Liquid biopsies in cancer

LUO Yang， MENG Min⁃Jie， DIAO Zhi⁃Hong （School of Biosciences and Biopharmaceutics， Guangdong Pharmaceutical U⁃niversity， Guangzhou 50006， China；Department of Medical Laboratory， Guangdong Second Provincial People's Hospital， Guangdong Provincial Emergency Hospital， Guangzhou 5037， China）

·综述·

Liquid biopsies in cancer

LUO Yang1， MENG Min⁃Jie1， DIAO Zhi⁃Hong2（1School of Biosciences and Biopharmaceutics， Guangdong Pharmaceutical U⁃niversity， Guangzhou 510006， China；2Department of Medical Laboratory， Guangdong Second Provincial People's Hospital， Guangdong Provincial Emergency Hospital， Guangzhou 510317， China）

Liquid biopsy， the detection of biomarkers from body fluids， has been widely used on circulating tumor cells （CTCs），circulating tumor DNA （ctDNA）， circulating tumor microRNA，exosomes and other kinds of molecular characterization．Cancer， a series of comprehensive mechanisms， presents in different molec⁃ular forms that could be used in clinic for diagnosis， treatment，drug monitoring and prognosis judgement via liquid biopsies．Liq⁃uid biopsies are available to tumor heterogeneity， resistance， pro⁃gression and metastases in clinic．Technologies for detection and analysis was improving for the obvious limitation of liquid biop⁃sies， that leads the future directions．In this review， we provided critical views of liquid biopsies on cancer，including different methods of liquid biopsies and applications of liquid biopsies in current clinical trials and practice．

liquid biopsy； cancer； CTCs； circulating tumor DNA；circulating tumor microRNA

Introduction

Liquid biopsy，also named fluid biopsy or fluid phase biopsy， is the sampling and analysis of non⁃solid biological tissue including blood samples or other body fluids［1］．Liquid biopsy refers to molecular analysis of tumor genetic features based on circulating genetic material in the peripheral blood［2］．Recently， liquid biopsy has become a hospot of research and attracted more attention on the potential biomarkers［3］．

Globally，cancer remains the greatest cause of non⁃accidental deaths in the past 20 years［4］．Cancer is a majorhealth problem worldwide， accounting for approximately 25%of death in United States in 2015［2］．Ways investigating people with suspected cancer include blood tests， X⁃rays， CT scans and endoscopy．Though medical tests to diagnosis are numerous，limitations are still obvious．Traditional tissue biopsies， with standard clinical protocols，sampling cells from human body by puncturing primary tumors，may introduce risks for patients［5］．Different to tissue biopsies， liquid biopsies remove invasive surgeries and procedures，reaching the signs of cancer from body fluid，which are easy to perform that giving hope to the patients that will lead to early diagnosis of cancer．Analysis of tumor⁃associated biomarkers is an increasing need for diagnostic and prog⁃nostic purposes［6］．

Biomarkers in cancer CTCs

CTCs are tumor cells that shed from primary and metastatic tumor and deposit into bloodstream including vasculature and lymphatics［7］．A large part of CTCs are destructed by mechanical factors as well as specific and nonspecific immune responses in the circulation system［8］．CTCs are one of the main elements of liquid biopsy，in an attempt to develop technological assays of detection，enrichment and molecular characterization in various cancers［2］．Modern cancer research has demon⁃strated that CTCs derive from clones in primary tumor［9］．CTCs can be detected in the blood of patients with various tumors and not be found in healthy people［10］．CTCs can be separated from blood cells by size or the tumor cell surface antigens［11］．The significant efforts put into un⁃derstanding the CTCs biological properties have demon⁃strated the critical role CTCs play in the metastatic spread of carcinoma［12］．CTCs reflect to the tumor cells that exist in the circulatory system and draw more attention on the belief of their responsibility for tumor metastasis and cancer⁃related deaths［13］．CTCs could be considered as a kind of liquid biopsy that reveals metastasis in action，providing live information about the disease status of patients［14］．CTCs are independent prognostic factors that were documented for cases with positive CTCs at diagnosis who had poorer prognosis in clinic［8］．CTCssomehow are divided into several types：traditional CTCs， Cytokeratin negative （ CK⁃） CTCs， apoptotic CTCs， small CTCs and CTC clusters．

Traditional CTCs are defined that cancer cells with an integrated，viable nucleus and with the expression of cytokeratins and the absence of CD45，which indicate that the cells are epithelial origin but not hematopoietic origin［15］．Cytokeratin negative （CK⁃） CTCs are cancer stem cells or cells undergoing epithelial⁃mesenchymal transition （EMT）．CK⁃CTCs have gene or protein expression of or genomics associated with cancer which may be related to resistant and with metastasis tendency．CK⁃CTCs have morphology similar to cancer cells with expression of neither cytokeratins nor CD45［16］．Apop⁃totic CTCs are traditional CTCs that are in the condition of apoptosis．They are identified as nuclear fragmentation or cytoplasmic blebbing related to apoptosis［17］．Small CTCs are cytokeratin positive and CD45 negative with the sizes and shapes similar to leukocyte．Importantly，small CTCs have cancer⁃specific biomarkers that identify them as CTCs．Small CTCs have been implicated in progres⁃sive disease and differentiation into small cell carcino⁃mas， which often require a different therapeutic course．

CTC clusters are two or more single CTCs bounding together．The CTC cluster may contain traditional， small，apoptotic or CK⁃CTCs．These clusters have cancer⁃spe⁃cific biomarkers that identify them as CTCs．Some studies have reported that these clusters are associated with increased metastatic risk and poor prognosis［18］．CTCs have been detected and investigated in most epithelial malignancies，where they represent cancer cells obtained as they transit through the bloodstream［19］．A variety of technologies have been applied to CTC enumeration and identification since modern cancer research began［20］．

CTCs thus could be considered as a “liquid biopsy”which reveals metastasis in action， providing live infor⁃mation about patients'disease status．CTCs isolation and enumeration is a non⁃invasive method that is easy to perform repetitively compared with traditional tissue biopsy［2］．Analysis of blood samples found a propensity for increased CTC detection as the disease progressed in individualpatients［21］． CTCs enable comprehensive perception of molecular status of a patient at a given time and capture molecular diversity of the disease［22］．The detection of CTCs may have important prognostic and therapeutic implications， however， for their num⁃bers are very small， which are not easily detected．Some efforts with remarkable results have been placed regard⁃ing the putative diagnostic and role of CTCs，but the lack of a standardized method makes it difficult to evaluate the role of CTCs as biomarkers in tumor patients［10］．

ctDNA There are two sources that tumor DNA can be assessed in the circulation：ctDNA and CTCs［23］．ctDNA or cell⁃free DNA （cfDNA） is tumor DNA circulating freely in the blood of cancer patients．ctDNA are mutant DNAs released to the circulation by tumor cells and constitute part of circulating cfDNA in cancer［24］．ctDNA can be present in a wide range of cancers but at varying levels and mutant allele fractions［25］．The ctDNA is highly fragmented to around 170 bp and is cleared rapidly after surgery to remove tumors or chemotherapeutic treatment．Beyond CTCs in clinical terms， ctDNA liquid biopsy with its specific that allow us to understand the kind of molecular changes happening in the tumor in re⁃al time［26］．Features of active ctDNA are the straightfor⁃ward isolations， ctDNA levels range increasingly or de⁃creasingly in response to the degree of tumor burden and ctDNA contains DNA mutations found in both primary and metastatic lesions［27］．For ctDNA can be serially collected to measure changes in quantity and composition over time，it can be method to monitor tumor development in real time［18］．Researches have found that ctDNA monitoring can assess response to treatment and identify mechanisms of resistance potentially，which may allow for detection of minimally residual disease，predicting recurrence months before clinical presentation［28］．In short，ctDNA analysis has demonstrated degrees of clinical utility in resistance mutation direction，tracking genetic heterogeneity， monitoring tumor burden，response to therapy and early detection of recurrence［20］．Current usage of ctDNA are reflected in several ways：①identifying mutations of interest（including resistance mutations） and／or characterizing tumor heterogeneity in primary and metastatic disease； ②serial ctDNA quanti⁃fication to assess tumor burden and response to treatment in primary and metastatic disease； ③early detection of disease recurrence in minimal residual disease； ④early detection of primary disease［28］．

ctDNA is recognized as liquid biopsy to profile tumor genome with increasing attention［29］．ctDNA has clear advantages to monitor tumor burden and therapeutic response compared with protein cancer markers and biomedical imaging that means highly sensitive and specific detection methods are required for ctDNA to serve as a clinically feasible approach［27］．It is reportedthat ctDNA is often present in patients with no detectable CTC levels，which has been shown that a ctDNA can better reflect clinical progression than CTCs［6］．Although ctDNA analysis is increasingly popular，the challenging is obvious on the detection of primary disease for the mutations present in the tumor are unknown［28］．ctDNA may be not detectable for its extremely low level presented in blood that influence its sensitivity and specificity［27］．With great prospects on tumor detection，ctDNA will be widely used in clinical approach with its technique approving．

Circulating tumor microRNA

miRNAs are small non⁃coding RNA molecule （con⁃taining about 22 nucleotides） found in plants， animals and some viruses，that function in RNA silencing and post⁃transcriptioanal regulation of gene expression［30］．

miRNAs may affect the development of cancer．miRNAs were first found to be associated with human disease such as chronic lymphocytic leukemia．MiRNAs participate in the B cell linked pathways， are fundamen⁃tal to B cell development like B cell receptor（BCR）signaling， B cell migration／adhesion， cell⁃cell interac⁃tions in immune niches， and the production and class⁃switching of immuonoglobulins，and also play critical regulatory roles in B cells in maturation，generation of pre⁃， marginal zone， follicular， B1， plasma and memo⁃ry B cells［31］．It has been reported that nucleosomes are released into plasma then cell death and apoptosis happen and capture by macrophages［32］．Current studies focus most on the miRNA contribution to regulation of significant signaling pathways．

Another role for miRNAs in cancers is to use their expression level for prognoses［33］．Optimal treatment for cancer involves accurately identifying patients for risk⁃stratified therapy．Those with a rapid response to initial treatment may benefit from truncating treatment regimens，showing the value of accurate disease response measures．Cell⁃free miRNAs are highly stable in blood，are overexpressed in cancer and are quantifiable within the diagnostic laboratory．Circulating miRNAs have the potential to assist clinical decision making and aid inter⁃pretation of positron emission tomography combined with computerized tomography．They can be performed at each consultation to assess disease response and detect relapse．

Patterns of gene activity that can distinguish types of cancerswere identified and miRNA profiling can determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer［31］．It has been found that a novel miRNA⁃profill⁃ing⁃based screening assay for the detection of early⁃stage，resectable（StageⅡ） colorectal cancer was undergoing a clinical trial［34］．With high stability， it has been found that circulating tumor miRNAs could be detectable and quantifiable in a range of body fluids［35］．miRNAs have the potential to be used as targets for treatment of differ⁃ent cancers．Circulating tumor miRNA might be a predic⁃tive biomarker on diagnosis and prognosis．

Extracellular vesicles or exosomes

Extracellular vesicles are produced by all domains of life including complex eukaryotes， both Gram⁃negative and Gram⁃positive bacteria， mycobacteria and fungi．Exosomes are cell⁃derived vesicles that are present in many and perhaps all eukaryotic fluids， including blood， urine， and cultured medium of cell cultures［36］．Exosomes are either released from the cell when multive⁃sicular bodies fuse with the plasma membrane or released directly from the plasma membrane［37］．Exosomes are membraneous vesicles of endocytic origin （30－100 nm）derived from endosomes with potential roles in both local and cell⁃cell signaling and enriched in CD63 and CD81［38］．Exosomes contain an abundance of DNA，RNA，proteins and other substance， they also resemble their cell of origin［39］．Evidence is accumulating that exosomes have specialized functions and play a key role in processes such as coagulation， intercellular signaling，and waste management［40］．Exosomes reflect the specialized funtions of their original cells via the molecular composition，owe the ability to bind target cells and participate in physiological and／or pathological processes［41］．

It is indicated that exosomes are secreted from almost all types of cells and play an important role in autocrine and paracrine communication in both normal and cancer cells［42］．It has been reported that exosomes influence many tumor properties， such as growth， angio⁃genesis， invasion and metastasis［43］．Tumor⁃derived exosomes are ubiquitously present in body fluids of cancer patients because tumor cells produce and release masses of exosomes［44］．Tumor⁃derived exosomes may have great impact both on tumor aggressiveness and metastasis even may interfere with therapeutic strategies against cancer［39］．

Discovery of exosomes is expected to bring major breakthrough in therapies for different diseases， inclu⁃ding cancer，for they are the most potent intercellular communicators［45］．Consequently， there is a growing interestin the clinicalapplications ofexosomes．Exosomes can potentially be used for prognosis， therapy，and as biomarkers for health and disease．

Application of liquid biopsy in clinical trails and practice

Liquid biopsies to tumor heterogeneity

Cancer is associated with genes，which leads analysis of tumor⁃linked genetic alteration and becomes more pop⁃ular on diagnostic， prognostic and treatment monitor［1］．Cancer is a multistep process originating from genetic alterations in normal proliferation and differentiation，triggering a network of processes that lead to carcinogen⁃esis［6］．Tumor heterogeneity describes the observation that different tumor cells can show distinct morphological and phenotypic profiles， including cellular morphology，gene expression， metabolism， motility， proliferation，and metastatic potential［46］．This phenomenon occurs both between tumors （inter⁃tumor heterogeneity） and within tumors （intra⁃tumor heterogeneity）．The hetero⁃geneity of cancer cells introduces significant challenges in designing effective treatment strategies．

Liquid biopsies can increase prediction accuracy and improve understanding of both the dynamics and heterogeneity of cancer［47］．An understanding that CTCs are highly heterogeneous for significant variability has been found from transcriptional profiling and surface marker analysis［48］．The heterogeneity of cells in primary cancer is visible in distinct mutations within the same slice of tumor tissue［49］．Single nucleotide variations on a specific genome can strongly indicate metastasis，which shows the additional layer of heterogeneity due to specific genomic mutations that can be found in CTCs［4］．

Detection of rare biomarkers has proven challenging because of the rarity and heterogeneity of targets in samples［4］． Biomarkers thatmay predicttreatment response or prognosis may not be widely available for the genetic differences within and between tumors．However，it has been suggested that the level of heterogeneity itself can be used as a biomarker since more heterogeneous tumors may be more likely to contain treatment⁃resistant subclones［50］．Further research into developing biomarkers that account for heterogeneity is still in progress．

Liquid biopsies for assessing resistance to treatment

Cancer genome undergoes several changes over the course of treatment，monitoring of oncogenic mutations could contribute to establishment of an effective treatment plan and management of drug⁃resistance mechanisms［51］．

Traditional methods might precede clinical or radio⁃logical signs but not identify the cause of resistance to therapy［2］．Liquid biopsies can effectively provide accu⁃rate information regarding the cancer's progression and intensity，gauge the body's responses to the chosen method of treatment，and detect early recurrence and identify possible causes behind resistance to treatment［15］．With real⁃time molecular characterization， liquidbiopsies allow rapid identification of a genomic alteration associ⁃ated with therapy resistance［52］．It has been reported that in the metastatic situation， CTC⁃based liquid biopsies might not only identify the right patients for effective therapies but also could help avoid futile treatment to resistant cancers［53］．Resistance to treatment is often correlated with the acquisition of new mutations and liquid biopsies method offers the opportunity for the identification of drug⁃resistance mutations［54］．

Liquid biopsies for studying tumor progression and metastases

Tumor progression is the third and last phase in tumor development．This phase is characterized by in⁃creased growth speed and invasiveness of the tumor cells．As a result of the progression， phenotypical chan⁃ges occur and the tumor becomes more aggressive and acquires greater malignant potential．Together with the progression， more and more aneuploidy occurs．This leads to expression in cell and nuclear polymorphism．

Metastasis is the spread of a tumor or other disease from one organ or part of the body to another without direct connection．Common sites of origin are lung，breast， skin， colon， kidney， prostate， liver and cervix．Malignancy allows for invasion into the circulation，followed by invasion to a second site for tumorigenesis．Some cancer cells acquire the ability to penetrate the walls of lymphatic and／or blood vessels， after which they are able to circulate through the bloodstream （CTCs） to other sites and tissues in the body．This process is known respectively as lymphatic or hematogenous spread．Possi⁃bly，each cell sheds off variety of CTCs with contrasting phenotypic and functional characteristics［4］．After the tumor cells come to rest at another site， they re⁃pene⁃trate the vessel or walls and continue to multiply， even⁃tually form another clinically detectable tumor．

Evaluation of genetic mutations has been most based on tissue biopsies and repeat biopsy has been used in an attempt to elucidate molecular biology of tumor progres⁃sion after initial response to treatment［2］．Liquid biopsy enables monitoring with real⁃time of ongoing changes in the tumor at the molecular level，which allow for more extensive insights into disease state and progression［25］．

Integrating liquid biopsies in clinical trails and practice

Mounting evidences suggest that molecular charac⁃terization of tumors can aid treatment decisions in clini⁃cal care with a significant impact on treatment outcome．Liquid biopsy has recently generated a strong interest among molecular oncologists as viable alternative in cases where tissue sampling is not available［1］．In comparison of traditional tissue biopsy，liquid biopsy is fast， convenient， less invasive， with limited injury and many other advantages［3］．With the property of real⁃time， high sensitivity and specificity， liquid biopsy is also used to monitor the drug efficacy［55］．In many cases，the target tissues are inaccessible to reach for the location in regions［4］．However， the important technical limitations of traditional tissue biopsies include the possible seeding of tumor cells to other sites［6］．Liquid biopsy analyses play a role with increasing importance in routine clinical practice in hospitals especially oncology departments worldwide［56］．Always acting as a basic blood test，liquid biopsy can reduce the stress of patients and is easily acceptable with low cost［57］．Liquid biopsy can serve as promising tools to track cancer evolu⁃tion［27］．With a combination with ongoing treatment to map molecular disease changes，liquid biopsy allow clinicians to chart the growth of secondary resistance and isolate heterogeneous subclonal populations of tumor cells that continue to grow［25］．This is important for selecting a personalized treatment to reach the goal that we describe as“the right patient with the right drug at the right dose at the right time”， which is suitable to the concept of“precision medicine”［11］．

Liquid biopsies in blood cancer

Blood cancer is also named tumors of hematopoietic and lymphoid tissues or haematopoietic and lymphoid malignancies，which are tumors that rooted from the blood， bone marrow， lymph and lymphatic system［58］．Current diagnostic and prognostic algorithms in blood cancer rely heavily on peripheral blood（PB） counts and bone marrow （BM） morphology， which often has inter⁃observer variability［59］．Morphologic， cytochemical，and／or immunophenotypic features are used for establis⁃hing the lineage of the neoplastic cells and for assessment of their maturation which the blast percentage remains a practical tool for categorizing myeloid neoplasms and jud⁃ging their progression［58］．In patients with hematologic malignancy， cell⁃free DNA reflects the underlying tumor mutational profile and circulating miRNA reflects genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immue effector cells［60］．Non⁃invasive genotyping tests can characterize mutations instead of an invasive biopsy［28］．

Technology of liquid biopsies

Technologies of liquid biopsy have made a great progress recently．Some advantages of liquid biopsy include the possibility for serial sampling，automation and reproducible results for diagnosis，monitoring and prognosis［4］．When designing liquid biopsy technolo⁃gies， it is ideal to integrate capture， detection and anal⁃ysis into simple platforms．

Enzyme⁃linked immunosorbent assay （ELISA） and polymerase chain reaction（PCR）are methods that have been routinely used for diagnosis and disease monitoring on RNA or DNA⁃protein level［60］．With the development of technology， the method has been improved．

Next⁃generation sequencing （ NGS）， also called high⁃throughput sequencing， applies in various gene areas such as genome sequecing， genome resequecing，transcriptome profilling （RNA⁃sequencing）， DNA⁃pro⁃tein interactions （CHIP⁃sequencing） and epigenome characterization［61］．For every individual is unique that indicates the genome variations， it is neccessary to rese⁃quence．The appliacation of NGS for CTCs can dignosis and guide therapy with real⁃time characteristic［55］．Whole⁃genome expression has been achieved in NGS methods［20］．NGS can identify ctDNA as potential liquid biopsies， such as in breast， coloretal， pancreatic， hae⁃motological， lung and urological malignancies［32］，which rise more interest in ctDNA as a tool for cancer diagnosis， prognosisand monitoring［27］．Researches have shown that NGS technology might increase the sensitivity on DNA level via ctDNA detection［1］．With the characteristics of highly specialized techniques，NGS are widely uesd on the detection of miRNA level，which is potential for novel miRNA discovery［60］．

It have been investigated that PCR⁃free biosensor technologies with the aim to obtain more stable，reliable and sensitive detection platforms were used for clinical diagnostic purposes which exploited the electrochemical or optical signal transduction，and most of them integrated nanostructured materials for the enhancement of the detected signal［6］．

Liquid biopsies on chip are low cost，less exhaustive of resources， superior at reproducing results， conducive to sequential sampling， and allow automation［4］．Emer⁃ging microchip based on technologies enables high throughput screening with single cell resolution andhelps understand tumor heterogeneity and metastatic process by performing genomics studies［62］．

Biomarker was defined by the National Cancer Institute as“a biological molecule found in blood， other body fluids or tissue that is a sign of a normal or abnormal process or of a condition or disease”［32］．

Conclusion

Biopsies have been used to diagnose and manage disease for over 1，000 years［5］．Currently， liquid biopsies are being integrated into preclinical trials to address many concerns of“tailored oncology”［10］．It is required more studies with long⁃term follow up to demonstrate a posi⁃tive diagnostic liquid test result as a starting point to initiate treatment［11］．Although liquid biopsies， arousing arguments for their correction，have not reached the standard goal as traditional tissue biopsy and imaging techniques on diagnosis［56］．Many problems remain to be addressed such as standardization of liquid biopsies technologies，evaluation of possible correlation between traditional methods and new methods，including liquid biomarkers［63］．Challenges associated with liquid biopsy are obvious， including the fact that the margins， grade，tumor size and exact location cannot be determined by liquid biopsy alone［26］．Although tumor biopsies remain the reference standard for the diagnosis and genotyping，they are invasive and not always feasible，liquid biopsies have the potential to overcome most of these barriers for their noninvasive manner，allowing a rapid and accurate identification and resistant genetic alterations and a real⁃time monitoring of treatment responses［64］．With limitations and challenges，technologies and concepts of biopsies will have to be renewed to achieve a brand⁃new goal．

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R730．4

A

2095⁃6894（2017）09⁃48⁃07

2017－04－29；接受日期：2017－05－12

广东省省级科技计划项目（2016A050502064）

骆 阳．硕士生．研究方向：核酸药物与肿瘤免疫．E⁃mail：sunnylok＠ qq．com

刁志宏．博士，副主任技师．研究方向：传染病诊断、免疫学诊断．Tel：020⁃86169131 E⁃mail：zhhdiao＠ 163．com

孟民杰（共同第一作者）．教授．研究方向：核酸药物与肿瘤免疫．E⁃mail：mminjie＠ 163．com