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Wu, H.; Chu, P. Liquid Biopsy Techniques for Breast Cancer. Encyclopedia. Available online: https://encyclopedia.pub/entry/22258 (accessed on 19 July 2025).
Wu H, Chu P. Liquid Biopsy Techniques for Breast Cancer. Encyclopedia. Available at: https://encyclopedia.pub/entry/22258. Accessed July 19, 2025.
Wu, Hsing-Ju, Pei-Yi Chu. "Liquid Biopsy Techniques for Breast Cancer" Encyclopedia, https://encyclopedia.pub/entry/22258 (accessed July 19, 2025).
Wu, H., & Chu, P. (2022, April 25). Liquid Biopsy Techniques for Breast Cancer. In Encyclopedia. https://encyclopedia.pub/entry/22258
Wu, Hsing-Ju and Pei-Yi Chu. "Liquid Biopsy Techniques for Breast Cancer." Encyclopedia. Web. 25 April, 2022.
Liquid Biopsy Techniques for Breast Cancer
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This entry is adapted from the peer-reviewed paper 10.3390/cancers14092052

Breast cancer is the most common cancer and leading cause of death worldwide. Therefore, it is important to diagnose and treat breast cancer early. Current diagnostic methods include mammography and tissue biopsy; however, they have limitations. Liquid biopsy is a less invasive tool for diagnosis.

breast cancer liquid biopsy circulating tumor cells

1. Introduction

Breast cancer is the most common female cancer in 2020, with an incidence of estimated 2.3 million, representing 11.7% of total cancer cases in the world, and the leading cause of cancer mortality in women, which was responsible for nearly 685,000 deaths worldwide [1]. In US, the American Cancer Society’s 2022 update estimated that approximately 287,850 new cases of breast cancer will be diagnosed in US women, with an estimated 43,250 deaths [2]. Based on immunohistochemistry classification, breast cancer is classified to five major molecular subtypes: luminal A (estrogen receptor (ER)+, progesterone receptor (PR)+, human epidermal growth factor receptor 2 (HER2)−, Ki-67 low), luminal B HER2− (ER+, PR+, HER2−, Ki-67 high), luminal B HER2+ (ER+, PR+, HER2+, Ki-67 high), HER2 (ER−, PR−, HER2+), and basal-like (triple-negative (TNBC), ER−, PR−, HER2−), which are related to the clinical outcomes [3][4].
Despite advances in diagnosis and treatments for breast cancer, the standard methods have several drawbacks, such as being invasive, expensive, not suitable for all patients, and low sensitivity and specificity [5]. Classical diagnostic/monitoring techniques include imaging (mammography, ultrasound, MRI, CT, PET, and X-ray) and tissue biopsy [6][7]. Mammography can lead to both false-positive and -negative results, unnecessary exposure to radiation, and the excessive use of biopsies, and it may fail to rapidly detect the changes in tumor burden [8]. Particularly, tissue biopsy is an invasive procedure that is neither extensive enough to capture the overall genomic landscape of breast tumors nor applicable for monitoring treatment response [9]. These limitations point to the urgent need for better and novel non-invasive methods for early detection, patient survival prediction, and treatment response monitoring. Recent advances in molecular testing and genomics have led the trend of personalized and precision medicine.
Liquid biopsy has attracted considerable attention and become an attractive alternative strategy, as it is a minimally invasive molecular procedure for advanced monitoring of cancer. It relies on the quantification of genetic materials derived from tumor cells and released into circulation, such as circulating tumor cells (CTCs), cell-free DNA (cfDNA)/circulating tumor DNA (ctDNA), circulating tumor RNA, extracellular vesicles (EVs), circulating tumor proteins, and tumor-educated platelets (TEPs) (Figure 1) by collecting body fluids, mostly peripheral blood [10][11]. In comparison with traditional tissue biopsy, liquid biopsy offers a number of notable advantages with easier and non-invasive sampling for serial evaluation [12]. A liquid biopsy, combined with highly sensitive molecular technologies and advance bioinformatics protocols, could reflect the intra-tumoral heterogeneity (spatial heterogeneity) and molecular evolution of a distant metastatic lesion (temporal heterogeneity), which is not possible for conventional tissue biopsies, as the biopsy specimen may not be representative of all the tumor cells [13][14][15][16]. Furthermore, it is possible for the early diagnosis and screening, prediction of prognosis, early relapse detection in localized and locally advance breast cancer, minimal residual disease (MRD) identification, and longitudinal monitoring of the disease progression and treatment response surveillance during adjuvant and neoadjuvant therapies upon sequential sampling, due to its minimally invasive nature [9][17][18] (Figure 1). Despite these, detection limits of liquid biopsy still exist. The low levels of CTCs and ctDNA found in early-stage breast cancer, along with the lack of ctDNA secreting from some tumors, can further complicate detection. Moreover, genetic patterns in primary tumors and metastases vary significantly from patient to patient [12][19]. More sensitive detection methods are urgently needed to improve the clinical application of liquid biopsy.
Cancers 14 02052 g001 550
Figure 1. Comparison of liquid biopsy and tissue biopsy. Liquid biopsy is a minimally invasive method and relies on quantification of genetic materials derived from tumor cells and released into circulation, such as circulating tumor cells (CTCs), cell-free DNA (cfDNA)/circulating tumor DNA (ctDNA), circulating tumor RNA, extracellular vesicles (EVs), and circulating tumor proteins. Liquid biopsy allows for early diagnosis and screening, prediction of prognosis, early relapse detection in localized and locally advance breast cancer, minimal residual disease (MRD) identification, and longitudinal monitoring of the disease progression and treatment response. Therefore, liquid biopsy can be applied in as many time points as required during tumor progression and treatments, in order to detect recurrence and monitor response to treatment (green arrows). In contrast, tissue biopsy is an invasive procedure and not applicable for monitoring treatment response; subsequently, tissue biopsy is mainly applied at the time points for diagnosis and detection of recurrence during tumor progression (purple arrows).

2. Tumor Components

Liquid biopsy components, termed tumor circulome, including CTCs, cfRNA, ctDNA, TEPs, EVs, proteins, and metabolites, are secreted from tumor (apoptotic or necrotic) cells [20][21] (Figure 1). These tumor components present novel and minimally invasive biosources that are clinically implicated in precision medicine [22]. Notably, CTCs and ctDNA have been approved by the US Food and Drug Administration (FDA) as biomarkers in clinical use for cancer management [23].

2.1. Circulating Tumor Cells (CTCs)

CTCs are cancer cells compromising of a heterogeneous population with the majority of cells being highly differentiated, while others have stem cell-like properties (CSCs). They are released from primary and metastatic tumors into the circulation by trans-endothelial transition as single cells or clusters. These cells, which are able to adapt and survive by epithelial-to-mesenchymal transition (EMT) in the bloodstream and different tissues, can form new tumors or metastases [24][25][26][27]. Interacting with blood components, such as platelets, is critical for promoting tumor cells for subsequent metastasis [28], and interaction with immune cells results in evasion from immune surveillance and formation of metastases [29][30].
There are a considerable number of studies demonstrating CTC detection as an effective technique for the evaluating treatment efficacy, early diagnoses, metastatic progresses, recurrence, and prognosis [31][32][33], and it was correlated with unfavorable prognosis, shorter disease-free survival (DFS) and overall survival (OS), lack of treatment efficacy with poor recurrence-free survival (RFS), and tumor progression [34][35][36][37]. Several researches showed that CTC enumeration could be an independent prognostic tool for early breast cancer patients, particularly for metastatic breast cancer [38][39]. CTCs are substantially less abundant in the blood of patients with early stage of tumors [34][35][36][37]. Cristofallini et al. applied CTCs, detected by CellSearch system, to stratify patients into Stage IV aggressive with ≥5 CTCs/7.5 mL and Stage IV indolent with <5 CTCs/7.5 mL. In a pooled analysis of 2436 metastatic breast cancer patients, Stage IV indolent patients had significantly longer median OS (36.3 months) than Stage IV aggressive patients (16.0 months, p < 0.0001), independent of metastasis localization, tumor subtype, and molecular variables [40]. Therefore, further demonstrated CTC count is an important prognostic tool for metastatic breast cancer. More recently, 1933 HER2- metastatic breast cancer patients who participated in DETECT III and IV trials were screened, and it was confirmed that the CTC count has a high prognostic relevance [41]. Intriguingly, patients with ER- and PR+ tumors were more likely to harbor ≥1 CTC with strong HER2 staining, and it was significantly associated with shorter OS (median OS: 9.7 vs. 16.5 months in patients with CTCs with negative-to-moderate HER2 staining, p = 0.013). CTC detection, in patients with HER2- breast cancer, is a strong prognostic factor, and it remains the largest study conducted in HER2- metastatic breast cancer.
In addition to the prognostic value, in the STIC CTC randomized, multicenter prospective, noninferiority phase 3 trial, 755 hormone receptor (HR)+, HER2- metastatic breast cancer patients were allocated into either clinician-driven group, where the decision to administer hormone therapy or chemotherapy was made clinically without the CTC results, or a CTC-driven group, where endocrine therapy was administered if CTC <5/7.5 mL and chemotherapy administered if CTC ≥5/7.5 mL. Median progression-free survival (PFS) was significantly longer in the CTC-driven arm (15.5 months, 95% CI: 12.7–17.3), compared with the clinically-driven arm (13.9 months, 95% CI: 12.2–16.3) [33]. CTC is promising to direct therapy. However, there is the need for more studies to validate this. Other studies also proved that CTCs can be applied in real-time monitoring treatment responses at different time points during the tumor progression and for the detection of relapses [42][43] (Figure 1). In another study of F.C. Bidard’s group, the CirCe01 trial evaluated the clinical utility of CTC-based monitoring of therapy [44]. In this prospective, multicentre, randomized phase III study (NCT01349842), patients with metastatic breast cancer, scheduled beyond the third line of chemotherapy, were randomized between the CTC-driven arm and standard arm. However, OS was not significantly different between two groups (p = 0.8). In subgroup analyses, patients with no CTC response who switched chemotherapy early nevertheless experienced longer median PFS and OS than those who did not.
Beside blood, as the most commonly studied and clinically used fluid in liquid biopsy, Malani et al. [45] recently applied the CTC count in cerebrospinal fluid (CSF) diagnose leptomeningeal metastases in HER2+ breast cancer patients. Their study also proved that CSF CTC enumeration could assess the tumor burden in the central nervous system during therapy for leptomeningeal metastasis and before detectable changes on MRI images or CSF cytology [45]. Importantly, these recent studies on CTC, as a liquid biopsy, confirmed its clinical value in prognosis and role in dynamic and real-time monitoring of treatment, although there is no current clinical application of CTC [39].

2.2. Cell-Free DNA (cfDNA) and Circulating Tumor DNA (ctDNA)

Like CTC, cfDNA and ctDNA play important roles in liquid biopsy. cfDNA refers to the double- or single-stranded fragmented DNA liberated into body fluids, such as blood, saliva, lymph, tear fluid, bile, urine, milk, sweat, mucous suspension, amniotic, cerebrospinal and pleural fluids, cervicovaginal secretion, and wound efflux, by both normal and tumor cells, whereas circulating tumor DNA (ctDNA) represents only a fraction of cfDNA derived from the tumor tissue [9][46][47]. Specific patterns of cfDNA can be analyzed ex vivo to characterize the targets of interest [48]. While cfDNA is present in healthy controls, its concentration is significantly lower in healthy subjects, compared to cancer patients, due to active nuclease degradation [49][50].
In addition to cfDNA gene sequence and mutation, cfDNA can be further analyzed for epigenetic alterations, such as DNA methylation, histone modifications, and expression of long and micro non-coding RNAs [51][52]. Methylation changes in DNA contribute to gene expression regulation and play a significant role in the etiology of early breast cancer [53][54]. The DNA methylation pattern is retained in the cfDNA released from its tissue origins of tumor cells [55][56]. Therefore, DNA methylation could serve as important biomarkers for diagnosis of cancer [57]. Indeed, DNA methylation has been assessed in cfDNA in several studies, both single and panels of genes have been demonstrated as diagnostic tools [58][59][60]. Furthermore, the methylation patterns of cfDNA could be also related to relapse, metastasis, and survival [5]. Panagopoulou et al. established a cfDNA methylation panel of five cancer-related genes (KLK10, SOX17, WNT5A, MSH2, and GATA3) and found that increased methylation of three or more and four or more genes (KLK10, SOX17, WNT5A, and MSH2) significantly correlated to OS (p = 0.042, 0.043, and 0.048) and the absence of pharmacotherapy response (p = 0.002), respectively. Subsequently, using machine learning combined clinical data and experimental findings, they developed multi-parametric prognostic signatures for the prediction of survival and treatment response to chemotherapy in metastatic breast cancer [19].
Correlations between elevated concentrations of cfDNA and tumor stage, tumor size, and nodal involvement were demonstrated [19]. In particular, Panagopoulou et al. showed that the metastatic breast cancer patients who had cfDNA levels > median value of 496.5 ng/mL had significantly shortened PFS, compared with those who had < median value of cfDNA (p = 0.036), indicating cfDNA quantification could serve as a prognostic marker for PFS. For the predictive value of cfDNA levels for the treatment response of metastatic breast cancer patients to first-line chemotherapy, the median value of cfDNA of the “non-responders” (970.0 ng/mL) was significantly higher than that of the “responders” (465.0 ng/mL, p = 0.026), thereby demonstrating cfDNA as a potent predictive marker for response to first-line chemotherapy [19]. The prognosis values of the combination of CTC and cfDNA were firstly evaluated by Ye et al. [61] by collecting blood samples from 117 metastatic breast cancer patients. High levels of CTC (CTC ≥ 5) and cfDNA, individually or jointly, had significantly higher risks of PFS and OS (CTC: p < 0.001 for PFS, p = 0.001 for OS; cfDNA: p = 0.001 for PFS, p = 0.002 for OS; joint effect: p < 0.001 for PFS, p = 0.002 for OS). In a similar result, Fernandez-Garcia et al. compared cfDNA and CTCs with conventional breast cancer blood biomarkers (CA15-3 and alkaline phosphatase (AP)) by analyzing blood samples from 194 metastatic breast cancer patients. Their results showed that both CTCs and total cfDNA levels are predictors for OS (p = 0.001 and 0.024, respectively), while only cfDNA correlated with PFS (p = 0.042), indicating their potential clinical application of liquid biopsy [62].
Generally, ctDNA can be released into the bloodstream by excretion and transport in exosomes or during the apoptosis and necrosis of tumor cells [47]. ctDNA is a small nucleic acid fragment of about 134–144 bp [50][63]. ctDNA is more abundant than CTCs, but it is more rapidly cleared from circulation, within hours, than CTCs. Moreover, ctDNA has been demonstrated to accurately represent the mutational profile of CTCs; Thierry et al. showed that ctDNA can capture the majority of mutations found in tissue biopsy, such as the PIK3CA and ESR1 mutations [64]. However, the evidence on the prognostic value of ctDNA in metastatic breast cancer is rather limited, especially compared with CTCs [65]. Specific somatic DNA mutations, loss of heterogeneity (LOH), and epigenetic alterations, such as methylations, are the valuable factors for precisely discriminating the cfDNA from normal cell and tumor cell [66]. LOH is a cross chromosomal event that results in the loss of one normal allele producing a locus with no normal function [67]. This is a common mechanism for cancer development as the inactivation of a tumor suppressor gene occurs [68]. ctDNA has been demonstrated to detect cancer in early stages [69][70], determine prognosis [13], real-time monitor treatment response [71], and determine therapeutic resistance [72], MRD after primary treatments, and relapse [73][74]. Minimally invasive serial measurement of ctDNA might, thus, monitor and predict treatment response, presenting an advantage over tissue biopsy [5][75][76][77] (Figure 1). Remarkably, increases in ctDNA levels could predict disease progression several months before standard imaging techniques [64]. However, ctDNA has not yet been validated to apply in clinical practice [78].
Prognostically, ctDNA detection was correlated with poor survival in early breast cancer [79][80][81][82]. As in early breast cancer, the quantity of ctDNA is associated with a worse outcome in metastatic breast cancer [75][76][77][83][84][85]. In both the INSPIRE phase II and LOTUS randomized phase II trials, ctDNA levels in TNBC were correlated with PFS, OS, and overall clinical response rate (ORR) [86].
In the aspect of recurrence, a prospective and multicenter study utilized serial plasma samples to assess patients with early-stage breast cancer [74]. Somatic mutations of primary tumors were identified by sequencing, and personalized tumor-specific digital polymerase chain reaction (digital PCR, dPCR) assays were applied to surveil these mutations. Plasma samples were collected every three months for the first-year follow-up and subsequently every six months. The results showed that the presence of ctDNA had a median lead time of 10.7 months before the development of clinical symptoms, indicating ctDNA could predict relapse. Moreover, the use of ctDNA could detect extracranial metastatic relapse in 96% of patients. This addressed that the use of ctDNA, as a surveillance technique, may improve survival.
A number of studies have evaluated ctDNA levels in both the neoadjuvant and adjuvant therapies [81][82][87][88][89]. In the phase 2 I-SPY 2 trial, Magbanua et al. examined the serial ctDNA test, in early breast cancer patients undertaking neoadjuvant chemotherapy, for predicting pathologic complete response (pCR) and risk of recurrence. Blood samples were collected at several time points, i.e., pretreatment, after therapy initiation, between regimens, or prior to surgery. Patients who remained ctDNA-positive after therapy initiation were significantly more likely to have residual disease after neoadjuvant chemotherapy (83% non-pCR) than those who were ctDNA-negative (52% non-pCR, p = 0.012). After neoadjuvant chemotherapy, the presence of ctDNA was associated with lower pCR rates, whereas ctDNA clearance after treatment was correlated with longer survival. Therefore, personalized monitoring of ctDNA during treatment may be a good predictor treatment response [81]. McDonald et al. also demonstrated nonmetastatic breast cancer patients with lower ctDNA concentrations achieve pCR than patients with higher ctDNA level after neoadjuvant therapy (p = 0.0057) [89], illustrating that personalized ctDNA panels could monitor breast cancer progression in the neoadjuvant setting. Most recently, Papakonstantinou et al. [90] performed a systematic review and meta-analysis to investigate the prognostic value of ctDNA in patients with early breast cancer treated with neoadjuvant therapy. The association between the detection of ctDNA, both at baseline and after completion of neoadjuvant therapy, and worse relapse-free survival (HR: 4.22, 95% CI: 1.29–13.82 and HR: 5.67, 95% CI: 2.73–11.75, respectively) and OS (HR: 19.1, 95% CI: 6.9–53.04 and HR: 4.00, 95% CI: 1.90–8.42, respectively) were observed, whereas the detection of ctDNA did not achieve a pCR. Therefore, this meta-analysis again supports the previous studies.
In metastatic breast cancer, Darrigues et al. also collected plasma samples of 61 patients at different time points, i.e., before treatment, at day 15, at day 30, and at disease progression, and proved that treatment with palbociclib and fulvestrant can be successfully monitored by serial ctDNA measurements before radiological evaluation [77]. However, more large, prospective, and randomized trials are needed. Interestingly, a study evaluated the predictive and prognostic values of ctDNA in 26 TNBC patients and observed a significant rise in ctDNA levels after neoadjuvant therapy was predictive of residual tumor and, thus, an incomplete pathologic response. This also indicated worse relapse-free survival (p = 0.046) and OS (p = 0.043) [79]. These studies support using serial monitoring of ctDNA for accurate assessment of tumor progression in real time, resulting therapeutic decision making. However, more clinical studies will be required before ctDNA monitoring can be implemented in a clinical setting [12][79].

2.3. Non-Coding RNAs

It is known that RNA, especially non-coding RNA (ncRNA), plays significant roles in the deregulation of cell function and cancer development. Like CTC and ctDNA, RNA can also be secreted from tumor cells into blood and other biological fluids of cancer patients and, thus, as a potential analyte in liquid biopsy [91][92]. However, RNA is less stable than CTC and DNA and hindered by the variability in the methodologies performed [93]. Despite these, there are growing evidences depicting the importance of circulating ncRNAs representing 80% of the total circulating RNA application in the field of oncology. They are involved in regulating transduction pathways, acting as tumor activators or suppressors [94]. There are a number of types of ncRNAs, including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs) [95][96].
microRNAs (miRNAs) are small ncRNAs (18~25 nt), capable of binding and regulating mRNA expression at the post-transcriptional level [97]. Additionally, miRNAs play important role in cellular communication, proliferation, programmed cell death, and differentiation [98]; thus, they have significant implications in cancer management [99] as potential biomarkers applied in liquid biopsy. miRNAs are derived from CTCs, cell-free miRNAs, apoptotic bodies, or from extracellular vesicles (EVs), either in their lumen or on their surface [100]. miRNAs are the most studied RNA types in tissues and the bloodstream, where several studies proved their clinical application in diagnosis, prognosis, detection of metastasis, and drug resistance [101][102][103][104]. However, little is known about their clinical utility as biomarkers in liquid biopsy, which requires more studies [101].

2.4. Extracellular Vesicles (EVs)

EVs refers to the cell-derived membranous vesicles released by all cells into the extracellular environment [105]. They play a role in intracellular communication among tumor cells [106]. EVs carry DNA, mRNA, ncRNA, lipids, metabolites, and proteins protecting and preventing degradation of their cargo from enzymes, such as plasma nucleases, and transferring their contents from a parental to different recipient cells [107][108]. Unlike CTCs, which are mostly released into blood, EVs exist in a variety of body fluids and can be more easily enriched for subsequent analysis than CTCs [109]. cfDNA is secreted into the bloodstream either as free DNA (unbound DNA), bound to protein or lipoprotein complexes (nucleosomes and vitrosomes) [110], or enclosed in EVs [111][112].
It has been proven that EVs, involve in the tumor development and initiating the formation of premetastasis niche, play a role in intracellular communication [113]. Tumor-derived vesicles also carry the molecular footprint reflecting the genetic status of parental tumor cells [114]. EVs have been demonstrated as diagnostic, prognostic, and therapeutic agents in clinical settings and have also been associated with drug resistance [115]. As a result, EVs are promising biomarkers in liquid biopsy. However, further studies are required to investigate their clinical validity in breast cancer [5].
EVs are generally heterogeneous and classified into microvesicles (MVs, also referred to as ectosomes or microparticles), exosomes, and apoptotic bodies, based on origin and size [116][117]. Apoptotic bodies are the largest vesicles (1~5 μm in diameter) derived from budding of apoptotic cells and usually contain nucleosomes, protecting tumor DNA and RNA from degradation by DNAses and RNAses [118][119].
The second largest EVs are microvesicles with large diameters (100–1000 nm) that are actively shed from protuberances in the plasma membrane [120][121]. Tumor-derived microvesicles (TDMs) contain DNA reflecting the genetic status of their original cell [5]; they also carry RNA that can be transferred to recipient cells [114]. It was found that the number of TDMs in the plasma of breast cancer patients was significantly associated to disease stages I-IV (p < 0.05 and p < 0.0001) [122], indicating a clinical value.
Exosomes, the best studied EVs, with small diameters (30–150 nm) derived from the endocytic pathway, are secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane [105][117][121]. Exosomes are secreted by almost all types of cells and can be transferred to recipient cells [123]. They also play critical roles in intercellular communication and can deliver their content to other cells in a paracrine fashion. Importantly, exosomes are also detected in biological fluids, including blood, saliva, urine, breast milk, and cerebrospinal fluid, indicating that they can act as mediators in long distance cellular signaling [124][125][126]. In particular, it has been demonstrated that exosomes contribute to cancer development and metastasis, preparation of the pre-metastatic niche, stem cell stimulation, apoptosis, angiogenesis, immunity, and drug resistance [117][127][128][129]. Tumor-derived exosomes also contain cancer-associated miRNA [130] and proteins [131] that could have diagnostic, prognostic, and therapy monitoring values. Exosomal miRNAs are also associated with tumor aggressiveness [132], angiogenesis [133], metastasis [134], and drug resistance [135] in breast cancer. Remarkably, it has been shown that tumor cells secrete more exosomes than normal cells in response to pathophysiological conditions, such as hypoxia in the tumor microenvironment [129]. Furthermore, exosomes from breast cancer patients contain distinct RNA and protein from healthy donors [136][137].
EVs represent one of the latest biomarkers in the liquid biopsy field; thus, the clinical application of EVs is still immature, and no standard detection method exists for breast cancer [23]. More clinical studies are required to confirm the clinical relevance of EVs, such as diagnosis and prognosis, and evaluate the sensitivity and specificity of EVs-based assays.

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Subjects: Oncology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Hsing-Ju Wu
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Pei-Yi Chu
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Update Date: 26 Apr 2022
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