Application of liquid biopsy in precision medicine: opportunities and challenges

Abstract

Precision medicine for cancer patients aims to adopt the most suitable treatment options during diagnosis and treatment of individuals. Detecting circulating tumor cell (CTC) or circulating tumor DNA (ctDNA) in plasma or serum could serve as liquid biopsy, which would be useful for numerous diagnostic applications. Liquid biopsies can help clinicians screen and detect cancer early, stratify patients to the most suitable treatment and real-time monitoring of treatment response and resistance mechanisms in the tumor, evaluate the risk for metastatic relapse, and estimate prognosis. We summarized the advantages and disadvantages of tissue and liquid biopsies. We also further compared and analyzed the advantages and limitations of detecting CTCs, ctDNAs, and exosomes. Furthermore, we reviewed the literature related with the application of serum or plasma CTCs, ctDNAs, and exosomes for diagnosis and prognosis of cancer. We also analyzed their opportunities and challenges as future biomarkers. In the future, liquid biopsies could be used to guide cancer treatment. They could also provide the ideal scheme to personalize treatment in precision medicine.

Keywords

liquid biopsy / circulating tumor cells / cell-free ctDNA / exosomes / precision medicine

Introduction

Diagnosing and screening tumors through non-invasive methods represent an important paradigm shift in precision medicine. Tumors are highly heterogeneous, and sampling in its entirety is challenging. Tissue biopsy is invasive and cannot reflect current tumor dynamics or sensitivity to the treatment. With the development of sensitive techniques that can detect rare mutations, the heterogeneous landscape of tumor can be determined using a blood sample [1].

Liquid biopsy refers to the analysis of circulating tumor cells (CTCs) and cell-free circulating nucleic acids (in particular, circulating tumor DNA (ctDNA) and exosomes) released in the peripheral blood from the primary tumor and/or metastatic deposits. A liquid biopsy or blood sample can provide the genetic landscape and epigenetic characteristics of all cancerous lesions and offer the opportunity to track genomic evolution systematically [2]. The response to treatments and the development of acquired resistance should also be predicted. Details of the tumor genetic profile can enable the prediction of disease progression and response to therapies. Liquid biopsy can be used in cancer screening, patient stratification, and monitoring [3]. In addition, short ctDNA fragments harbor the footprints of transcription factors, which could indicate the cell-type origin of pathological states, such as cancer [4].

Table 1 provides a summary of the advantages and disadvantages of tissue and liquid biopsies.

CTCs

CTCs were first detected in cancer patients in 1869 [7]. CTCs, ctDNAs, and exosomes are released from the primary tumor and/or metastatic sites into the bloodstream, as shown in Fig. 1. Numerous studies in the past decade showed that CTCs may be used as a biomarker to predict cancer progression and survival in metastatic [7–9] and possibly in early-stage cancer patients [5,10].

Detection and clinical application of CTC

An average metastatic carcinoma patient shows 5–50 CTCs for approximately 7.5 ml of blood. High numbers of CTC correlate with aggressive disease, increased metastasis, and decreased time to relapse [11]. CTCs potentially carry valuable information about tumor composition, invasiveness, drug susceptibility, and resistance to therapy [12]. Given that high CTC burden can predict poor prognosis in metastatic disease, efforts regarding aggressive chemotherapeutic intervention are also being tested [13].

Therefore, CTCs could serve as a real-time marker for disease progression and survival. CTCs can also screen and detect cancer early, guide therapeutic management, determine therapy effectiveness or necessity even in the absence of detectable metastases, and provide insights into the mechanisms of drug resistance.

Opportunities and challenges of CTCs

The potential clinical value of CTCs is clear: early detection and treatment of metastatic spread are key for disease outcome, and CTCs can target metastasis in real time [14]. CTC enumeration and characterization with certified systems offer reliable information on prognosis, and liquid biopsy may be considered to identify therapeutic targets or mechanisms of resistance on metastatic cells, such as mutations in KRAS [15]. CTC enumeration correlates with poor clinicopathological features and exhibits prognostic and predictive values.

Despite this considerable potential, CTC application is confronted with many challenges [15]. The possibility of using tumor metastasis with CTCs for detection should also be assessed. The few CTCs in serum and current detection technologies limit the further clinical application of CTCs. The genetic and epigenetic variation data collected by analyzing solid tumor biopsies provide valuable insights into a patient’s disease and therapy options. In the near future, CTCs may significantly improve our ability to monitor malignancies and indicate whether or not a treatment may work, thereby providing the complementary roles of conventional and liquid biopsies [16].

ctDNA

ctDNA can be released from primary tumors, CTCs, micrometastasis, or overt metastases into the blood of cancer patients. The majority of ctDNA originates from apoptotic and necrotic tumor cells that release their fragmented DNA in the blood. ctDNA concentration in serum or blood is significantly increased compared with that of healthy individuals [17]; this change is correlated with malignancy [18].

Detection and clinical application of ctDNA

Increased concentrations of ctDNA fragments are observed in blood plasma and serum of cancer patients with various tumor types, and they are correlated with unfavorable outcome [19]. Analysis of tumor-linked genetic alterations is increasingly used for diagnostic, prognostic, and treatment purposes. ctDNA can be a promising biomarker in the liquid biopsy of cancer [20]. In addition, ctDNA methylation assay can significantly promote diagnoses of cancer of unknown primary origin and guide remarkably precise therapies associated with improved outcomes [21].

DNA methylation profiling can also be applied in clinic, but confronted with challenges [22]. DNA methylation profiling can be used for disease diagnosis. Non-invasive DNA methylation markers for diagnosis of many cancers, such as GSTP1 hypermethylation as a biomarker of prostate and colorectal cancers with a sensitivity of 82% and a specificity of 95% [23], frequently show hypermethylation of APC, MGMT, RASSF2, and WNT inhibitory factor 1 [22].

Opportunities and challenges of ctDNA

Analyses of ctDNA may replace CTC detection for monitoring cancer progression in the future [6,24–26]. The major challenge with ctDNA analysis is assay sensitivity and specificity. Multigene panel analysis of ctDNA may increase test sensitivity. Many researchers indicated that ctDNA is more sensitive than CTCs in early detection; ctDNA also presents large dynamic range and considerable relation with changes in tumor burden [24,27].

The challenges of genome-wide DNA methylation analysis are as follows [28]: whether ctDNA reflects a representative characteristic of a cancer, whether metastatic tumors release the same amount of DNA as the primary tumors, whether all the cells in a tumor release the same amount of ctDNA as the other, and whether an accurate profile of tumor burden or a real-time monitoring of emerging or epigenetic mutations saves patients or improves their quality of life [29].

However, results regarding these biomarkers should be validated in independent cohorts of cancer patients. Furthermore, the clinical relevance in daily practice is difficult to determine. The dynamic changes in the ability to classify different subtypes of cancers and measure their evolution functional properties by non-invasive blood monitoring strongly relate cancer therapeutics and diagnostics.

Exosomes

Exosomes are lipid-bilayer-enclosed extracellular vesicles (carrying RNA, DNA, and protein) secreted by cells and circulated in the blood; these vesicles can also function as intercellular messengers, which contribute to their increased recognition in the scientific field [30–35]. Microvesicles, such as exosomes, contain miRNAs, proteins, and DNAs; they have also become promising diagnostic tools of liquid biopsy [36,37]. In addition, exosomes mediate tumor metastasis [38,39].

Detection and clinical application of exosomes

Exosomes present considerable application prospects, such as depiction and capture of tumorigenesis, tumor progression, and predictive markers, as shown in Fig. 2. The isolation of cancer exosomes from cancer patients remains a challenge owing to the lack of specific markers that can differentiate cancer from noncancer exosomes. Melo et al. provided evidence that GPC1 may serve as a pan-specific marker of cancer exosomes. Therefore, GPC1 may be an attractive candidate for detection and isolation of exosomes in the blood serum of patients with cancer for genetic analysis of specific alterations [36].

Opportunities and challenges of exosomes

Detection of exosomes regarding somatic genetic alterations in the blood has been challenging, but new approaches for such analyses have improved the sensitive and specific detection at low levels.

Table 2 presents a summary of the advantages and limitations of detecting CTCs, cell-free ctDNAs, and exosomes [3,40].

Perspectives

CTCs, ctDNAs, and circulating exosomes [36,42] might serve as promising novel candidates as blood-based biomarkers. However, when and how the liquid biopsy will be used as a routine method for cancer patient care should be further determined. Currently, a real gap exists between the gained attraction of liquid biopsy in media and the increased number of publications in this field and its application to the patients. Finally, future blueprint of liquid biopsy in precision medicine should include the affordable cost, reproducibility and reliability of the results, and performance comparison of liquid biopsy and the existing solid biopsy among cancer patients. On the basis of this perspective, we can adopt the optimal strategy or remarkably effective drugs based on patient characteristics. Nevertheless, in the absence of subsequent treatment strategies or drugs, significant benefit from the early detection and precision medication is not obtained, despite that many risk factors related with the cancer or other diseases are screened.

In summary, the molecular and functional analyses of CTCs and circulating nucleic acids could be used as promising diagnostics and prognostics to enhance the stratification of therapies and acquire insights into precision therapy.

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