Tumour biomarkers—Tracing the molecular function and clinical implication

In recent years, with the increase in cancer mortality caused by metastasis, and with the development of individualized and precise medical treatment, early diagnosis with precision becomes the key to decrease the death rate. Since detecting tumour biomarkers in body fluids is the most non‐invasive way to identify the status of tumour development, it has been widely investigated for the usage in clinic. These biomarkers include different expression or mutation in microRNAs (miRNAs), circulating tumour DNAs (ctDNAs), proteins, exosomes and circulating tumour cells (CTCs). In the present article, we summarized and discussed some updated research on these biomarkers. We overviewed their biological functions and evaluated their multiple roles in human and small animal clinical treatment, including diagnosis of cancers, classification of cancers, prognostic and predictive values for therapy response, monitors for therapy efficacy, and anti‐cancer therapeutics. Biomarkers including different expression or mutation in miRNAs, ctDNAs, proteins, exosomes and CTCs provide more choice for early diagnosis of tumour detection at early stage before metastasis. Combination detection of these tumour biomarkers may provide higher accuracy at the lowest molecule combination number for tumour early detection. Moreover, tumour biomarkers can provide valuable suggestions for clinical anti‐cancer treatment and execute monitoring of treatment efficiency.     

肿瘤液态活检的研究进展及其临床应用

液态活检是一类新兴的病理检测手段,其研究内容包括肿瘤循环细胞、肿瘤循环DNA、外泌体等物质,其中的信息为肿瘤患者的个性化医疗提供有力的依据。它凭借无创、便捷等优势在临床应用中展现出广大前景。近年来,随着液态活检研究内容的扩大及其捕获与检测技术的发展,液态活检在临床中的应用日益广泛。本文旨在探讨近年来液态活检的主要研究对象、检测技术以及在临床应用中的发展前景与所面临的挑战,以期为肿瘤患者获得更佳的治疗,推动肿瘤精准医学的发展。     

The proteomics of prostate cancer exosomes

Exosomes and other microvesicles are emerging as rich reservoirs of tumor-specific proteins and biomarkers for cancer detection and progression. For prostate cancer, exosomes secreted by the prostate can be isolated from prostatic secretions, seminal fluid, tissue, urine or blood for further proteomic analysis. Structurally, prostate-derived exosomes are distinct in size, membrane composition and specific prostate protein content, potentially providing a novel and easily isolatable source of biomarkers from clinical biofluids. The key to these isolation strategies will be the targeting of specific prostatic proteins expressed in these exosomes, thus requiring detailed proteomic characterizations. A summary of ongoing efforts to characterize the proteome of these unique prostate cancer-associated exosomes and their potential applications for use in biomarker assays is presented.     

On a chip

The future of clinical and POC BioMEMS is very bright. With an increasing emphasis on the personalization of medicine and the rising costs of health care, early detection and diagnostics at the POC will be even more important. Early detection implies early intervention, resulting in the saving of lives and reducing overall spending. The potential impact of these technologies on the early diagnosis and management of both communicable and noncommunicable diseases is very high. Many grand challenges applications are possible, e.g., routine tests such as complete blood cell count on a chip that an individual can perform at home; detection of cardiac markers from blood after a perceived heart attack; detection of cancer markers such as exosomes, CTCs from blood, or protein biomarkers in serum; and detection of infectious agents such as virus and bacteria for public health. These applications are expected to result in new diagnostic assays for home, doctor's office, clinical laboratories, and various POC settings.     

Circulating tumor cells as therapy-related biomarkers in cancer patients

Carcinomas (tumors of epithelial origin) are responsible for most of all new cancers in the industrialized countries. Due to the high mortality rate caused by the metastatic spread of aggressive cancer cells, there is an urgent demand in finding new biomarkers, which should detect early formation of metastases and monitor efficacy of systemic adjuvant therapy in a timely manner. It has been considered that the molecular analysis of cells which are shed from tumors into the blood system (circulating tumor cells (CTCs)) might provide new insights for the clinical management of cancer, probably far earlier than using traditional high-resolution imaging technologies. Clinical trials indicated that CTCs can be deployed for diagnostic, monitoring, and prognostic purposes. Furthermore, these cells are discussed to be suitable as predictive markers. In any case, identification of CTCs requires innovative and challenging technologies as detection methods should be specific, sensitive, standardized, and highly reproducible. Although many different approaches have been developed until now, only the CellSearch? method has been cleared by the American Food and Drug Administration. Although the detection of CTCs has already shown to have a prognostic impact in many tumor entities including breast, prostate, lung and colon cancer, ongoing and future studies are aimed to explore whether CTCs can be used for an individual therapy decision making including novel immunotherapeutic approaches. This review discusses (1) different detection strategies for CTCs, (2) their clinical impact, and (3) the potential use of CTCs guiding the treatment of individual cancer patients.     

The role of Exosomes in the Pathogenesis of Nasopharyngeal Carcinoma and the involved Clinical Application

Exosomes are nanoscale membrane vesicles, which carry biologically active substances of their cell of origin and play an important role in signal transduction and intercellular communication. At present, exosomes have been identified as a promising non-invasive liquid biopsy biomarker in the tissues and circulating blood of nasopharyngeal carcinoma (NPC) and found to participate in regulating pathophysiological process of the tumor. We here review recent insights gained into the molecular mechanisms of exosome-induced cell growth, angiogenesis, metastasis, immunosuppression, radiation resistance and chemotherapy resistance in the development and progression of NPC, as well as the clinical application of exosomes as diagnostic biomarkers and therapeutic agents. We also discuss the limitations and challenges in exosome application. We hope this review may provide some references for the use of exosomes in clinical intervention.     

Implementing biological markers as a tool to guide clinical care of patients with pancreatic cancer

A major obstacle for the effective treatment of pancreatic ductal adenocarcinoma (PDAC) is its molecular heterogeneity, reflected by the diverse clinical outcomes and responses to therapies that occur. The tumors of patients with PDAC must therefore be closely examined and classified before treatment initiation in order to predict the natural evolution of the disease and the response to therapy. To stratify patients, it is absolutely necessary to identify biological markers that are highly specific and reproducible, and easily measurable by inexpensive sensitive techniques. Several promising strategies to find biomarkers are already available or under development, such as the use of liquid biopsies to detect circulating tumor cells, circulating free DNA, methylated DNA, circulating RNA, and exosomes and extracellular vesicles, as well as immunological markers and molecular markers. Such biomarkers are capable of classifying patients with PDAC and predicting their therapeutic sensitivity. Interestingly, developing chemograms using primary cell lines or organoids and analyzing the resulting high-throughput data via artificial intelligence would be highly beneficial to patients. How can exploiting these biomarkers benefit patients with resectable, borderline resectable, locally advanced, and metastatic PDAC? In fact, the utility of these biomarkers depends on the patient''s clinical situation. At the early stages of the disease, the clinician''s priority lies in rapid diagnosis, so that the patient receives surgery without delay; at advanced disease stages, where therapeutic possibilities are severely limited, the priority is to determine the PDAC tumor subtype so as to estimate the clinical outcome and select a suitable effective treatment.     

Circulating tumor cells: approaches to isolation and characterization

Circulating tumor cells (CTCs) shed from primary and metastatic cancers are admixed with blood components and are thus rare, making their isolation and characterization a major technological challenge. CTCs hold the key to understanding the biology of metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. Improvements in technologies to yield purer CTC populations amenable to better cellular and molecular characterization will enable a broad range of clinical applications, including early detection of disease and the discovery of biomarkers to predict treatment responses and disease progression.     

Current and Future Perspectives of Cell-Free DNA in Liquid Biopsy

A liquid biopsy is a minimally invasive or non-invasive method to analyze a range of tumor material in blood or other body fluids, including circulating tumor cells (CTCs), cell-free DNA (cfDNA), messenger RNA (mRNA), microRNA (miRNA), and exosomes, which is a very promising technology. Among these cancer biomarkers, plasma cfDNA is the most widely used in clinical practice. Compared with a tissue biopsy of traditional cancer diagnosis, in assessing tumor heterogeneity, a liquid biopsy is more reliable because all tumor sites release cfDNA into the blood. Therefore, a cfDNA liquid biopsy is less invasive and comprehensive. Moreover, the development of next-generation sequencing technology makes cfDNA sequencing more sensitive than a tissue biopsy, with higher clinical applicability and wider application. In this publication, we aim to review the latest perspectives of cfDNA liquid biopsy clinical significance and application in cancer diagnosis, treatment, and prognosis. We introduce the sequencing techniques and challenges of cfDNA detection, analysis, and clinical applications, and discuss future research directions.     

Multifaceted modifications for a cell size-based circulating tumor cell scope technique hold the prospect for large-scale application in general populations

Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear−cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.     

Circulating tumor cells: the 'leukemic phase' of solid cancers

It is well known that malignant cells circulate in the bloodstream of patients with solid tumors. However, the biological significance of circulating tumor cells (CTCs) and the clinical relevance of their detection are still debated. Besides technical issues regarding CTC-detection methods, discontinuous shedding of CTCs from established cancer deposits, genomic instability and metastatic inefficiency might underlie the conflicting results currently available. Nevertheless, technological advances and recent clinical findings are prompting researchers to dissect CTC biology further. Here, we review these recent findings, and discuss the prospects for the identification and molecular characterization of the CTC subset that is responsible for metastasis development. This would provide a formidable tool for prognosis evaluation, anticancer-drug development and, ultimately, cancer-therapy personalization.     

循环microRNAs的外泌形成机制及其对乳腺癌侵袭和转移的影响

目前乳腺癌的临床诊疗主要依赖影像学和相对较少的预后/预测指标(如雌激素受体、孕激素受体、HER2等).这些生物标志物主要是基于原发肿瘤病灶的生物学检测,可用于转移或复发的检测指标很少,尤其是在切除肿瘤原发灶后,复发监测很困难.循环cell-free microRNAs(circulating cf-miRNAs,或简称circulating miRNAs)的发现为改变现有乳腺癌临床诊疗模式提供了可能.Cell-free miRNA通过外泌体、微囊或转运蛋白的主动外泌机制,可能在循环miRNA的形成中起着重要作用.Cell-free miRNA特别是circulating miRNA不仅自身可以作为信号分子影响肿瘤细胞和组织微环境,而且还可以与其他信号通路发生交互通讯来调控肿瘤部位新生血管的形成和肿瘤细胞表型的上皮-间质转换,影响乳腺癌的侵袭和转移.本文综述了循环miRNA的特征与分泌机制,特别是乳腺癌相关的循环miRNA参与作为一种液体活检生物标志物在乳腺癌诊断、预后评价和疗效评估的临床意义.     

cfRNAs as biomarkers in oncology – still experimental or applied tool for personalized medicine already?

Currently, the challenges of contemporary oncology are focused mainly on the development of personalized medicine and precise treatment, which could be achieved through the use of molecular biomarkers. One of the biological molecules with great potential are circulating free RNAs (cfRNAs) which are present in various types of body fluids, such as blood, serum, plasma, and saliva. Also, different types of cfRNA particles can be distinguished depending on their length and function: microRNA (miRNA), PIWI-interacting RNA (piRNA), tRNA-derived RNA fragments (tRFs), circular RNA (circRNA), long non-coding RNA (lncRNA), and messenger RNA (mRNA). Moreover, cfRNAs occur in various forms: as a free molecule alone, in membrane vesicles, such as exosomes, or in complexes with proteins and lipids. One of the modern approaches for monitoring patient's condition is a "liquid biopsy" that provides a non-invasive and easily available source of circulating RNAs. Both the presence of specific cfRNA types as well as their concentration are dependent on many factors including cancer type or even reaction to treatment. Despite the possibility of using circulating free RNAs as biomarkers, there is still a lack of validated diagnostic panels, defined protocols for sampling, storing as well as detection methods.In this work we examine different types of cfRNAs, evaluate them as possible biomarkers, and analyze methods of their detection. We believe that further research on cfRNA and defining diagnostic panels could lead to better and faster cancer identification and improve treatment monitoring.     

循环肿瘤细胞在女性实体器官肿瘤中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)指的是从实体的肿瘤或转移的病灶进入外周血液循环的恶性肿瘤细胞。自发现以来,随着其检验技术日趋成熟,循环肿瘤细胞(CTCs)日渐成为肿瘤学炙手可热的研究对象。因为它将通过外周血的检验来实现监测肿瘤的发生、发展、转移、复发等情况,相对于肿瘤实体活检,"液体活检"不仅让患者易于接受,更有利于医务工作者监测病情变化。本文综述了循环肿瘤细胞(CTCs)的检测方法并综述了循环肿瘤细胞在女性实体肿瘤--乳腺癌、卵巢癌、宫颈癌、子宫内膜癌中的研究进展。其中着重介绍了其在早期乳腺癌及复发转移性乳腺癌中的重大意义以及在评价治疗效果中的分子学特征。实践表明,循环肿瘤细胞(CTCs)与HE-4、CA125的联合应用在评估卵巢癌化疗敏感性中也具有重要的临床意义。     

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery

IntroductionExosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current “gold standard” surrogate biomarker of myocardial damage.ConclusionsThe plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.     

Liquid biopsy for early diagnosis of non-small cell lung carcinoma: recent research and detection technologies

Liquid biopsy, an innovative method for early diagnosis of cancer, has changed the traditional method of diagnosing lung cancer and is considered a feasible auxiliary diagnostic tool. To date, various reports emphasize the need for non-small cell lung carcinoma (NSCLC), both with higher incidence and mortality and less effective treatments; thus, emphasizing the need for early detection of NSCLC for improved patient outcomes. Invasive tissue biopsy is a common diagnostic tool that is usually extracted from the primary tumor to indicate molecular composition. In comparison, liquid biopsy taken from body fluid reflects extensive malignant features nonexistent in primary tumors. Owing to new detection technologies, liquid biopsy reduces the need for invasive treatments and enhances the accuracy and specificity of early detection of cancer in clinical settings. This review summarizes some latest research on the diagnosis of early-stage NSCLC via liquid biopsy, including circulating DNA, circulating tumor cells, exosomes, and tumor-educated platelets, as well as their detection technologies, such as fluorescence in-situ hybridization-based, polymerase chain reaction-based, next-generation sequencing-based, Chip-based, and microfluidic methods. Additionally, we outline the existing challenges and possible solutions for liquid-biopsy biomarkers. Our study mainly highlights the merits of liquid biopsy as a promising biomarker for non-invasive detection in the future, particularly for the early detection of NSCLC, thereby benefitting human health.     

Circulating tumour cells and cancer stem cells: A role for proteomics in defining the interrelationships between function,phenotype and differentiation with potential clinical applications

Research on the discovery and implementation of valid cancer biomarkers is one of the most challenging fields in oncology and oncoproteomics in particular. Moreover, it is generally accepted that an evaluation of cancer biomarkers from the blood could significantly enable biomarker assessments by providing a relatively non-invasive source of representative tumour material. In this regard, circulating tumour cells (CTCs) isolated from the blood of metastatic cancer patients have significant promise. It has been demonstrated that localised and metastatic cancers may give rise to CTCs, which are detectable in the bloodstream. Despite technical difficulties, recent studies have highlighted the prognostic significance of the presence and number of CTCs in the blood. Future studies are necessary not only to detect CTCs but also to characterise them. Furthermore, another pathogenically significant type of cancer cells, known as cancer stem cells (CSCs) or more recently termed circulating tumour stem cells (CTSCs), appears to have a significant role as a subpopulation of CTCs.     

Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies

Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development.     

Blood-based DNA methylation signatures in cancer: A systematic review

DNA methylation profiles are in dynamic equilibrium via the initiation of methylation, maintenance of methylation and demethylation, which control gene expression and chromosome stability. Changes in DNA methylation patterns play important roles in carcinogenesis and primarily manifests as hypomethylation of the entire genome and the hypermethylation of individual loci. These changes may be reflected in blood-based DNA, which provides a non-invasive means for cancer monitoring. Previous blood-based DNA detection objects primarily included circulating tumor DNA/cell-free DNA (ctDNA/cfDNA), circulating tumor cells (CTCs) and exosomes. Researchers gradually found that methylation changes in peripheral blood mononuclear cells (PBMCs) also reflected the presence of tumors. Blood-based DNA methylation is widely used in early diagnosis, prognosis prediction, dynamic monitoring after treatment and other fields of clinical research on cancer. The reversible methylation of genes also makes them important therapeutic targets. The present paper summarizes the changes in DNA methylation in cancer based on existing research and focuses on the characteristics of the detection objects of blood-based DNA, including ctDNA/cfDNA, CTCs, exosomes and PBMCs, and their application in clinical research.     

Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.