg JL, et al. Colorectal cancer screening using stool DNA analysis in clinical practice:early clinical experience with respect to patient acceptance and colonoscopic follow-up of abnormal tests [J]. Clin Colorectal Cancer,2006,5(5):338-43.
[35]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer[J]. J Natl Compr Canc Netw,2010,8(1):81-92.
[36]Itzkowitz SH, Jandorf L, Brand R, et al. Improved fecal DNA test for colorectal cancer screening[J]. Clin Gastroenterol Hepatol,2007,5(1):111-7.
[37]Ahlquist DA. Next-generation stool DNA testing:expanding the scope [J]. Gastroenterology,2009,136(7):2068-73.
[38]Lansdorp-Vogelaar I, Kuntz KM, Knudsen AB, et al. Stool DNA testing to screen for colorectal cancer in the medicare population:a cost-effectiveness analysis[J]. Ann Intern Med,2010,153(6):368-77.
[39]刘志贞,李佩珍,韩晓立,等.大肠癌病人粪便和组织中APC基因突变的研究[J].现代预防医学,2007,(03)：446-7+451.
[40]Rengucci C, Maiolo P, Saragoni L, et al. Multiple detection of genetic alterations in tumors and stool[J]. Clin Cancer Res,2001,7(3):590-3.
[41]Hasegawa Y, Takeda S, Ichii S, et al. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA)[J]. Oncogene, 1995,10(7):1441-5.
[42]Traverso G, Shuber A, Levin B, et al. Detection of APC mutations in fecal DNA from patients with colorectal tumors [J]. N Engl J Med, 2002,346(5):311-20.
[43]Krypuy M, Ahmed AA, Etemadmoghadam D, et al. High resolution melting for mutation scanning of TP53 exons 5-8[J]. BMC Cancer,2007,7:168.
[44]Gundry CN. Vandersteen JG, Reed GH. et al. Amplicon melting analysis with labeled primers:a closed-tube method for differentiating homozygotes and heterozygotes[J]. Clin Chem,2003,49(3):396-406.
[45]Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus[J]. BMC Genet,2009,10:5.
[46]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling[J]. Hum Mutat, 2008,29(5):757-64.
[47]Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[48]Ma ES, Wong CL, Law FB, et al. Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis[J]. J Clin Pathol, 2009,62(10):886-91.
[49]De Leeneer K, Coene I, Poppe B, et al. Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting:comparison of two high-resolution melting platforms[J]. Clin Chem,2008,54(6):982-9.
[50]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
[51]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn,2009.
[52]Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics [J]. Pharmacogenomics, 2007,8(6):597-608.
[53]Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine[J]. Exp Mol Pathol,2008,85(1):50-8.
[54]Taylor CF. Mutation scanning using high-resolution melting[J]. Biochem Soc Trans,2009,37(Pt 2):433-7.
[55]Vossen RH, Aten E, Roos A, et al. High-resolution melting analysis (HRMA): more than just sequence variant screening[J]. Hum Mutat,2009,30(6):860-6.
[56]Wittwer CT. High-resolution DNA melting analysis:advancements and limitations[J]. Hum Mutat,2009,30(6):857-9.
[57]Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics [J]. Expert Rev Mol Diagn, 2010,10(2):219-40.
[58]Li BS, Wang XY, Ma FL, et al. Is high resolution melting analysis (HRMA) accurate for detection of human disease-associated mutations? A meta analysis[J]. PLOS ONE,2011,6(12):e28078.
[59]李琛,王新颖,李丙生,等.高分辨率熔解曲线分析在大肠癌筛查中的应用[J].广东医学,2011,32(1)：10-3.
1 Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol,2010,24(12):705-14.
2 Levin B, Lieberman DA, McFarland B. et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
3 Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
4 Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
5 Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol,2006,126(4):608-15.
6 Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
7 Fuster O, Barragan E, Bolufer P, et al. Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis[J]. J Mol Diagn,2009,11(5):458-63.
8 Heideman DA, Thunnissen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes[J]. Cell Oncol, 2009,31(5):329-33.
9 Dagar V, Chow CW, Ashley DM, et al. Rapid detection of SMARCB1 sequence variation using high resolution melting[J]. BMC Cancer,2009,9:437.
10 Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[1]Fu X, Deng Y, Fan X, et al. Detecting KRAS mutations in colorectal carcinoma patients with high resolution melting curve analysis[J]. Chin Arch Gen Surg (Electronic Edition),2009,3(5):376-8.
[2]Fukui T, Ohe Y, Tsuta K, et al. Prospective study of the accuracy of EGFR mutational analysis by high-resolution melting analysis in small samples obtained from patients with non-small cell lung cancer[J]. Clin Cancer Res, 2008,14(15):4751-7.
[1]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening[J]. Gut,2008,57(8):1166-76.
[2]许岸高,姜泊,钟旭辉,等.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志,2006,45(1)：9-12.
[3]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology[J]. CA Cancer J Clin,2008,58(3):130-60.
[4]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer[J]. J Natl Compr Canc Netw,2010,8(1):81-92.
[5]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors [J]. Science (80-),1992,256(5053):102-5.
[6]Andreyev HJ, Norman AR, Cunningham D, et al. Kirsten ras mutations in patients with colorectal cancer:the multicenter "RASCAL" study[J]. J Natl Cancer Inst,1998,90(9):675-84.
[7]Russo A, Bazan V, Agnese V, et al. Prognostic and predictive factors in colorectal cancer:Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies[J]. Ann Oncol,2005,16 Suppl 4:iv44-9.
[8]Iacopetta B. TP53 mutation in colorectal cancer[J]. Hum Mutat, 2003,21(3):271-6.
[9]Iacopetta B, Russo A, Bazan V, et al. Functional categories of TP53 mutation in colorectal cancer:results of an International Collaborative Study [J]. Ann Oncol,2006,17(5):842-7.
[10]刘伟,王丽,余英豪,等k-ras基因在中国结直肠癌患者中的突变状态[J].世界华人消化杂志,2011,(13)：1367-74.
[1]曲利园,王亚东,王贵齐等.国内外大肠癌筛查现状分析及对我国大肠癌筛查的建议[J].中国全科医学.2007.(19)：1584-1586.
[2]李明.中国结直肠癌20年来发病模式的变化趋势[J].中华胃肠外科杂志.2004.(03)：214-217.
[3]许岸高,姜泊,钟旭辉,刘集鸿.广东地区3870例大肠癌的临床流行病学特征[J].中华内科杂志.2006.45(1)：9-12.
[4]Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics,2009[J]. CA Cancer J Clin.2009.59(4):225-49.
[5]Zisman AL, Nickolov A, Brand RE, Gorchow A, Roy HK. Associations between the age at diagnosis and location of colorectal cancer and the use of alcohol and tobacco:implications for screening[J]. Arch Intern Med.2006. 166(6):629-34.
[6]Forde KA. Colonoscopic screening for colon cancer[J]. Surg Endosc.2006.20 Suppl2:S471-4.
[7]Leddin DJ, Enns R, Hilsden R, et al. Canadian Association of Gastroenterology position statement on screening individuals at average risk for developing colorectal cancer:2010[J]. Can J Gastroenterol.2010.24(12):705-14.
[8]Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology [J]. CA Cancer J Clin.2008.58(3):130-60.
[9]Sung JJ, Lau JY, Young GP, et al. Asia Pacific consensus recommendations for colorectal cancer screening [J]. Gut.2008.57(8):1166-76.
[10]3rd SPC, Lal S, Glick JT, Robinson PA, Zamor P, Heeren TC. Patient preferences for colorectal cancer screening:how does stool DNA testing fare[J]. Am J Manag Care.2007.13(7):393-400.
[11]许岸高,余志金,钟旭辉等.自然人群大肠癌筛查方案的比较研究[J].中华健康管理学杂志.2009.3(3)：155-158.
[12]Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population[J]. N Engl J Med.2004.351(26):2704-14.
[13]Ahlquist DA, Sargent DJ, Loprinzi CL, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia[J]. Ann Intern Med.2008. 149(7):441-50, W81.
[14]Scholefield JH. Moss S, Sufi F, Mangham CM, Hardcastle JD. Effect of faecal occult blood screening on mortality from colorectal cancer:results from a randomised controlled trial [J]. Gut.2002.50(6):840-4.
[15]Hardcastle JD, Chamberlain JO, Robinson MH, et al. Randomised controlled trial of faecal-occult-blood screening for colorectal cancer[J]. Lancet.1996. 348(9040):1472-7.
[16]Parekh M, Fendrick AM, Ladabaum U. As tests evolve and costs of cancer care rise:reappraising stool-based screening for colorectal neoplasia[J]. Aliment Pharmacol Ther.2008.27(8):697-712.
[17]Davies RJ, Miller R, Coleman N. Colorectal cancer screening:prospects for molecular stool analysis[J]. Nat Rev Cancer.2005.5(3):199-209.
[18]Markowitz SD, Bertagnolli MM. Molecular origins of cancer:Molecular basis of colorectal cancer[J]. N Engl J Med.2009.361(25):2449-60.
[19]Osborn NK, Ahlquist DA. Stool screening for colorectal cancer:molecular approaches [J]. Gastroenterology.2005.128(1):192-206.
[20]Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors[J]. Science (80-).1992.256(5053):102-5.
[21]Potack J, Itzkowitz SH. Practical advances in stool screening for colorectal cancer. J Natl Compr Canc Netw.2010.8(1):81-92.
[22]Ahlquist DA, Skoletsky JE. Boynton KA, et al. Colorectal cancer screening by detection of altered human DNA in stool:feasibility of a multitarget assay panel[J]. Gastroenterology.2000.119(5):1219-27.
[23]Diehl F, Schmidt K, Durkee KH, et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients[J]. Gastroenterology.2008. 135(2):489-98.
[24]Zou H, Taylor WR, Harrington JJ, et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay[J]. Gastroenterology.2009.136(2):459-70.
[25]Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics[J]. Expert Rev Mol Diagn.2010. 10(2):219-40.
[26]李丙生,许岸.高分辨率熔解曲线分析用于大肠肿瘤分子检测[J].广东医学.2011.(01)：6-7.
[27]Li BS, Wang XY, Ma FL, Jiang B, Song XX, Xu AG. Is high resolution melting analysis (HRMA) accurate for detection of human disease-associated mutations? A meta analysis[J]. PLOS ONE.2011.6(12):e28078.
[28]李琛,王新颖,李丙生等.高分辨率熔解曲线分析在大肠癌筛查中的应用[J].广东医学.2011.32(1)：10-13.
[29]Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer.2008.8:142.
[30]Fassina A, Gazziero A, Zardo D, Corradin M, Aldighieri E, Rossi GP. Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis[J]. J Clin Pathol.2009.62(12): 1096-102.
[31]Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples:KRAS codon 12 and 13 mutations in non-small cell lung cancer[J]. BMC Cancer.2006.6:295.
[32]傅新晖,邓艳红,范新娟等.利用高分辨率熔解曲线分析法检测结直肠癌中KRAS基因突变[J].中华普通外科学文献(电子版).2009.(05)：376-378.
[33]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation call ing [J]. Hum Mutat.2008.29(5):757-64.
[34]van ER, van PM, Chhatta AR, et al. Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues[J]. J Mol Diagn.2010.12(1):27-34.
[35]Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol.2006.126(4):608-15.
[36]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol.2009.31(3):161-7.
[37]Fuster O, Barragan E. Bolufer P, et al. Rapid detection of KIT mutations in core-binding factor acute myeloid leukemia using high-resolution melting analysis[J]. J Mol Diagn.2009.11(5):458-63.
[38]Heideman DA, Thunnissen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes[J]. Cell Oncol.2009. 31(5):329-33.
[39]Dagar V, Chow CW, Ashley DM, Algar EM. Rapid detection of SMARCB1 sequence variation using high resolution melting[J]. BMC Cancer.2009.9:437.
[40]Pichler M, Balic M, Stadelmeyer E, et al. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors[J]. J Mol Diagn.2009.11(2):140-7.
[41]Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing[J]. J Mol Diagn.2005.7(3): 413-21.
[42]Rapado I, Grande S, Albizua E, et al. High resolution melting analysis for JAK2 Exon 14 and Exon 12 mutations:a diagnostic tool for myeloproliferative neoplasms[J]. J Mol Diagn.2009.11(2):155-61.
[43]Grand RJ, Owen D. The biochemistry of ras p21[J]. Biochem J.1991.279 (Pt 3):609-31.
[44]Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer:the multicenter "RASCAL" study [J]. J Natl Cancer Inst.1998.90(9):675-84.
[45]Puig P, Urgell E, Capella G. et al. A highly sensitive method for K-ras mutation detection is useful in diagnosis of gastrointestinal cancer [J]. Int J Cancer.2000. 85(1):73-7.
[46]Hasegawa Y, Takeda S, Ichii S, et al. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA) [J]. Oncogene.1995.10(7):1441-5.
[47]范如英,李世荣,武子涛.结肠癌患者癌组织及其粪便K-ras基因突变的检测[J].中华消化杂志.2001.21(7)：445-446.
[48]Vogelstein B, Lane D, Levine AJ. Surfing the p53 network[J]. Nature.2000. 408(6810):307-10.
[49]Petitjean A, Mathe E, Kato S, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype:lessons from recent developments in the IARC TP53 database[J]. Hum Mutat.2007.28(6):622-9.
[50]Eguchi S, Kohara N, Komuta K, Kanematsu T. Mutations of the p53 gene in the stool of patients with resectable colorectal cancer [J]. Cancer.1996.77(8 Suppl): 1707-10.
[51]Rengucci C, Maiolo P, Saragoni L, Zoli W, Amadori D, Calistri D. Multiple detection of genetic alterations in tumors and stool [J]. Clin Cancer Res.2001. 7(3):590-3.
[52]占强,蒋志阳,陈涛,陶国清.粪便p53基因突变检测在结直肠癌诊断中的应用[J].中华内科杂志.2004.(07)：26-29.
[53]Russo A, Bazan V, Agnese V, Rodolico V, Gebbia N. Prognostic and predictive factors in colorectal cancer:Kirsten Ras in CRC (RASCAL) and TP53CRC collaborative studies[J]. Ann Oncol.2005.16 Suppl 4:iv44-49.
[54]Iacopetta B. TP53 mutation in colorectal cancer[J]. Hum Mutat.2003.21(3): 271-6.
[55]Iacopetta B, Russo A, Bazan V, et al. Functional categories of TP53 mutation in colorectal cancer:results of an International Collaborative Study[J]. Ann Oncol. 2006.17(5):842-7.
[56]刘伟,王丽,余英豪等k-ras基因在中国结直肠癌患者中的突变状态[J].世界华人消化杂志.2011.(13)：1367-1374.
[57]占强,蒋志阳,陈涛,陶国清.粪便p53基因突变检测在结直肠癌诊断中的应用[J].中华内科杂志.2004.(07)：26-29.
[58]Al-Kuraya KS. KRAS and TP53 mutations in colorectal carcinoma[J]. Saudi J Gastroenterol.2009.15(4):217-9.
[59]Berg M, Danielsen SA, Ahlquist T, et al. DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset[J]. PLOS ONE.2010.5(11):e13978.
[60]Powell SM, Petersen GM, Krush AJ, et al. Molecular diagnosis of familial adenomatous polyposis[J]. N Engl J Med.1993.329(27):1982-7.
[61]Kronborg O, Fenger C, Olsen J, Jorgensen OD, Sondergaard O. Randomised study of screening for colorectal cancer with faecal-occult-blood test[J]. Lancet. 1996.348(9040):1467-71.
[62]Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping[J]. Clin Chem.2007.53(8):1544-8.
[63]Er TK, Chang YS, Yeh KT, Chang TJ, Chang JG. Comparison of two different screening methods for the KRAS mutation in colorectal cancer[J]. Clin Lab. 2010.56(5-6):175-86.
[64]Haug U, Hillebrand T, Bendzko P, et al. Mutant-enriched PCR and allele-specific hybridization reaction to detect K-ras mutations in stool DNA: high prevalence in a large sample of older adults[J]. Clin Chem.2007.53(4): 787-90.
[65]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn.2009.
[66]Takano EA, Mitchell G, Fox SB, Dobrovic A. Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis[J]. BMC Cancer.2008.8:59.
[67]der Stoep N v, van PCD, Janssens T, et al. Diagnostic guidelines for high-resolution melting curve (HRM) analysis:an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner[J]. Hum Mutat. 2009.30(6):899-909.
[68]Thomas RK, Nickerson E, Simons JF. et al. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing[J]. Nat Med.2006.12(7):852-5.
[1]Krypuy M, Ahmed AA, Etemadmoghadam D, et al. High resolution melting for mutation scanning of TP53 exons 5-8[J]. BMC Cancer,2007,7:168.
[2]Gundry CN, Vandersteen JG, Reed GH, et al. Amplicon melting analysis with labeled primers:a closed-tube method for differentiating homozygotes and heterozygotes[J]. Clin Chem,2003,49(3):396-406.
[3]Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen[J]. Clin Chem,2003,49(6 Pt 1):853-60.
[4]Liew M, Pryor R, Palais R, et al. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons[J]. Clin Chem, 2004,50(7):1156-64.
[5]Zhou L, Myers AN, Vandersteen JG, et al. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye[J]. Clin Chem, 2004,50(8):1328-35.
[6]Montgomery J, Wittwer CT, Palais R, et al. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis[J]. Nat Protoc, 2007.2(1):59-66.
[7]Zhou L, Wang L, Palais R, et al. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution[J]. Clin Chem, 2005,51(10):1770-7.
[8]Worm J, Aggerholm A, Guldberg P. In-tube DNA methylation profiling by fluorescence melting curve analysis[J]. Clin Chem,2001,47(7):1183-9.
[9]Malentacchi F, Forni G, Vinci S, et al. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol[J]. Nucleic Acids Res,2009,37(12):e86.
[10]Chateigner-Boutin AL, Small I. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons[J]. Nucleic Acids Res,2007,35(17):e114.
[11]Mader E, Lukas B, Novak J. A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM)[J]. BMC Genet, 2008,9:69.
[12]Zhou L, Vandersteen J, Wang L, et al. High-resolution DNA melting curve analysis to establish HLA genotypic identity [J]. Tissue Antigens, 2004,64(2):156-64.
[13]Takano T, Ohe Y, Tsuta K, et al. Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non small cell lung cancer treated with gefitinib[J]. Clin Cancer Res,2007,13(18 Pt 1):5385-90.
[14]Willmore-Payne C, Layfield LJ, Holden JA. c-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens[J]. Cancer,2005,105(3):165-70.
[15]Pichler M, Balic M, Stadelmeyer E, et al. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors[J]. J Mol Diagn,2009,11(2):140-7.
[16]Whitehall V, Tran K, Umapathy A, et al. A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting[J]. J Mol Diagn,2009.
[17]Hollstein M. Sidransky D, Vogelstein B, et al. p53 mutations in human cancers[J]. Science (80-),1991,253(5015):49-53.
[18]Vogelstein B, Lane D, Levine AJ. Surfing the p53 network[J]. Nature, 2000,408(6810):307-10.
[19]Aylon Y, Oren M. Living with p53, dying of p53[J]. Cell, 2007,130(4):597-600.
[20]Vousden KH. Outcomes of p53 activation--spoilt for choice[J]. J Cell Sci, 2006,119(Pt 24):5015-20.
[21]Horn HF, Vousden KH. Coping with stress:multiple ways to activate p53[J]. Oncogene,2007,26(9):1306-16.
[22]Laframboise S, Chapman W, McLaughlin J, et al. p53 mutations in epithelial ovarian cancers:possible role in predicting chemoresistance[J]. Cancer J, 2000,6(5):302-8.
[23]Olivier M, Eeles R, Hollstein M, et al. The IARC TP53 database:new online mutation analysis and recommendations to users[J]. Hum Mutat, 2002,19(6):607-14.
[24]Petitjean A, Mathe E, Kato S, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype:lessons from recent developments in the IARC TP53 database[J]. Hum Mutat, 2007,28(6):622-9.
[25]Bastien R, Lewis TB, Hawkes JE, et al. High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling[J]. Hum Mutat, 2008,29(5):757-64.
[26]Tindall EA, Petersen DC, Woodbridge P, et al. Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments [J]. Hum Mutat,2009,30(6):876-83.
[27]Garritano S, Gemignani F, Voegele C, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus[J]. BMC Genet,2009,10:5.
[28]Milbury CA, Li J, Makrigiorgos GM. COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations[J]. Clin Chem,2009.
[29]Zou H, Taylor WR, Harrington JJ. et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay [J]. Gastroenterology,2009,136(2):459-70.
[30]Adjei A A. Blocking oncogenic Ras signaling for cancer therapy [J]. J Natl Cancer Inst,2001,93(14):1062-74.
[31]Krypuy M, Newnham GM, Thomas DM, et al. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer[J]. BMC Cancer,2006,6:295.
[32]Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies[J]. BMC Cancer,2008,8:142.
[33]Ma ES, Wong CL, Law FB, et al. Detection of KRAS mutations in colorectal cancer by high-resolution melting analysis[J]. J Clin Pathol, 2009,62(10):886-91.
[34]Simi L, Pratesi N, Vignoli M, et al. High-resolution melting analysis for rapid detection of gene mutations in colorectal cancer [J]. Am J Clin Pathol, 2008,130(2):247-53.
[35]Liu ZM, Liu LN, Li M, et al. Mutation detection of KRAS by high-resolution melting analysis in Chinese with gastric cancer[J]. Oncol Rep, 2009,22(3):515-20.
[36]Seth R, Crook S, Ibrahem S, et al. Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer[J]. Gut, 2009.58(9):1234-41.
[37]Li M. Liu L, Liu Z, et al. The status of KRAS mutations in patients with non-small cell lung cancers from mainland China[J]. Oncol Rep, 2009,22(5):1013-20.
[38]Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers[J]. J Natl Cancer Inst,2005,97(5):339-46.
[39]Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib[J]. N Engl J Med,2004,350(21):2129-39.
[40]Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy [J]. Science (80-), 2004,304(5676):1497-500.
[41]Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis[J]. Am J Clin Pathol,2006,126(4):608-15.
[42]Fukui T, Tsuta K, Furuta K, et al. Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lung[J]. Cancer Sci,2007,98(11):1714-9.
[43]Takano T, Fukui T, Ohe Y, et al. EGFR mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma:a historical comparison of patients treated before and after gefitinib approval in Japan[J]. J Clin Oncol,2008,26(34):5589-95.
[44]Fukui T, Ohe Y, Tsuta K, et al. Prospective study of the accuracy of EGFR mutational analysis by high-resolution melting analysis in small samples obtained from patients with non-small cell lung cancer[J]. Clin Cancer Res, 2008,14(15):4751-7.
[45]Ikenoue T, Hikiba Y, Kanai F, et al. Functional analysis of mutations within the kinase activation segment of B-Raf inhuman colorectal tumors[J]. Cancer Res,2003,63(23):8132-7.
[46]Willmore-Payne C, Volmar KE, Huening MA, et al. Molecular diagnostic testing as an adjunct to morphologic evaluation of pancreatic ductal system brushings:potential augmentation for diagnostic sensitivity[J]. Diagn Cytopathol,2007,35(4):218-24.
[47]Friedenson B. BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian[J]. MedGenMed,2005,7(2):60.
[48]der Stoep N v, van PCD, Janssens T, et al. Diagnostic guidelines for high-resolution melting curve (HRM) analysis:an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner[J]. Hum Mutat, 2009,30(6):899-909.
[49]Takano EA, Mitchell G, Fox SB, et al. Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis[J]. BMC Cancer,2008,8:59.
[50]Jimenez IJ, Esteban CE, Palanca SS, et al. Advantages of the high resolution melting in the detection of BRCA1 or BRCA2 mutation carriers[J]. Clin Biochem,2009,42(15):1572-6.
[51]Vorkas PA, Christopoulos K, Kroupis C, et al. Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis[J]. Clin Biochem,2009.
[52]De Leeneer K, Coene I, Poppe B, et al. Genotyping of frequent BRCA1/2 SNPs with unlabeled probes:a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden[J]. J Mol Diagn, 2009,11(5):415-9.
[53]Holden JA, Willmore-Payne C, Coppola D, et al. High-resolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors[J]. Am J Clin Pathol,2007,128(2):230-8.
[54]Er TK, Lin SF, Chang JG, et al. Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation[J]. Clin Chim Acta,2009,408(1-2):39-44.
[55]Do H, Solomon B, Mitchell PL, et al. Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting[J]. BMC Res Notes,2008,1:14.
[56]Rouleau E, Lefol C, Bourdon V, et al. Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements:application to MLH1 germline mutations in Lynch syndrome[J]. Hum Mutat,2009,30(6):867-75.
[57]Seth R, Keeley J, Abu-Ali G, et al. The putative tumour modifier gene ATP5A1 is not mutated in human colorectal cancer cell lines but expression levels correlate with TP53 mutations and chromosomal instability[J]. J Clin Pathol,2009,62(7):598-603.
[58]Nguyen-Dumont T, Calvez-Kelm FL, Forey N, et al. Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis[J]. Hum Mutat,2009,30(6):884-90.
[59]Sinthuwiwat T, Poowasanpetch P, Wongngamrungroj A, et al. High-resolution melting curve analysis for genotyping of common SNP in MTHFR gene using fixed-cell suspension [J]. Mol Cell Probes, 2008,22(5-6):329-32.
[60]Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations[J]. Cell Oncol,2009,31(3):161-7.
[61]Kristensen LS, Dobrovic A. Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free high-resolution melting analysis[J]. Cancer Epidemiol Biomarkers Prev,2008,17(5):1240-7.
[62]Margraf RL, Mao R, Highsmith WE, et al. RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis[J]. J Mol Diagn,2007,9(2):184-96.
[63]Seipp MT, Durtschi JD, Liew MA, et al. Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting[J]. J Mol Diagn,2007,9(3):284-9.
[64]Illingworth R, Kerr A, Desousa D, et al. A novel CpG island set identifies tissue-specific methylation at developmentalgene loci[J]. PLoS Biol, 2008,6(1):e22.
[65]Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation[J]. N Engl J Med,2003,349(21):2042-54.
[66]Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM):a new approach for sensitive and high-throughput assessment of methylation[J]. Nucleic Acids Res,2007,35(6):e41.
[67]Snell C, Krypuy M, Wong EM, et al. BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype[J]. Breast Cancer Res,2008,10(1):R12.
[68]Balic M, Pichler M, Strutz J, et al. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis [J]. J Mol Diagn, 2009,11(2):102-8.
[69]Li Z, Zhang W, Shao Y, et al. High-resolution melting analysis of ADAMTS18 methylation levels in gastric, colorectal and pancreatic cancers[J]. Med Oncol,2009.[70] Wittwer CT. High-resolution DNA melting analysis:advancements and limitations[J]. Hum Mutat, 2009,30(6):857-9.
[71]Taylor CF. Mutation scanning using high-resolution melting[J]. Biochem Soc Trans,2009,37(Pt 2):433-7.
[72]Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine[J]. Exp Mol Pathol,2008,85(1):50-8.
[73]Do H, Dobrovic A. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies[J]. Mol Cancer,2009,8:82.