结直肠癌循环肿瘤细胞及DNA的研究进展

结直肠癌是最常见的恶性肿瘤之一,尽管联合应用手术、化疗、放疗可提高患者的生存率,但仍有大量患者因术后转移和复发死亡。循环肿瘤细胞及循环肿瘤DNA对结直肠癌的早期诊断、疗效监测、预后评估以及个体化治疗方案制定均有重要意义,已成为近年来研究的热点。本文对近年来结直肠癌循环肿瘤细胞及循环肿瘤DNA的检测技术及其在早期诊断、疗效监测和预后判断中的应用进展作一综述。     

与晚期结直肠癌EGFR单克隆抗体靶向药物选择相关的分子标志物

对于转移性结直肠癌而言,如何在标准化疗的基础上进行个体化靶向治疗是目前关注的热点。KRAS基因突变被认为是转移性结直肠癌对抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单克隆抗体(anti-EGFR monoclonal antibody,anti-EGFR)靶向治疗反应不佳的独立预测因素。35%～45%的转移性结直肠癌患者存在KRAS基因的突变,且对anti-EGFR治疗抵抗;同时只有50%的野生型KRAS患者对anti-EGFR治疗有效,提示EGFR下游信号通路其他分子的激活和变异可能影响了其治疗反应。EGFR依赖的2条主要信号通路RAS-RAF-MAPK和PI3K-PTEN-AKT均与anti-EGFR治疗失败有关。EGFR基因拷贝数(gene copy number,GCN)增加与结肠癌anti-EGFR的疗效相关,但EGFR GCN增加并不意味着EGFR蛋白表达增加,且EGFR表达与anti-EGFR疗效无相关性。在野生型KRAS转移性结直肠癌患者中增加对BRAF和PIK3CA基因突变以及PTEN基因表达缺失的检测可能有助于筛选anti-EGFR抵抗患者。本研究对近年来转移性结直肠癌研究中所涉及的疗效预测和预后分子标志物进行综述,以进一步指导转移性结直肠癌的个体化分子靶向治疗。     

CTC、ctDNA在结直肠癌诊断中的研究进展

结直肠癌（colorectal cancer,CRC）是世界上第三大最常见的癌症类型,也是导致癌症死亡的第四大原因。CRC的常规治疗策略包括手术、新辅助治疗和辅助治疗。不幸的是,大约50％的CRC患者仅在晚期被诊断,因此显著降低了不同治疗方案的可用性。学者们不断寻找更准确的诊断结直肠癌的方法,除了通过蛋白质组学技术研究新的血清肿瘤生物标记物外,还引入循环肿瘤细胞（CTC）和循环肿瘤DNA（ctDNA）等新概念。本文就血液中CTC、ctDNA在结直肠癌诊断中的研究进展作一综述。     

Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment

BackgroundWhile tumor-tissue remains the ‘gold standard’ for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples. Here, we broaden our previous study on the clinical validation of plasma DNA in metastatic colorectal cancer patients, by evaluating its clinical utility under standard management care.Patients and methodsConcordance and data turnaround-time of ctDNA when compared with tumor-tissue analysis were studied in a real-time blinded prospective multicenter clinical study (n = 140 metastatic colorectal patients). Results are presented according to STARD criteria and were discussed in regard with clinical outcomes of patients.ResultsMuch more mutations were found by ctDNA analysis: 59%, 11.8% and 14.4% of the patients were found KRAS, NRAS and BRAF mutant by ctDNA analysis instead of 44%, 8.8% and 7.2% by tumor-tissue analysis. Median tumor-tissue data turnaround-time was 16 days while 2 days for ctDNA analysis. Discordant samples analysis revealed that use of biopsy, long delay between tumor-tissue and blood collection and resection of the tumor at time of blood draw, tumor site, or type of tissue analyzed seem to affect concordance. Altogether, the clinical data with respect to the anti-epidermal growth factor receptor response (RAS status) and the prognosis (BRAF status) of those discordant patients do not appear contradictory to the mutational status as determined by plasma analysis. Lastly, we present the first distribution profile of the RAS and BRAF hotspot mutations as determined by ctDNA analysis (n = 119), revealing a high proportion of patients with multiple mutations (45% of the population and up to 5 mutations) and only 24% of WT scored patients for both genes. Mutation profile as determined from ctDNA analysis with using various detection thresholds highlights the importance of the test sensitivity.ConclusionOur study showed that ctDNA could replace tumor-tissue analysis, and also clinical utility of ctDNA analysis by considerably reducing data turnaround time.     

Clinical utility of circulating tumor DNA for colorectal cancer

Colorectal cancer (CRC) is currently the most common type of cancer in Japan, and its prognosis has improved because of development of diagnosis and advancement in treatments including surgery and chemotherapy. However, because of intratumor heterogeneity and clonal evolution, tumors often develop resistance to treatment. Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine examination in clinical practice. However, the inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens only. Circulating tumor DNA (ctDNA) carrying tumor‐specific genetic or epigenetic alterations is released into the circulation from tumor cells undergoing apoptosis or necrosis. Analysis of ctDNA has the potential to change clinical practice by exploiting blood rather than tissue, as a source of information. Here, we provide an overview of the characteristics of ctDNA and focus on detection methods for ctDNA, and the feasibility of use of ctDNA to monitor tumor dynamics for patients with colorectal cancer.     

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.     

RAS mutation analysis in circulating tumor DNA from patients with metastatic colorectal cancer: the AGEO RASANC prospective multicenter study

BackgroundRAS mutations are currently sought for in tumor samples, which takes a median of almost 3 weeks in western European countries. This creates problems in clinical situations that require urgent treatment and for inclusion in therapeutic trials that need RAS status for randomization. Analysis of circulating tumor DNA might help to shorten the time required to determine RAS mutational status before anti-epidermal growth factor receptor antibody therapy for metastatic colorectal cancer. Here we compared plasma with tissue RAS analysis in a large prospective multicenter cohort.Patients and methodsPlasma samples were collected prospectively from chemotherapy-naive patients and analyzed centrally by next-generation sequencing (NGS) with the colon lung cancer V2 Ampliseq panel and by methylation digital PCR (WIF1 and NPY genes). Tumoral RAS status was determined locally, in parallel, according to routine practice. For a minimal κ coefficient of 0.7, reflecting acceptable concordance (precision ± 0.07), with an estimated 5% of non-exploitable data, 425 subjects were necessary.ResultsFrom July 2015 to December 2016, 425 patients were enrolled. For the 412 patients with available paired plasma and tumor samples, the κ coefficient was 0.71 [95% confidence interval (CI), 0.64–0.77] and accuracy was 85.2% (95% CI, 81.4% to 88.5%). In the 329 patients with detectable ctDNA (at least one mutation or one methylated biomarker), the κ coefficient was 0.89 (95% CI, 0.84–0.94) and accuracy was 94.8% (95% CI, 91.9% to 97.0%). The absence of liver metastases was the main clinical factor associated with inconclusive circulating tumor DNA results [odds ratio = 0.11 (95% CI, 0.06–0.21)]. In patients with liver metastases, accuracy was 93.5% with NGS alone and 97% with NGS plus the methylated biomarkers.ConclusionThis prospective trial demonstrates excellent concordance between RAS status in plasma and tumor tissue from patients with colorectal cancer and liver metastases, thus validating plasma testing for routine RAS mutation analysis in these patients.Clinical Trial registrationClinicaltrials.gov, NCT02502656.     

Perspectives for circulating tumor DNA in clinical management of colorectal cancer

Growing evidence has demonstrated that circulating tumor DNA (ctDNA) detection in colorectal cancer might be a promising approach to address current important clinical questions. During chemotherapy for metastatic colorectal cancer, tumor cells acquire potential resistance by generating additional somatic mutations related to therapeutic resistance. ctDNA can capture the tumor landscape, including heterogeneity, which might provide the opportunity for additional treatment options. Moreover, ctDNA detection is advantageous, because it can monitor tumor heterogeneity serially, in a non-invasive manner. ctDNA is considered valid for detecting minimal residual disease after a curable resection. By utilizing ctDNA detection, adjuvant chemotherapy for patients with stage II–III colorectal cancer might be omitted for patients at low risk of recurrence; or conversely, adjuvant chemotherapy might be highly recommended for patients at high risk, based on ctDNA findings. During multidisciplinary treatments for locally advanced rectal cancer, it is essential to monitor the responses to sequential treatments to make appropriate decisions. Currently, these decisions are mainly based on radiological or pathological findings. ctDNA can add value by providing the real-time status of locally advanced rectal cancer. In this review, we summarized the current evidence and discussed future strategies for using ctDNA in the treatment of colorectal cancer.

     

Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer

Background: We demonstrated that circulating tumor cell (CTC) number at baseline and follow-up is an independent prognostic factor in metastatic colorectal cancer (mCRC). This analysis was undertaken to explore whether patient and treatment characteristics impact the prognostic value of CTCs.Patients and methods: CTCs were enumerated with immunomagnetic separation from the blood of 430 patients with mCRC at baseline and on therapy. Patients were stratified into unfavorable and favorable prognostic groups based on CTC levels of ≥3 or <3 CTCs/7.5 ml, respectively. Subgroups were analyzed by line of treatment, liver involvement, receipt of oxaliplatin, irinotecan, or bevacizumab, age, and Eastern Cooperative Oncology Group performance status (ECOG PS).Results: Seventy-one percent of deaths have occurred. Median follow-up for living patients is 25.8 months. For all patients, progression-free survival (PFS) and overall survival (OS) for unfavorable compared with favorable baseline CTCs is shorter (4.4 versus 7.8 m, P = 0.004 for PFS; 9.4 versus 20.6 m, P < 0.0001 for OS). In all patient subgroups, unfavorable baseline CTC was associated with inferior OS (P < 0.001). In patients receiving first- or second-line therapy (P = 0.003), irinotecan (P = 0.0001), having liver involvement (P = 0.002), ≥65 years (P = 0.0007), and ECOG PS of zero (P = 0.04), unfavorable baseline CTC was associated with inferior PFS.Conclusion: Baseline CTC count is an important prognostic factor within specific subgroups defined by treatment or patient characteristics.     

循环肿瘤DNA检测在结直肠癌临床诊疗中的研究进展

结直肠癌是我国第三大常见癌症，现有的结直肠癌早期筛查和术后监测手段均存在局限性。循环肿瘤（DNAcirculating tumor DNA，ctDNA）通过肿瘤细胞凋亡、坏死或分泌等途径释放到外周血，可反映肿瘤组织相关变异信息。随着二代测序（next generation sequencing，NGS）技术快速发展，血浆中微量ctDNA的精准检测为结直肠癌的早期筛查提供机遇。同时，纵向监测结直肠癌患者的ctDNA可以较好地反映肿瘤负荷的动态变化。本文拟从ctDNA检测在结直肠癌早期筛查、个性化用药指导、疗效监测以及预后评估等方面的潜在临床价值进行综述。     

循环肿瘤细胞监测在结直肠癌肝转移微波消融治疗中临床价值的评估

目的 探讨利用循环肿瘤细胞（CTCs）评估结直肠癌肝转移微波消融治疗的疗效及其与预后的关系。方法 收集2014年1月至2018年5月在南京市第二医院因结直肠癌肝转移行肝脏病灶微波消融治疗的29例患者的临床资料,并纳入术前与术后CTCs检测结果。计数资料分析采用χ2检验或Fisher确切概率法;生存分析采用Kaplan-Meier法和Log-rank检验。结果 经影像学评估,29例患者局部病灶微波消融的近期疗效为完全消融者8例、部分消融16例和进展5例。29例患者微波消融术后CTCs测量值较术前升高,差异有统计学意义（Z=-2.489,P=0.013）;患者的年龄、性别、术后CTCs升高与否、结直肠癌原发部位、同时性或异时性肝转移与微波消融近期疗效均无关（P＞0.05）。微波消融术后CTCs数目＜7个／ml患者局部肝转移病灶的近期疗效优于术后CTCs数目≥7个／ml者（P=0.031）。29例患者的中位生存时间（OS）为30.0个月（95％CI：10.7～49.3个月）。入组患者的年龄、结直肠癌原发部位及肝转移后的治疗方式与中位OS相关（P＜0.05）,而性别、同时性或异时性肝转移、术后CTCs升高与否及术后CTCs值／ml与中位OS均无关（P＞0.05）。结论 微波消融是结直肠癌肝转移局部治疗的有效手段,治疗前后CTCs数量的监测可能有助于评估其近期疗效。     

Molecular predictive factors of response to anti-EGFR antibodies in colorectal cancer patients

Monoclonal antibodies against EGFR represent one of the most important recent advancements in the treatment of colorectal cancer. Anyway, to date, only a subset of patients can really take advantage from this kind of drugs. The identification of predictive factors that are able to stratify patients who potentially can benefit by these biological treatment, is an important aim of anticancer research both in clinical and in pre-clinical fields. Many studies evaluating classical IHC analysis of protein expression failed in this purpose. So in the last years there was the need to look forward to a new class of molecular predictive factors. Indeed a number of biomarkers have been evaluated in their potential to predict the response to anti-EGFR-based therapies. These include marker related to EGFR amplification, activation and phosphorylation, EGFR polymorphisms, but also markers related to Ras/Raf/MAPK and the PI3K/Akt signalling pathways and angiogenesis. This review will focus the attention to these new genetic and molecular markers.     

DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps

Colorectal cancer (CRC) screening rates are currently suboptimal. Blood-based screening could improve rates of earlier detection for CRC and adenomatous colorectal polyps. In this study, we evaluated the feasibility of plasma-based detection of early CRC and adenomatous polyps using array-mediated analysis methylation profiling of 56 genes implicated in carcinogenesis. Methylation of 56 genes in patients with Stages I and II CRC (N=30) and those with adenomatous polyps (N=30) were compared with individuals who underwent colonoscopy and were found to have neither adenomatous changes nor CRC. Composite biomarkers were developed for adenomatous polyps and CRC, and their sensitivity and specificity was estimated using five-fold cross validation. Six promoters (CYCD2, HIC1, PAX 5, RASSF1A, RB1 and SRBC) were selected for the biomarker, which differentiated CRC patients and controls with 84% sensitivity and 68% specificity. Three promoters (HIC1, MDG1 and RASSF1A) were selected for the biomarker, which differentiated patients with adenomatous polyps and controls with sensitivity of 55% and specificity of 65%. Methylation profiling of plasma DNA can detect early CRC with significant accuracy and shows promise as a methodology to develop biomarkers for CRC screening.     

循环DNA在大肠癌中的应用

循环DNA是一种存在于血浆、脑脊液及滑液等体液中的细胞外DNA.肿瘤患者循环DNA的含量明显高于健康人群,并具有与肿瘤发生、发展相关的特征性改变,例如基因突变、DNA甲基化、微卫星不稳定性等.循环DNA的定量与定性检测,在肿瘤的筛查、诊断、病情的监测以及预后评价中均有潜在的价值.     

结直肠癌患者循环肿瘤细胞与临床病理特征的关系

目的:探讨外周血循环肿瘤细胞（circulating tumor cells,CTCs）在结直肠癌患者检测中的临床意义。方法:收集陕西省人民医院结直肠癌患者外周血标本57例,良性病变和健康受试者外周血标本29例。应用CyttelTM-CTC平台检测技术（即阴性富集技术结合免疫荧光原位杂交法）检测受试者外周血CTCs,搜集结直肠癌患者临床病理特征,采用统计学方法分析与其外周血CTCs之间的关系。结果:在Cut-off值≥2的结直肠癌患者中CTCs检出率为78.9%（45/57）,远高于良性病变及健康受试者中CTCs检出率的6.9%（2/29）（P＜0.05）。在Cut-off值≥3的结直肠癌患者中,发生淋巴转移的患者CTCs检出率为62.1%（18/29）,高于未发生淋巴转移患者CTCs检出率的32.1%（9/28）（P=0.024）。在Cut-off值≥4的结直肠癌患者中,周围组织侵犯患者CTCs检出率为53.8%（7/13）,高于周围组织未侵犯患者CTCs检出率的25.0%（11/44）（P=0.049）。在不同的病理分期中,与癌胚抗原（CEA）检测比较,CTCs检出率均高于CEA检出率。结论:CTC平台检测技术可有效检测结直肠癌患者外周血CTCs,CTCs计数分布与结直肠癌患者的病理分期显著相关,该技术敏感性高,准确性好,对于结直肠癌患者的临床诊断具有重要指导意义。     

Markers of resistance to anti-EGFR therapy in colorectal cancer

Epidermal growth factor receptor (EGFR) is a therapeutic target in colorectal cancer (CRC). The benefit from EGFR inhibitors appears to be limited to a subset of patients with CRC. Mechanisms of resistance to EGFR inhibitors are being identified. KRAS codon 12 activating mutation is a predominate mechanism of resistance to EGFR inhibitors in around 40% of patients with advanced CRC. Other potential mechanisms of resistance include ligand expression, increased EGFR number, mutations of BRAF and activation of alternate signaling pathways.Key Words: Resistance, anti-epidermal growth factor receptor (anti-EGFR), colorectal cancer     

Cell-free circulating tumor DNA in cancer

Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason under-lying difculties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive“liquid biopsy”from the blood has been attempted to characterize tumor heterogeneity. This review focuses on cell-free circulating tumor DNA (ctDNA) in the bloodstream as a versatile biomarker. ctDNA analysis is an evolving field with many new methods being developed and optimized to be able to successfully extract and analyze ctDNA, which has vast clinical applications. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. In addition, ctDNA has the potential to accurately monitor tumor burden and treatment response, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse. There are still many challenges that need to be overcome prior to this biomarker getting wide adoption in the clinical world, including optimization, standardization, and large multicenter trials.     

循环肿瘤细胞评估转移性结直肠癌微波消融治疗的初步研究

目的:利用循环肿瘤细胞（circulating tumor cells,CTCs）评估转移性结直肠癌微波消融治疗及其预后的相关性。方法:研究分析了2015年1月至2018年4月在东南大学附属第二医院因转移性结直肠癌行肺部或肝脏病灶微波消融治疗的34例患者的临床资料。术前及术后均进行CTCs检测,并搜集患者临床特征。应用统计方法进一步分析研究。结果:微波消融术后,患者的CTCs测量值较术前升高,差异具有统计学意义(P=0.003)。对性别、年龄、微波治疗部位（同转移部位）、微波术前后CTCs变化状况、微波术后CTCs数目与随访截止时期患者生存状态进行分析后得出:女性患者在随访截止时期的生存状态优于男性,差异具有统计学意义（P=0.011）;微波术后CTCs数目＜7个/ml的患者生存状态优于术后CTCs数目≥7个/ml者,差异具有统计学意义（P=0.030）。而年龄（P=0.274）、微波治疗部位（同转移部位）（P=0.271）、微波术前后CTCs变化状况（P＝0.214）与随访截止时期患者的生存状态之间无统计学差异。结论:转移性结直肠癌的患者CTCs数目在微波消融术后会增加;女性患者在随访截止时期的生存状态优于男性;微波术后CTCs数目＜7个/ml的患者生存状态优于术后CTCs数目≥7个/ml者;年龄、微波治疗部位（同转移部位）、微波术前后CTCs变化状况与随访截止时期患者疾病状态不相关。     

Characterizing DNA methylation patterns in pancreatic cancer genome

We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.