Parallel Evaluation of Circulating Tumor DNA and Circulating Tumor Cells in Metastatic Colorectal Cancer

Background

Tissue biopsy is the gold standard for tumor genotyping, but it is an invasive procedure providing a single snapshot into tumor heterogeneity. Liquid biopsy approaches, encompassing the analysis of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), have been proposed as an alternative, with the potential of providing a comprehensive portrait of the tumor molecular landscape. In metastatic colorectal cancer (mCRC), both CTCs and ctDNA analysis have been investigated, but comparative analyses are limited.

A headlight on liquid biopsies: a challenging tool for breast cancer management

Breast cancer is the most frequent carcinoma and second most common cause of cancer-related mortality in postmenopausal women. The acquisition of somatic mutations represents the main mechanism through which cancer cells overcome physiological cellular signaling pathways (e.g., PI3K/Akt/mTOR, PTEN, TP53). To date, diagnosis and metastasis monitoring is mainly carried out through tissue biopsy and/or re-biopsy, a very invasive procedure limited only to certain locations and not always feasible in clinical practice. In order to improve disease monitoring over time and to avoid painful procedure such as tissue biopsy, liquid biopsy may represent a new precious tool. Indeed, it represents a basin of “new generation” biomarkers that are spread into the bloodstream from both primary and metastatic sites. Moreover, elevated concentrations of circulating tumor DNA (ctDNA) as well as circulating tumor cells (CTCs) have been found in blood plasma of patients with various tumor types. Nowadays, several new approaches have been introduced for the detection and characterization of CTCs and ctDNA, allowing a real-time monitoring of tumor evolution. This review is focused on the clinical relevance of liquid biopsy in breast cancer and will provide an update concerning CTCs and ctDNA utility as a tool for breast cancer patient monitoring during the course of disease.     

循环肿瘤细胞在早期肺癌诊疗中的研究进展

循环肿瘤细胞(circulating tumor cells, CTCs)作为液体活检的一种重要类型,在肺癌的筛查诊断、疗效评估、术后监测与预后判断等方面显示出越来越丰富的临床价值.随着对肺癌高危人群筛查工作的进展,大量肺小结节患者被检出,但是肺小结节不等于肺癌,而且据统计良性比例达90%-95%,这使得该部分患者在首次就诊时的良恶性鉴别诊断成为临床医生面临着的新的机遇与挑战.CTCs检测技术的不断进步与完善,是否可以在早期肺癌的鉴别诊断中发挥更大的作用,此外,它是否对早期肺癌手术治疗时的操作具有指导意义,这还需要进一步科研探索,以期将来实现临床转化.     

Circulating tumor DNA/circulating tumor cells and the applicability in different causes induced hepatocellular carcinoma

In 2015, liquid biopsy was rated one of the top 10 breakthrough technologies of the year by MIT Technology Review. Liquid biopsy is a type of in vitro diagnostic method involving a noninvasive blood test. It is also a breakthrough technology used to detect tumors and cancers and assist in therapeutic strategies. The most widely used markers are circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Primary carcinoma of the liver is a malignancy of hepatocytes or intrahepatic biliary epithelial cells. The most common type of liver cancer is hepatocellular carcinoma (HCC), the causes of which mainly include infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV), alcohol abuse, aflatoxicosis, and nonalcoholic fatty liver disease/ nonalcoholic steatohepatitis. As there are few typical clinical characteristics during the early stage of the disease, early diagnosis of HCC is very challenging. However, CTCs and ctDNA carry tumor-specific information. Therefore, the detection and analysis of CTCs and ctDNA can provide evidence for the early diagnosis of HCC and guide treatment. Furthermore, several studies have indicated that different inducers of HCC cause different DNA mutations, and accordingly, detection of specific mutations in ctDNA will facilitate the determination of the HCC type and help physicians provide distinctive therapies.     

The Clinical Potential of Circulating Tumor Cells and Circulating Tumor-Associated Cellular Elements in Colorectal Cancer

Colorectal cancer (CRC) remains one of the commonest types of cancer in the USA. Effective treatment and potential curative resection is dependent on early detection, prior to the development of distant metastases. Radiologic and endoscopic evaluations are operator-dependent and limited by macroscopic detection when the relative cellular tumor burden is already high. Also, use of serum biomarkers such as carcinoembryonic antigen may not correlate with the extent of disease involvement. Circulating tumor cells (CTCs) continue be a prominent area of investigation, despite having been described first by Ashworth in 1869. As methods for detection expand and improve, CRC is increasingly characterized by the properties of CTCs and tumor-associated cellular elements (TACEs), such as circulating tumor DNA, cell-free DNA, and microRNA. This review serves to highlight the clinical implications of CRC CTCs and TACEs for prognostication, monitoring for disease recurrence, and response to therapy.     

Genotype-Specific Differences in Circulating Tumor DNA Levels in Advanced NSCLC

IntroductionPlasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease.MethodsWe retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients.ResultsThere was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding.ConclusionsLevels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.     

Liquid biopsy in central nervous system tumors: the potential roles of circulating miRNA and exosomes

The Central nervous system (CNS) tumor still remains the most lethal cancer, and It is hard to diagnose at an earlier stage on most occasions. It is found that recurrent disease is finally observed in patients who occurred chemo-resistance after completely primary treatment. It is a challenge that monitoring treatment efficacy and tumor recurrence of CNS tumors are full of risks and difficulties by brain biopsies. However, the brain biopsies are considered as an invasive technique with low specificity and low sensitivity. In contrast, the liquid biopsy is based on blood and cerebrospinal fluid (CSF) test, which is going to acceptable among the patients through it’s minimally invasive and serial bodily fluids. The advantages of liquid biopsy are to follow the development of tumors, provide new insights in real time, and accurate medical care. The major analytical constituents of liquid biopsy contain the Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free microRNAs (cfmiRNAs), and circulating exosomes. Liquid biopsy has been widely utilized in CNS tumors in recent years, and the CTCs and ctDNA have become the hot topics for researching. In this review, we are going to explain the clinical potential of liquid biopsy biomarkers in CNS tumor by testing circulating miRNAs and exosomes to evaluate diagnose, prognosis, and response to treatment.     

肺癌循环肿瘤细胞的研究进展

转移和复发是肺癌患者死亡的主要原因。研究发现循环肿瘤细胞(circula tingtumo rcells,CTCs)在肺癌转移和复发中起着重要作用。而且随着靶向治疗的不断进步,对于晚期无法取得肺癌实体组织的患者,CTCs作为一种肺癌组织替代物可以决定治疗方案。所以CTCs在早期发现肺癌患者的微转移、检测肿瘤复发、评估预后和选择个体化治疗方案方面有着重要作用。本文针对CTCs的研究进展及肺癌领域的应用进行综述。     

循环肿瘤细胞检测技术的研究进展

循环肿瘤细胞（circulating tumor cells,CTCs）是导致发生肿瘤远处转移和复发的必要前提。随着靶向治疗的不断进步,对于无法取得肿瘤组织的晚期癌症患者,CTCs可作为一种肿瘤组织替代物指导患者治疗方案的选择。精确计数以及分子生物学分析对于肿瘤患者的预后判断、疗效评价以及个体化治疗均有重要的指导作用。随着CTCs检测技术不断发展,CTCs检测的准确性明显提高。本文针对CTCs的检测技术进行综述。      

Circulating Tumor Cells and Colorectal Cancer

The significance of circulating tumor cells (CTCs) has been discussed for more than a century. The advent of modern technology has allowed for more reliable detection of CTCs, and recent studies have provided compelling evidence that CTCs predict clinical response in metastatic colorectal cancer (mCRC). Combination of CTC analysis with independent prognostic factors has demonstrated powerful synergy in some studies. The ability of CTCs to predict metastasis and therapy-specific response has high potential clinical utility, with early studies showing promising results in colorectal cancer (CRC). Reliable CTC detection has also allowed for examination of tumor cell dissemination during surgery, and there appears to be a heavy dependence on the approach chosen. This review discusses the evidence for CTC significance, with particular focus on detection methods, novel markers, and clinical outcomes in CRC. Numerous opportunities exist for preclinical, clinical, and translational studies to explore molecular determinants within CTCs, as well as the value of CTCs in directing targeted therapeutics.     

肺癌循环肿瘤细胞的研究进展

转移是导致肺癌患者死亡的主要因素,积极防治转移的发生是提高患者疗效的关键。循环肿瘤细胞（CTCs）是启动远处转移的重要环节,自原发灶脱落开始即触发一系列转移级联反应,最终在远处靶器官定植和增殖。近年来,肺癌CTCs的检测技术手段不断优化,不仅可以对CTCs进行计数,还可鉴定不同亚群。肺癌CTCs检测可用于早期筛查、手术及放化疗后的预后预测、免疫靶向治疗的疗效评价等方面,动态监测肿瘤异质性,有助于调整个体化治疗方案,达到精准治癌的目的。田建辉研究组建立世界首例人肺腺癌循环肿瘤细胞系,将其融入肺癌亚临床核心病机“正虚伏毒”学说的探索,并积极促进肺癌特异性研究平台的构建,以期提高肺癌转移的研究和防控效率。     

Circulating Tumor BRAF Mutation and Personalized Thyroid Cancer Treatment

Circulating tumor DNA (ctDNA) is now being extensively studied as it is a noninvasive “real-time” biomarker that can provide diagnostic and prognostic information before, during treatment and at progression. These include DNA mutations, epigenetic alterations and other forms of tumor-specific abnormalities such as microsatellite instability (MSI) and loss of heterozygosity (LOH). ctDNA is of great value in the process of cancer treatment. However, up to date, there is no strict standard considering the exact biomarker because the development and progression of cancer is extremely complicated. Also, results of the studies evaluating ctDNA are not consistent due to the different detection methods and processing. The major challenge is still the exact position of ctDNA in cancer management and the exact place of it versus tissue biopsy. actually it is still under the debate that circulating tumor markers will take the place of standard tissue biopsy or will support it to guide us to the more effective interventions?     

Mutated circulating tumor DNA as a liquid biopsy in lung cancer detection and treatment

Over the past decade, substantial developments have been made in the detection of circulating tumor DNA (ctDNA)—cell‐free DNA (cfDNA) fragments released into the circulation from tumor cells and displaying the genetic alterations of those cells. As such, ctDNA detected in liquid biopsies serves as a powerful tool for cancer patient stratification, therapy guidance, detection of resistance, and relapse monitoring. In this Review, we describe lung cancer diagnosis and monitoring strategies using ctDNA detection technologies and compile recent evidence regarding lung cancer‐related mutation detection in liquid biopsy. We focus not only on epidermal growth factor receptor (EGFR) alterations, but also on significant co‐mutations that shed more light on novel ctDNA‐based liquid biopsy applications. Finally, we discuss future perspectives of early‐cancer detection and clonal hematopoiesis filtering strategies, with possible inclusion of microbiome‐driven liquid biopsy.     

The feasibility of using mutation detection in ctDNA to assess tumor dynamics

For many decades it has been known that tumor DNA is shed into the blood. As a consequence of technological limitations, researchers were unable to comprehensively characterize circulating DNA. The advent of ultrasensitive and highly specific molecular assays has provided a comprehensive profile of the molecular characteristics and dynamics of circulating DNA in healthy subjects and cancer patients. With these new tools in hand, significant interest has been provoked for an innovative type of tumor biopsy termed a “liquid biopsy”. Liquid biopsies are obtained by minimal invasive blood draws from cancer patients. Circulating cancer cells, exosomes and a variety of molecules contained within the liquid biopsy including cell‐free circulating tumor DNA (ctDNA) can serve as promising tools to track cancer evolution. Attractive features of ctDNA are that ctDNA isolation is straightforward, ctDNA levels increase or decrease in response to the degree of tumor burden and ctDNA contains DNA mutations found in both primary and metastatic lesions. Consequently, the analysis of circulating DNA for cancer‐specific mutations might prove to be a valuable tool for cancer detection. Moreover, the capacity to screen for ctDNA in serial liquid biopsies offers the possibility to monitor tumor progression and responses to therapy and to influence treatment decisions that ultimately may improve patient survival. Here we focus on mutation detection in ctDNA and provide an overview of the characteristics of ctDNA, detection methods for ctDNA and the feasibility of ctDNA to monitor tumor dynamics. Current challenges associate with ctDNA will also be discussed.     

Patient monitoring through liquid biopsies using circulating tumor DNA

Tumors release components such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor‐derived extracellular vesicles into the circulation. Multiple studies have demonstrated that molecular information about tumors and metastases can be extracted from these factors, which are therefore frequently referred to as “liquid biopsies.” Liquid biopsies allow the longitudinal monitoring of tumor genomes non‐invasively and may hence ensure that patients receive appropriate treatments that target the molecular features of their disease. Accordingly, the number of studies employing liquid biopsy based assays has been skyrocketing in the last few years. Here, we focus on three important issues, which are of high relevance for monitoring tumor genomes. First, we analyze the relation between the allele frequency of somatic tumor‐specific mutations and the tumor fraction within plasma DNA. Second, we ask how well current tumor evolution models correlate with findings in longitudinal liquid biopsy studies. And, finally, as sensitivity is one of the key challenges of mutation detection, we address the challenge of detecting mutations occurring at very low allele frequencies in plasma DNA.     

The Evolving Role of Circulating Tumor Cells in the Personalized Management of Breast Cancer: From Enumeration to Molecular Characterization

Circulating tumor cells (CTCs) represent tumor cells in the blood stream dislodged from the primary tumor. The presence of CTCs in the bloodstream provides a unique opportunity to sample cancer tissue by means of a relatively less-invasive “liquid biopsy.” Over the past decade, there has been a tremendous increase in the amount of research examining the potential clinical utility of CTCs in the management of cancer. A number of techniques to refine the sensitivity and range of CTC assays are also in development. In this article, we review the recent developments in the current and potential clinical applications of CTCs in breast cancer. CTC enumeration already has an established role as a prognostic biomarker in metastatic breast cancer, whereas molecular characterization of CTCs can serve as a potential predictive biomarker for therapy selection, pharmacodynamic evaluation, and identification of novel actionable targets for novel therapies. The role of CTCs in breast cancer screening and detection of recurrence is currently limited. Further development in techniques will be pivotal in enhancing the broad applicability of CTCs and advancing the field of personalized breast cancer therapy.     

The CTC-Chip: An Exciting New Tool to Detect Circulating Tumor Cells in Lung Cancer Patients

Circulating tumor cells (CTCs) are rare cells that originate from a malignancy and circulate freely in the peripheral blood. The ability to capture and study CTCs is an emerging field with implications for early detection, diagnosis, determining prognosis and monitoring of cancer, as well as for understanding the fundamental biology of the process of metastasis. Here, we review the development and initial clinical studies with a novel microfluidic platform for isolating these cells, the CTC-chip, and discuss its potential uses in the study of lung cancer.     

Role of Circulating Tumor Cells in Metastatic Colorectal Cancer: Clinical Challenges and Opportunities

Circulating tumour cells (CTCs) have been presumed critical to the metastatic process since 1800. These epithelial cells are disseminated from the primary or metastatic tumor site and can be identified in the peripheral blood of patients. Nowadays, technical advances have rendered it easier to reproducibly and repeatedly sample this population of cells with an acceptable degree of accuracy. Therefore, these cells could become an attractive surrogate for phenotypic and genotypic markers in correlation with the development of molecular targeted therapies. In metastatic colorectal cancer several prospective studies have demonstrated the independent prognostic significance of CTCs and identified the number of CTCs before and during treatment as a predictor for overall survival and disease free survival. However, the underlying molecular characteristics of CTCs associated with outcome remain largely unknown. In this review, the role of CTCs in metastatic colorectal cancer is discussed. The variety of assays that can be used for enrichment and detection steps in CTC detection will be described and the clinical utility of these cells for assessing prognosis and monitoring response to therapy will be analyzed. We will also address the shortcomings of current detection methods that fail to identify a mesenchymal subgroup of CTCs, and briefly address how characterization of these cells can help elucidate the biology of cancer metastases.     

Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.     

循环肿瘤细胞的体外培养与应用

循环肿瘤细胞（circulating tumor cells, CTCs）是原发灶或转移灶中的癌细胞播散到循环系统所形成,在肿瘤早期诊断、转移复发监控、疗效评价中都有重要价值,尤其可成为转移干预的重要靶点。CTCs体外培养可为转移干预药物的筛选提供模型以及提高CTCs表型分析的精度。但目前CTCs的体外培养难度较大,成为制约该领域研究的瓶颈。未来本领域研究的首要任务是鉴别出真正导致转移的CTC亚群的分子特征,进而以之构建转移干预药物的体外筛选平台,从而提高转移干预的疗效。本文对CTCs的提取,培养方法,以及体外研究进展进行了系统综述,以期推动该领域的进展。