Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer.

Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma- or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.     

大肠癌患者血清肿瘤标志物含量测定与临床研究

目的评价血清肿瘤标志物(CEA、CA199和CA242)单项或多项联合检测对大肠癌患者的临床诊断价值,探讨其在病理分期、淋巴结转移、侵袭程度及肿瘤大体形态等临床特征方面的意义。方法应用酶联免疫法检测134例大肠癌患者和200名健康人血清中CEA、CA199和CA242含量。结果大肠癌患者血清3种肿瘤标志物含量明显高于健康人(均P<0.01);单项检测中,CEA和CA242的阳性率无差异,但均显著高于CA199;CEA+CA242联合检测和CEA+CA242+CA199联合检测的阳性率显著高于单项和CEA+CA199的联合及CA199+CA242的联合,但特异性低于单项检测。在dukesA、B、C及D期中,3项肿瘤标志物含量及检测的阳性率依次增高,总体水平差异均有统计学意义(P<0.05～0.01);淋巴结转移患者的3项标志物含量及CA199、CA242的阳性率均高于无淋巴结转移的患者;3项标志物含量随肿瘤侵袭程度的加深显著增高,但在组织病理分类和肿瘤大体形态中均无明显的差异。结论CEA、CA199及CA242肿瘤标志物联合检测可以提高大肠癌诊断的敏感度,并对临床分期、判断淋巴结转移、肿瘤侵袭程度、进而进行有效临床治疗,具有一定的指导意义。     

Detection of mitochondrial DNA alterations in primary tumors and corresponding serum of colorectal cancer patients.

We previously examined colorectal cancer patients using mutation-specific mismatch ligation assay for genetic alterations in primary tumors and paired serum samples and proved that genetic alterations present in the tumors of cancer patients can be detected in the serum of those same patients. Recent evidence has proved that various cancers frequently have mutations in the D-loop region of mitochondrial DNA (mtDNA). Therefore, we thought that mutations in the mitochondrial genome might also become a genetic marker of colorectal cancer to detect tumor DNA in the serum of patients. We first sequenced the D-loop region of mtDNA in colorectal cancers. We then proceeded with a sensitive method, i.e., mismatch ligation assay to examine the possibility that mtDNA alterations can be found in the serum DNA. We analyzed the D-loop region of mtDNA in 77 primary colorectal cancers, 7 of which (9%) contained true somatic mutations in this region. We then examined whether mtDNA alterations can be found in the serum DNA using mismatch ligation assay. Of 7 alterations that were examined, 1 (14%) could be detected in the serum. This result suggested that the mtDNA alteration could also be used as a tumor marker to detect tumor DNA in the serum.     

DETECTION OF TUMOR MUTANT DNA IN PLASMA OF PATIENT WITH COLORECTAL CANCER

Colorectal cancer accounts for more than 90% of malignancies of the large bowel and is the second most common cause of cancer mortality in the adult American population[1]. Also the incidence of colorectal cancer increases by 4.2% per year in China[2]. Approximately 40% of affected individuals will ultimately die from the cancer. Early diagnosis and prompt treatment are mandatory to prevent this devastating disease. Thus, the search for markers which might provide reliable information for ear…     

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

PURPOSE: This study was designed to detect aberrant p16 promoter methylation in the serum of patients with colorectal cancer (CRC) and to explore the possibility of using this assay in early detection or as a prognostic marker of CRC patients. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and matching serum samples and control serum samples from 34 patients with adenomatous polyps and 10 healthy individuals. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was then analyzed. RESULTS: P16 hypermethylation was found in 20 of 52 (38%) CRCs. Among the 20 cases with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) cases. No methylated p16 sequences were detected in the peripheral serum of the other 32 CRC cases without these changes in the tumor, in 34 patients with adenomatous polyps, or in 10 healthy control subjects. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (P = 0.03). CONCLUSIONS: This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.     

用三种生物标志物对大肠癌进行的检测

 目的 寻找一种切实可行的检测肿瘤的生物标志物及其方法。方法 对100例结直肠癌患者取血清，另取献血员19例，非肿瘤患者13例为对照；并另取癌组织13份，癌旁组织11份作对比观察，同时用三种方法进行检测。一为对p16基因甲基化分析；二为对其进行缺失分析，三为对其进行点突变的分析。结果 患者血清异常甲基化为58 %；缺失为14 %；点突变为25 %；癌组织之结果与血清相似；而癌旁组织显然较低。SSCP阳性者经序列分析证实有3例为错义突变，1例为移位，1例为同义突变。其中4例造成p16蛋白的缺失。作为肿瘤的生物标志物，三项联合应用其灵敏度为75 %，特异性为96.87 %，准确率为80.30 %。比文献用CEA+CA19-9+CA72-4+CA242 四项指标联合之灵敏度、特异性、准确率皆高，尤其是特异性。如果仅用甲基化加突变亦与之相当。结论 血清DNA含量不高但能反映人体对肿瘤的负荷，实验对DNA的质和量的要求不是太高，能检出10-3的DNA片段，可用于临床诊断及大面积普查，并可用于对出院患者的长期随访。     

结直肠癌患者血清中CDKN2／p16基因异常的检测

 目的 寻找一种切实可行的检测肿瘤的生物标志物及其方法。方法 对100例结直肠癌患者取血清，另取献血员19例，非肿瘤患者13例为对照；并另取癌组织26份，癌旁组织22份及非恶性组织29份作对比观察，p16基因甲基化分析；并对其进行缺失分析及点突变的分析。结果 患者血清异常甲基化为69 %；缺失为9 %；点突变为45 %；癌组织之结果与血清相似；而癌旁组织显然较低。SSCP阳性者经序列分析证实有3例为错义突变，1例为移位，1例为同义突变。其中4例造成p16蛋白的缺失。作为肿瘤的生物标志物，3项联合应用其灵敏度为88 %，特异性为96.87 %，准确率为90.15 %。比文献用CEA+CA19-9+CA72-4+CA242 4项指标联合之灵敏度、特异性、准确率皆高。结论 血清DNA含量不高但能反映人体对肿瘤的负荷，本实验对DNA的质和量的要求不是太高，能检出10-3的DNA片段，可用于临床诊断及大面积普查，并可用于对出院患者的长期随访。并提示P16基因异常甲基化在结直肠癌癌变机制中起决定性作用。     

Detection of TFPI2 methylation in the serum of colorectal cancer patients

We examined whether TFPI2 methylation can be used as a molecular marker for colorectal cancers by detecting TFPI2 methylation in colorectal cancer patients’ sera by using quantitative methylation-specific polymerase chain reaction (qMSP). The qMSP analysis showed that 39 of 215 (18%) patients exhibited TFPI2 methylation in their serum DNA, suggesting that TFPI2 methylation frequently existed in colorectal cancer patients’ sera. After completion of qMSP analysis, clinicopathological data were correlated with molecular data. TFPI2 methylation was significant in the sera of patients with large (p = 0.0022), poorly differentiated carcinoma (p = 0.0164), deep invasion (p = 0.0002), lymph node metastasis (p = 0.0147), or distant metastasis (p < 0.0001). Moreover, TFPI2 methylation was observed more frequently according to the progression of TNM stage, suggesting that serum TFPI2 methylation could be detected more easily in patients with advanced colorectal cancer. We also examined whether serum TFPI2 methylation would be useful in the detection of colorectal cancer, compared to the conventional tumor markers. Detection rates of colorectal cancer using the tumor markers TFPI2 methylation, CEA and CA19-9, in the serum were 18%, 33%, and 17%, respectively. In cases where we combined all three markers, the detection rate was 42%. High sensitivity of qMSP enables detection of smaller amounts of serum tumor DNA. In principle, the methylation status of a primary tumor is not required in advance to detect circulating tumor DNA, suggesting the potential of qMSP as a cancer screening method.     

DNA tumor viruses and colorectal cancer

Colorectal cancers (CRCs) are characterized by forms of genomic or epigenetic instability that generate a large number of genetic variations, some of which provide the growth and survival advantages necessary for neoplastic growth. The mechanistic basis of these fundamental processes is currently unknown. Herein, we review evidence that links the human polyomavirus JC virus mechanistically with these processes in CRC. We propose a model in which JC virus is ubiquitously present in the gastrointestinal tracts of healthy people and speculate that through a process that remains to be discovered, the virus becomes activated, expresses a potent oncogene, and triggers chromosomal instability and the methylator phenotype resulting in CRC.     

肿瘤病人外周血DNA检测的意义

肿瘤病人血循环中DNA的量较一般人明显增多,而且从中可检测到肿瘤相关基因的特征性改变,可用于协助诊断,并作为预测预后及疗效观察的指标,具有重要的临床意义.     

乳腺癌患者外周血浆中游离的肿瘤相关DNA检测及其临床价值

目的探讨外周血浆中游离DNA变异在乳腺癌早期诊断、疗效评估和复发监控中应用的可行性。方法采用甲基化特异性PCR(MSP)方法,检测84例乳腺癌患者肿瘤组织、癌旁正常腺体组织及外周血浆中游离的肿瘤相关DNA钙黏素E(E-cadherin)和APC基因启动子甲基化畸变状况,选择10例乳腺良性疾病患者的血浆作为正常对照。结果乳腺癌肿瘤组织E-cadherin和APC基因启动子甲基化畸变频率分别为52.4%和45.2%,相应外周血浆中相同的DNA变异阳性检出率分别为33.3%和31.0%。外周血浆中DNA甲基化变异与肿瘤组织的甲基化畸变状况显著相关(E- cadherin,P<0.001;APC,P=0.002)。血浆DNA E-cadherin和APC基因甲基化畸变检测的灵敏度分别为63.6%和63.2%,特异度分别为100%和95.7%。肿瘤组织及外周血浆中游离DNA甲基化畸变与临床分期、病理类型、肿块大小及受体状况无相关性(P>0.05)。癌旁正常腺体组织及健康对照血浆中均未检测到E-cadherin和APC基因甲基化变异。结论在受检乳腺癌患者中,约1/3外周血浆中可检测到与原发肿瘤相同的E-cadherin和(或)APC基因甲基化畸变,与临床分期无相关性。在乳腺癌早期诊断、疗效评估和复发监控中,检测外周血浆中游离的肿瘤相关DNA变异有一定的可行性。     

Detection of APC and k-ras mutations in the serum of patients with colorectal cancer

Detection of tumor DNA in peripheral blood of patients with colorectal cancer (CRC) may allow early diagnosis of tumor disease and be of prognostic value. However, a reliable tumor marker detectable in the serum of patients with this disease is missing. Because k-ras and APC mutations occur frequently and at an early stage in CRCs, these mutations might also be detected in the serum of CRC patients and serve as tumor markers. Hence, tumor tissues of CRC patients were examined for the presence of mutations in the k-ras and APC genes. If a mutation was detected in the tumor, the serum of the patient was screened subsequently for the presence of this mutation. K-ray mutations were detected in 22 of 30 colorectal tumor tissues, but only in six patients was the mutation identified in their serum samples. Mutations of the APC gene were identified in 25 of 65 tumors: 20 of these 25 patients showed the respective mutation in their serum. Given their higher detection rate, APC mutations could be a more informative serum marker than k-ras in CRC patients.     

Circulating tumor cells in colorectal cancer patients

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.     

改进MSP法对肿瘤患者血清DNA的探测

Objective To search for circulating DNA as a biomarker for the cancer burden of patients. Methods Methylation specific PCR (MSP), according to Herman et al, was used to identify this epigenetic modification of DNA from serum and tissue of 111 patients with different cancers. In parallel the mutant K-ras gene was analyzed in 55 cases for comparison. Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference to healthy controls (P<0.05). There was no difference between the DNA from the tumor and from the neighboring normal tissue or from serum in early cancer. The positivity rate increased with progression of the tumor. 20 days after the operation methylated DNA nearly disappeared. The positivity rate for mutant K-ras was only 30% and thus much lower than that of methylated DNA. The combination of the two markers increased the positivity rate of tumors by 13%. Conclusion The detection of methylation of the p16 gene by MSP combined with mutant K-ras in serum of cancer patients would be a very effective method to estimate the cancer burden.     

Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results:KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.     

Methylation of serum DNA is an independent prognostic marker in colorectal cancer.

PURPOSE: Aberrant CpG island hypermethylation is a feature of a subgroup of colorectal cancers, which can be detected in the serum of affected patients. This study was designed to identify methylation targets with prognostic significance in the serum of patients with colorectal cancer. EXPERIMENTAL DESIGN: In a gene evaluation set consisting of sera from 24 patients with local colorectal cancers, 14 with metastasized disease, and 20 healthy controls, the genes HPP1/TPEF, HLTF, and hMLH1 were identified as potential serum DNA methylation markers. These genes were further analyzed in a test set of sera of 104 patients with colorectal cancer. RESULTS: Methylation of HLTF, HPP1/TPEF, and hMLH1 was found to be significantly correlated with tumor size, and methylation of HLTF and HPP1/TPEF was significantly associated with metastatic disease and tumor stage. Moreover, methylation of HPP1/TPEF was also associated with serum carcinoembryonic antigen. The prognostic relevance of methylation of these genes was tested in pretherapeutic sera of 77 patients with known follow-up. Patients with methylation of HPP1/TPEF or HLTF were found to have unfavorable prognosis (P = 0.001 and 0.008). In contrast, serum methylation of hMLH1 was not associated with a higher risk of death. Multivariate analysis showed methylated HPP1 and/or HLTF serum DNA to be independently associated with poor outcome and a relative risk of death of 3.4 (95% confidence interval, 1.4-8.1; P = 0.007). CONCLUSIONS: These data show that the methylation status of specific genes in the serum of patients with colorectal cancer has the potential to become a pretherapeutic predictor of outcome.     

Detection of DNA hypermethylation in remote media of patients with colorectal cancer: new biomarkers for colorectal carcinoma

Colorectal cancer is the third most common cancer and a major cause of cancer-related mortality. The lifetime risk to develop colorectal cancer is 6% in the Western world, and one third of the affected people will ultimately die from this disease. Colorectal carcinomas develop slowly over a period of several years. Therefore, identifying people with precancerous lesions holds the potential to reduce the incidence and mortality of colorectal cancer. Apart from mutations and chromosomal imbalances, inactivation of tumor suppressor genes by DNA hypermethylation has been recognized as an important mechanism driving colorectal carcinogenesis. In recent years, screening tests have been developed that rely on the detection of DNA hypermethylation in remote media to identify people suffering from asymptomatic colorectal cancers at early stages. Apart from their diagnostic value, methylation markers hold the potential to be used as prognostic markers. Here, we summarize the recent development of DNA methylation markers for colorectal cancer in remote media with regard to their diagnostic and prognostic value.     

结直肠癌患者血清外泌体代替肿瘤组织检测K-Ras基因突变的研究*

目的 探讨结直肠癌患者血清外泌体代替肿瘤组织进行DNA中K-Ras基因12密码子突变检测的价值。方法 收集2014年5月至2015年9月于我院病理组织学诊断为局部晚期或转移性结直肠癌患者的外周血标本90例,使用ExoQuick试剂提取血清外泌体,盐析法提取外泌体及血细胞DNA,采用PCR方法扩增K-Ras基因12密码子并通过高通量测序法检测突变,比较血清外泌体DNA与血细胞DNA突变的特异性,比较外泌体与组织学检测的K-Ras基因12密码子突变情况。结果 90例结直肠癌患者中检测出外泌体K-Ras基因12密码子突变43例,突变率为47.8％。全组共检出4种突变类型,以G12D突变率为最高。与组织学检测比较,灵敏度为90.6％,一致性为81.1％（Kappa值=0.62,P＜0.05）。结论 血清外泌体DNA用于检测肿瘤相关突变,与肿瘤组织相比一致性较高,可作为液体活检的来源指导肿瘤的个体化治疗。