Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review)
- Authors:
- Published online on: August 6, 2018 https://doi.org/10.3892/ijo.2018.4516
- Pages: 1395-1434
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Copyright: © Finotti et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
1. The concept of liquid biopsy
Liquid biopsy investigates circulating tumor cells (CTCs) and/or cell-free nucleic acids in the peripheral blood of cancer patients (Fig. 1) and is considered one of the most advanced non-invasive diagnostic systems with which to obtain key molecular information relevant to clinical decisions and the practice of precision medicine (1-5). Diagnostic actions include, but are not limited to, early diagnosis, staging, prognosis, the prediction of therapeutic responses, and follow-up during therapeutic intervention (5-13). Historically, the applications of liquid biopsy for the characterization of cancer patients have been focused on CTCs (1). Looking for CTCs in peripheral blood has generated a very large number of reports focusing on diagnosis, prognosis and therapeutic management (6). The downstream characterization of CTCs, including the identification of possible therapeutic targets (e.g., mutations or other traits of aggressiveness) in this peculiar tumor cell subset not only has had a great impact on diagnosis and prognostication, but also has an impact on clinical protocols, charting the route to precision medicine (14-16). In this respect, an excellent example is colorectal cancer (CRC), one of the most frequent malignancies worldwide (17). As is known, the transformation of normal colonic epithelium into CRC is punctuated by the progressive accumulation of acquired genetic and epigenetic alterations deeply altering morphological parameters, cell growth potential and differentiation, and shutting down apoptosis. Recent basic and clinical research on CTCs in patients with CRC has underlined that the molecular detection of CTCs in peripheral blood is feasible, and their phenotypic characterization drives therapeutic protocols for tailored clinical interventions (18). Moreover, the real-time monitoring of CTCs in patients with CRC has been extensively applied for a better mechanistic understanding of the factors determining clinical outcome and the efficacy of therapeutic treatment, as well as the stability of therapeutic effects over time (18-21).
In addition to CTCs, the formal demonstration that free nucleic acids are present (although short-lived) in biological fluids (plasma being investigated by most authors), has led to the development of a large wealth of studies aimed at circulating DNA and RNA (22-24). This strategy, similar to CTC detection, allows for non-invasive diagnosis, and at the same time it represents a convenient method for directly interrogating tumor aberrations, addressing tumor heterogeneity and metastatic dissemination across multiple, longitudinally collected clinical specimens (6,7). On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of circulating free DNA (cfDNA), the instability of RNA, low analyte concentrations, and the confounding, variable presence of DNA and RNA from normal tissues and mutated cells from the hematopoietic compartment (clonal hematopoiesis) (25). Limitations notwithstanding, the analysis of cfDNA has successfully identified mutations arising in, and the pathognomonicity of, tumor DNA, while the analysis of circulating free RNA (cfRNA) has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression (22). Examples of the detection of tumor cfDNA are presented in Table I (18-20,26-52), while examples of the detection of circulating miRNAs are presented in Table II (53-104).
 | Table IISelected examples of liquid biopsy based on the analysis of circulating microRNAs (miRNAs or miRs). |
2. Analytes in plasma: Examples of biomedical applications
Molecular targets: Cancer genetic aberrations
One of the most robust evidence supporting the application value of liquid biopsies is the detection of circulating genomic aberrations, mainly mutations. The topic is extensive, and is the subject of a number of excellent reviews. Therefore, in this review, we focus on very specific examples, particularly in early-stage tumors. KRAS mutations are a case in point, since they serve as an actionable marker for EGFR blockade therapy, are highly prevalent, and have been thoroughly investigated. For instance, Brychta et al compared plasma and paired tumor samples from early-stage pancreatic cancer patients (105) by chip-based digital PCR. Their major aim was to identify selected KRAS codon 12 mutations (G12D, G12V and G12C) in circulating tumor DNA (ctDNA). Remarkably, circulating KRAS mutations were demonstrated in 72% of the patients, were associated with tumor burden, and were undetectable in the healthy controls. This study supports the use of liquid biopsy for early cancer diagnosis. Other studies focusing on KRAS mutations in ctDNA were reported by Kinusaga et al (pancreatic cancer) (106), Couraud et al (lung cancer) (107), Perez-Carbonell et al (CRC) (108) and Case et al (lymphoblastic leukemia) (109). Table I summarizes the applicative examples of liquid biopsy for the identification of oncogene mutations, including the detection of EGFR mutations in the blood of lung cancer patients, now approved by regulatory bodies. These assays are of outmost interest and exemplify the profound difference between non-invasive liquid biopsy and invasive tumor tissue biopsy. Tissue biopsy may not reflect the genomic profile of the tumor in its entirety due to intra-tumor heterogeneity, multiple foci poorly accessible to sampling, and/or changes occurring during tumor development and/or therapy. On the contrary, the non-invasive liquid biopsy of plasma, urine or saliva samples may more effectively recapitulate the mutational complexity of the many populations (cryptic and clinically evident) accounting for tumor burden in a given patient. This makes liquid biopsy particularly suitable to identify truncal aberrations that, when targeted, may result in a considerably greater systemic clinical benefit, as compared to targeting site-specific aberrations (Lin et al, 2015) (110).
Molecular targets: Gene methylation
A variation on the theme is to look at non-mutational events marking the cancer genome. Particularly relevant in this context is DNA methylation. It has been known for quite some time that tumor progression is associated with the abnormal methylation of cancer genes. Both hypomethylation and hypermethylation have been reported. Most often, the specific DNA hypermethylation of tumor suppressor genes is observed in the context of widespread DNA hypomethylation. Since aberrant DNA methylation at specific promoter regions is a defined molecular feature of cancer, ctDNA methylation has been developed into a very promising molecular marker. DNA methylation is to date one of the preferred consensus circulating biomarkers in pre-symptomatic and symptomatic patients with CRC. This has been discussed by Warton et al (111) and by Mitchell et al (112), who have considered methylation-specific PCR assays as a novel approach for the assessment of low levels of DNA methylation in 29 regions of 17 genes. Eight differentially-methylated regions (DMRs) residing in the BCAT1, GRASP, IKZF1 and IRF4 genes, exhibited low positivity in the plasma of healthy subjects and high positivity (>59%) in ctDNA from colonoscopy-confirmed patients with CRC.
Molecular targets: Circulating microRNAs
MicroRNAs (miRNAs or miRs) are a family of small (19 to 25 nucleotides in length) non-coding RNAs which play important roles in controlling post-transcriptional gene expression. Regulatory miRNAs reduce protein synthesis through selective interactions with complementary sequences of target messenger RNAs (mRNAs) (113,114). Single or multiple mRNAs can be targeted at their 3′untranslated region (UTR), coding sequence (CDS), 5′UTR sequences, and it is calculated that >60% of human mRNAs are miRNA targets (114). The miRNA/mRNA interaction occurs at the level of RNA-induced silencing complex (RISC) and is associated with the repression of translation or mRNA degradation, depending on the levels of complementarity with nucleotide sequences on the target mRNAs (115-118). Since their discovery and first characterization, the number of human miRNAs identified and deposited in the miRBase databases (miRBase v.22, www.mirbase.org) has been steadily increasing and is now >2,500 (119,120). Research on miRNAs has confirmed the complexity of this expanding miRNA/RNA network (117-122).
Alterations in miRNA expression have been associated with different human diseases. The guided alteration of specific miRNAs may potentially lead to innovative therapeutic protocols (123,124). miRNAs function both as tumor promoters (oncomiRNAs and metastamiRNAs) and tumor suppressors (125,126), depending on their regulatory preference for oncoproteins with opposing influences on cancer cells. Based on this, it is unsurprising that circulating cell-free miRNAs have been actively investigated as liquid biopsy analytes. OncomiRNAs are abundant in several extracellular body fluids (127-132), where they are protected and stabilized by exosome-like structures and small intraluminal vesicles produced by a variety of cells (including cancer cells) (127). Hence, elevated levels of several miRNAs (including miR-221, miR-222, miR-141, miR-92a, miR-21, miR-155, miR-506 and miR4316, miR-4772 and miR-29a) are present in the blood from patients with CRC (53-62,133,134) and may contribute to the diagnosis and prognosis of patients with CRC (134). Furthermore, miRNAs may aid in the monitoring of therapeutic approaches. For instance, Ogata-Kawata et al reported that serum exosomal miRNA levels (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223 and miR-23a) were higher in patients with CRC than in the controls, that this abnormally high levels were already detectable at early disease stages, and that they were significantly downregulated following surgical resection (60).
3. Technologies
In order to identify specific DNA mutations and quantify miRNA levels in plasma and other body fluids of cancer patients, several types of technologies for DNA/RNA analysis have been proposed. For cfDNA analysis, the golden standards are possibly quantitative PCR (qPCR) and digital PCR; however, several additional technologies have been proposed (Table I), such as polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis (27), multiplex digital PCR (dPCR), allele-specific qPCR (18,39), whole genome sequencing (WGS) (28), cancer personalized profiling deep sequencing (Capp-Seq) (30), methylation-specific PCR (31,37,44), the Discrimination of Rare EpiAlleles by Melt qPCR (DREAMing) (33), bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) real-time PCR (40) and tagged-amplicon deep sequencing (TAm-Seq) (42). For miRNA analysis, qPCR and reverse transcription (RT)-PCR (53-58), NGS RNA sequencing (63), miRNA microarray analysis (60) and digital PCR (65) are the most commonly used technologies (Table II).
A common step, and under many respects a complication of all the above-mentioned technologies, is the need to amplify the minute amounts of target analytes by an enzymatic reaction with DNA modifying enzymes, most often Taq polymerase and its derivatives. Biosensing platforms hold great promise for the simple and rapid detection of cfDNA and cfRNA (135), since they skip this time-consuming, analyte-dependent, PCR amplification step. Novel PCR-free biosensing approaches are able to detect KRAS and BRAF mutations in the serum of patients with lung cancer and melanoma (136).
Digital PCR (137) is based on the limiting dilution of DNA, and single molecule detection to identify and quantify the target mutated DNA in a given sample (138,139). This experimental approach is very useful for the identification of rare variants and in non-invasive diagnosis on peripheral blood, since only a small concentration of template is required for the analysis. Next generation sequencing (NGS) is a high throughput DNA sequencing technology which allows for the analysis, in a single reaction, a large variety of different DNA aberrations across multigene panels (140,141), although comprehensiveness may somewhat detract from sensitivity. Different commercial NGS platforms are available, such as Genome Analyzer and HiSeq 2000 (Illumina), HeliScope (Helicos BioSciences), SOLiD and Ion Torrent (Life Technologies), Roche/454 (Roche). In these instruments, templates, primers or polymerase enzymes are immobilized on a solid support or on microbeads before sequencing, allowing the process of millions of microreactions carried out in parallel on each spatially distinct template.
However, as already pointed out, several challenges are related to liquid biopsy, the most important of which is the amount of target molecules to be detected and quantified. As far as cfDNA detection is concerned, these target molecules are so diluted by normal DNA that existing sequencing methods, such as Sanger sequencing, were not considered sufficiently sensitive to detect tumor-associated DNA mutation. As shown in Table I, the most commonly used approach was based on mutation-specific PCR, a technology proven to exhibit sufficient specificity and sensitivity allowing for the detection of the weak tumor signal present in the patient's circulation. This technology may be associated with important drawbacks when the quantification of miRNAs is considered, suffering from biases in the template-to-product ratios of the amplified target sequences (141). In addition, differential RT efficiency on different miRNA targets may also introduce variability when miRNA patterning is considered. Once again, PCR-free detection strategies are of great interest (142,143).
4. Experimental model systems for technological validation
Liquid biopsy is a complex strategy requiring pre-analytical steps, post-analytical optimization, and the careful selection of optimal analytes for specific biological queries. In vivo model systems may be very useful in addressing and isolating these numerous individual variables (that are both technical and biological), and validate complex multi-step approaches. It is surprising, in this respect, that only few reports are available focusing on the use of animal models. For example, Garcia-Olmo et al directly compared the tumor ctDNA concentration and the number of circulating cancer cells in rats with xenograft tumors during the spread of CRC (144). Of note, they found that high ctDNA levels preceded the presence of CTCs. Rago et al (145) developed an elegant and highly sensitive qPCR test to quantify ctDNA by targeting LINE-1 in mouse xenografts, demonstrating that this experimental system enables the monitoring of systemic tumor burden and close examination of the therapeutic management on a variety of animal tumor models. These studies demonstrate the importance of ctDNA and how it intertwines with CTCs. In a more recent study, Thierry et al (146) evaluated the relative quantitative contributions of non-tumor, tumor and mutated ctDNA, as well as ctDNA integrity, in an animal model. In this case, they found differences between patients with CRC and nude mice xenografted with human colon cell lines, suggesting that further research is necessary to validate in vivo model systems based on mice xenografted with tumor cell lines.
As for miRNAs, different independent studies have firmly demonstrated that miRNAs released into the circulation by tumor xenografts are distinct from 'background' mouse miRNAs. This is a key point, since pre-existing miRNAs present in mouse body fluids may be a powerful confounding parameter, possibly altering conclusions and implications of any circulating miRNA signature. In this respect, the use of laboratory mouse strains has the advantage that it sets a 'background' mouse miRNA pattern that is stable and easily quantifiable. Mitchell et al demonstrated that several miRNAs originating from xenografted human prostate cancer cells are present in the circulation (one of the most interesting being miR-141), and are readily measured in plasma, allowing a clear distinction between tumor-xenografted mice and controls (147). Selth et al (148) performed global miRNA profiling and identified a set of miRNAs exhibiting significantly altered serum levels in transgenic mice bearing prostate adenocarcinoma tumors. Among the most interesting miRNAs, they focused their attention on miR-141 and miR-375. Waters et al observed a complex miRNA dysregulation in the circulation of athymic nude mice subcutaneously injected with MDA-MB-231 cells. Some miRNAs (such as miR-10b) were undetectable in the circulation, some others (miR-195 and miR-497) were significantly decreased, the miR-221 content was not altered, and a positive correlation was observed between miR-497 and miR-195. That study highlighted the distinct roles of miRNAs in the circulation and in disease dissemination and progression, all of which may be candidates as molecular targets for diagnosis, as well as for systemic therapy (149). More recently, Greystoke et al developed a robust protocol that allowed for the specific profiling of human tumor miRNAs in microliters of tail vein plasma (150). In a recent study, Gasparello et al presented the analysis of KRAS variants and the content of miR-141, miR-221 and miR-222 in mice xenografted with colon cancer cell lines (151). These results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models (Fig. 2).
5. Specific biomedical applications
ctDNA and miRNAs find application in a variety of clinical cancer settings.
Early diagnosis
Liquid biopsy for early lung cancer detection has been reviewed by Hofman (152) and by Pérez-Ramírez et al (153). Coupled with thoracic imaging, liquid biopsy is a powerful diagnostic tool, and potentially provides surveillance recommendations for high-risk populations without a detectable nodule. In a study on patients with CRC, Bedin et al (154) examined a large cohort of patients with CRC in comparison to healthy subjects and patients with adenomatous lesions. In their study, the presence and integrity of plasma cfDNA and the methylation profile of two gene promoters were evaluated. The cfDNA concentration and cfDNA integrity were found to be increased in patients with CRC, and were associated with a poor prognosis. A lower extent of DNA methylation was observed in cfDNA as compared to tissue DNA.
With respect to alterations affecting cancer drivers, a high prevalence was previously described by Allenson et al of mutant KRAS in circulating exosome-derived DNA from patients with early-stage pancreatic cancer (155). One very interesting observation of their study was that exosomes from viable cancer cells may reflect a different biology than cfDNA shed from dying tissues, including normal tissues. The information that the analyzed ctDNA is derived from actively metabolizing cancer cells with invasive potential, and not from normal cells, is certainly a crucial issue for early diagnosis. In this respect, Allenson et al (155) observed that the size of ctDNA differs depending on whether it is released from necrotic and dying cells or from live cells. The former is contained in cfDNA, the latter is included in the exosome-DNA fraction. The conclusion of their study was that exosomes should be considered as distinct sources of tumor DNA that may be complementary to other liquid biopsy DNA sources. In addition, circulating mutant KRAS was found in a minority of healthy samples, suggesting that care should be exercised when proposing liquid biopsy as a broad cancer-screening method.
As far as miRNA-based early diagnosis, an interesting study by Shimomura et al employed a highly sensitive micro-array assay for the evaluation of serum miRNA expression profiles (156). In this large study, a total of 1,280 serum samples from patients with breast cancer were tested. In addition, 2836 serum samples were obtained from non-cancer controls, 451 from patients with other types of cancers, and 63 from patients with non-breast benign diseases. The expression of miRNAs was compared between breast cancer and non-breast cancer patients. The conclusion was that a set of five miRNAs (miR-1246, miR-1307-3p, miR-4634, miR-6861-5p and miR-6875-5p) discriminated breast cancer from healthy control and non-breast cancer patients.
Staging and prognosis
Schröck et al (157) presented a study on free-circulating methylated DNA for the diagnosis, staging and prognosis of head and neck squamous cell carcinoma patients. In their study, the DNA methylation of two genes [short stature homeobox 2 (SHOX2) and septin (SepT9)] was quantified in plasma before treatment, and thereafter longitudinally during follow-up. The methylation levels were associated with the tumor and nodal category, and increased DNA methylation levels were associated with a shorter survival. On the whole, the data independently obtained in different laboratories support the hypothesis that the testing of DNA methylation in plasma is a powerful diagnostic tool for staging, risk stratification and disease monitoring. Patients with initially high biomarker levels may benefit from intensified treatment and surveillance. The marker-driven, timely detection of recurrent/metastatic disease may guide successive lines of treatment, thereby improving patient outcomes.
Therapeutic outcome
One example demonstrating the possible role of liquid biopsy in predicting therapeutic outcome has been reported by Quandt et al (158) who discussed how information obtained from liquid biopsies may contribute to the clinical decision-making process for cancer immunotherapy. This issue is of great interest since the application of immune checkpoint blockade over the past decade has revolutionized the treatment of a number of malignancies, leading to significantly improved survival. In this context, liquid biopsies are proposed to monitor treatment efficacy, acquired resistance to therapy and assign prognosis. A second example was published by Goodall et al (159) on cfDNA to guide prostate cancer treatment with poly(ADP)-ribose polymerase (PARP) inhibition. They reported whole exome sequencing of serial cfDNA samples collected during the treatment of patients with prostate cancer with the PARP inhibitor, olaparib. Decreases in the cfDNA concentration were found to be associated with a favorable outcome. All tumor tissue somatic DNA repair mutations were detectable in cfDNA, and allelic frequencies of somatic mutations decreased selectively in responding patients. At disease progression, following response to olaparib, multiple sub-clonal aberrations and somatic mutations in DNA repair genes (BRCA2 and PALB2) emerged as mechanisms of resistance. These data support the role of liquid biopsies as predictive, prognostic, response and resistance biomarkers in prostate cancer.
Final considerations on the management of cancer patients, follow-up and treatment monitoring
The results obtained thus far have indicated that liquid biopsy considerably affects systemic cancer therapy in metastatic cancer, due to the relevant information it provides to the medical oncologist. This is expected to improve key clinical parameters, such as patients overall survival and quality of life. This should be considered a major advantage of liquid biopsy (as outlined in Fig. 3), since the tissue biopsy of metastatic foci, is not only invasive, but is limited to certain locations, does not reflect clonal heterogeneity and multiple biopsies (even assuming they are feasible) may not be easily accepted, and may ingenerate doubts and contradictory diagnostic reports. Along this line, a droplet digital PCR (ddPCR) study by He et al (160) on 120 patients with a diverse EGFR mutational status supported an association between liquid biopsy and outcome. Of note, it was found that the mutant signature was stable and marked dynamic changes during the treatment allowing efficient and continuous disease profiling, which is expected to greatly facilitate the clinical decision-making process.
6. PCR-free detection strategies
Despite the fact that the majority of the analytical technologies are based on PCR and RT-PCR (see the Technologies section above and Tables I and II), PCR-free methods have attracted great interest in biomedicine. In fact, several articles have been published dealing with PCR-free methods for the detection of point mutations. In addition to the already cited limitation of PCR-based approaches, the need for repeated steps involving heating and cooling is an important limitation of all the PCR-based technologies, particularly when the PCR steps for the amplification of nucleic acids are associated with procedures performed in microfluidic-based devices (143,161-164). Several alternative isothermal-amplification methods (which do not require thermal cycling) have been developed to overcome this limitation, including nucleic-acid-sequence-based polymerization (NASBA), loop-mediated amplification (LAMP), helicase-dependent amplification (HAD), rolling-circle amplification (RCA), recombinase-polymerase amplification (RPA) and multiple-displacement amplification (MDA) (142). Recently, isothermal circular-strand-displacement polymerization (ICSPD) has emerged as a novel and promising method for nucleic-acid amplification and detection (163). Finally, promising opportunities are offered by selected isothermal amplification approaches that are based on a simple design of the amplification process and can be integrated in microfluidic devices (142). Such methods allow for the minimization of potential sample contaminations and minimize the sample volume required for the analysis (143). In this respect, the direct detection of point mutations in non-PCR-amplified human genomic DNA has been recently demonstrated by surface plasmon resonance imaging (SPR-I). Attomolar concentrations of target genomic DNA have been detected, demonstrating the ultra-sensitivity of the new method and its potential application in several biomedical fields, including liquid biopsy methods (164).
7. Patents and clinical trials on liquid biopsy and ultrasensitive detection systems
Tables III (165-173) and IV (174-182) summarize patents and patent applications related to the development of liquid biopsy protocols in cancer diagnosis. It is of interest to go through the claims of these patents, as they reflect the consideration given to liquid biopsy by a large part of the scientific community. Several patents build on the concept that mutations of tumor-associated genes (present in cfDNA from body fluids) and/or miRNA profiles are prognostic (associated with outcome), and/or predictive (associated with susceptibility to specific treatments). Examples of patents related to cfDNA and miRNAs are numerous and have steadily increased over the years. In fact, CTC counts, and molecular signatures originating from or associated with CTCs have been shown to be associated with conventional cancer molecular genotyping in tissues.
| Table IIIExamples of patents and patent applications on circulating free DNA (cfDNA) based liquid biopsy in oncology. |
For instance, in EP2426217A1 (166) a method is described for detecting cell free nucleic acids, preferably cfDNA in a body fluid sample from an individual or a patient. A general claim, present in many other similar patent applications, relates to a method that comprises the step of accurately and sensitively determining the concentration of cell free nucleic acids in the sample and/or the index of integrity of said cell free nucleic acid and/or the determination of the presence of genetic polymorphisms [such as known single nucleotide polymorphisms (SNPs) or mutations]. The invention encompasses also a method to discriminate body fluid individuals where cfDNA are highly released. The majority of the approaches described in Table III have been validated on a variety of body fluids (urine, saliva, serum, plasma, bone marrow, lymphatic fluid, lacrimal fluid, serous fluid, peritoneal fluid, pleural fluid, ductal fluid from breast, gastric juice, or pancreatic juice) and cancers [breast cancer, CRC, periampullary cancer, melanoma, prostate cancer, gastric cancer, leukemia/lymphoma, renal cell carcinoma, hepatocellular carcinoma (HCC), neural-derived tumor, head and neck cancer, lung cancer, or sarcoma]. In addition to SNPs and tumor-associated mutations (presented in EP2426217A1, US7718364B2, WO2016168844A1 and US20160053301A1) (166,168,171,173), another interesting marker is DNA methylation. In US7718364B2 (168), a method is provided for assessing allelic losses and the hypermethylation of genes in the CpG tumor promoter region on specific chromosomal regions in patients suffering from melanoma, neuroblastoma, breast, colorectal and prostate cancer. The method is based on the evidence that free DNA and hyper-methylation of genes in the CpG tumor promoter region may be identified in the bone marrow, serum, plasma and tumor tissue samples of cancer patients. Table III lists examples of patents and patent applications focusing on cfDNA analysis in the body fluids of cancer patients.
As far as miRNAs are concerned, US8216784B2 (175) and EP2806273B1 (176) deal with cancer-derived microvesicle-associated miRNAs as a diagnostic marker for the detection of cancer. The method is based on the analysis of one or more miRNAs selected from a group comprising miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205 and miR-214. The method can be applied to a variety of biologicals, including milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof. EP3011058A1 (178) is an example of patents focusing on total miRNA analysis. In addition, in this case, a shortlist of candidate miRNAs is provided, such as the combination of miR-365, miR-425, miR-143, miR-133a, miR-15a and miR-18a. The alteration in the level of the miRNA in the test sample (such as serum, plasma, or whole blood), relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk of developing, cancer, or the response of a subject to any treatment for the cancer. Further examples of miRNAs identified as cancer biomarkers are shown in Table IV.
| Table IVExamples of patents and patent applications on circulating microRNAs (miRNAs or miRs) for non-invasive liquid biopsy in oncology. |
Liquid biopsy is implemented (and is technologically tested) in several ongoing clinical trials (Tables V and VI) (183-204). For instance, NCT02639832 (183) is focused on the presence of tumor-derived CTCs or ctDNA using an investigational medical device known as the LiquidBiopsy. Using the LiquidBiopsy platform, recovered cells or DNA can also be investigated to obtain genetic information that may be useful to physicians for treating and understanding disease. The LiquidBiopsy device is able to purify the tiny numbers of tumor cells or ctDNA in blood. Even if a tumor is too small to be found by other means, such as an X-ray, it is possible that ctDNA or CTCs may be found in the blood. Genetic information can then be recovered from these cells or DNA to identify genetic alterations that are related to abnormal growth in a tumor. The proponents claim that this will potentially allow researchers to study tumor cells or tumor DNA from a blood sample instead of a biopsy sample, and may influence cancer diagnosis, treatment and drug selection in the future. In NCT02784639 (195) a method is employed which simultaneously allows the determination of three parameters: The specific quantification of tumor-derived ccfDNA, the ccfDNA fragmentation index and SNP or point mutation detection. The evaluation and validation of the method will be performed by determining the KRAS/BRAF mutational status prior to anti-EGFR therapy in patients with CRC. The protocol will detect the six most frequent KRAS mutations in CRC (G12D, G12V, G13D, G12S, G12C and G12A) and BRAF V600E. The goal of this multicenter prospective study is to validate, and ultimately translate in routine clinical practice, the use of plasma analysis of cfDNA for the determination of KRAS mutation status in patients with CRC. Table V lists several examples of ongoing clinical trials based on the analysis of cfDNA in breast, lung, colorectal, stomach, gastric and pancreatic cancers, HCC, non-Hodgkin lymphoma and melanoma.
As regards clinical trials related to miRNAs, one example is NCT02928627 (198), which is focused on miRNAs, such as miR-221 and miR-222, that are both dysregulated in the tumor tissues in approximately 80% of patients with HCC. The aim of that study was to evaluate whether these two miRNAs are overexpressed not only in tumor tissues, but also in the blood of cancer patients. An association between tumor tissue and blood levels will also be evaluated. Table VI lists additional examples of ongoing clinical trials in breast, esophageal and prostate cancer, HCC, lymphoma and leukemias.
8. International Networks focusing on liquid biopsy
The interest in non-invasive tumor diagnosis by liquid biopsy is demonstrated by several international projects supported by public funding. A few selected examples (LIQBIOPSENS, CANCER-ID, PRECISE, BILOBA and ULTRAPLACAD) are reported in the following sections irrespectively of their success in terms of delivered products (manuscripts, validated protocols and platforms, or patents).
LIQBIOPSENS (Reliable Novel Liquid Biopsy technology for early detection of CRC)
This Horizon-2020 project is based on a reliable novel liquid biopsy technology for the early detection of CRC using multidisciplinary approaches involving microelectronics, microfluidics, nanomaterials and genomics. The overall aim of LIQBIOPSENS is the further development and validation in real settings of a novel diagnostic platform for the early and rapid detection of ctDNA and their KRAS and BRAF mutations associated with CRC through blood samples. This sensitive (in the zM range) assay includes 27 KRAS and BRAF mutations, to be simultaneously analyzed. Other main features of LIQBIOPSENS are reliability, low-cost and short analysis time. Furthermore, a user-friendly and flexible interface is provided. Among the participants are DestiNA Genomics Ltd. (UK); the Foundation for Research and Technology Hellas, Greece; the Servicio Andaluz del Salud, Spain; BEABLE SL, Spain; Université Catholique de Louvain, Belgium and Sistemas Genomicos SL, Spain (http://liqbiopsens.com).
CANCER-ID (cancer treatment and monitoring through the identification of CTCs and tumor-related nucleic acids in blood)
This is a newly formed European consortium funded by the Innovative Medicines Initiative (IMI) with currently 33 partners from 13 countries aiming at the establishment of standard protocols for, and the clinical validation of, blood-based biomarkers. It brings together experts from academic and clinical research, innovative small-to-medium sized enterprises (SMEs), diagnostics companies and the pharmaceutical industry, thus providing a unique setting for establishing clinical utility of liquid biopsy. The academic leads of the CANCER-ID consortium are Professor Klaus Pantel, Head of the Department of Tumor Biology at the University Medical Center Hamburg-Eppendorf, Hamburg, Germany, and Professor Leon Terstappen, Head of the Department of Medical Cell Biophysics at the University of Twente, Enschede, The Netherlands. The lead companies of the CANCER-ID consortium are Bayer HealthCare and Silicon Biosystems, A Menarini Group Company (http://www.cancer-id.eu).
PRECISE (Personalized Engine for Cancer Integrative Study and Evaluation)
This project is an Horizon-2020 pilot project that combines hypothesis-driven strategies with data-driven analysis in a novel mathematical and computational methodology for the integration of genomic, epigenetic, transcriptomic, proteomic and clinical data with the goal of risk-stratifying patients and suggesting personalized therapeutic interventions. As far as motivation is concerned, the PRECISE project is based on the concept that, despite their great promise, high-throughput technologies in cancer research have often failed to translate to major therapeutic advances in clinical practice. One challenge has been tumor heterogeneity, where multiple competing subclones co-exist within a single tumor. Genomic heterogeneity renders it difficult to identify all driving molecular alterations, and thus results in therapies that only target subsets of aggressive tumor cells. Another challenge lies in the integration of multiple types of molecular data into mathematical disease models that can make actionable clinical statements. PRECISE aims to develop predictive computational technology that can exploit molecular and clinical data to improve our understanding of disease mechanisms and to inform clinicians about optimized strategies for therapeutic intervention. The Precise project will focus on two urgent clinical needs in prostate cancer: i) Distinguishing the many indolent tumors from the minority of lethal ones; and ii) providing rationally selected treatment options for patients with advanced disease (http://www.precise-project.eu).
BILOBA (Bloch electromagnetic surface wave biosensors for early cancer diagnosis)
BILOBA is a collaborative project funded by the European Commission through its Seventh Framework Program. The major goal of the project is to explore, design and set-up systems optimized for analytical sensing, associated with the development of a corresponding analytical instrument. For this purpose, the immobilization protocols and biochemical assays have been established to ensure an optimized binding site density at the surface of SPR sensors and to enable the detection of the target biomarkers. Furthermore, a fluidic system has been developed for handling the aqueous analyte solutions ensuring a high signal-to-noise ratio and robust results even in the case of ultralow concentrations. The BILOBA multifunctional point-of-care platform is expected to be capable of performing real-time cancer biomarker detection in a tandem configuration. The BILOBA project consists of 9 participants from different European countries, including the Department of Basic and Applied Sciences for Engineering (Università degli Studi di Roma 'La Sapienza', Rome, Italy); the Department of Applied Sciences and Technology (Politecnico di Torino, Torino, Italy); Fraunhofer Gesellschaft, Munich, Germany; the Department of Materials (Imperial College London, London, UK), the Department of Oncology (Università degli Studi di Torino, Torino, Italy); Labor Srl, Rome, Italy; Biotray SAS, Lyon, France; Horiba Jobin Yvon SAS, Paris, France; KDS Radeberg GmbH, Dresden, Germany (http://www.biloba-project.eu/).
ULTRAPLACAD (ULTRAsensitive PLAsmonic devices for early CAncer Diagnosis)
With respect to the issue of the detection of cfDNA and miRNAs in tumor liquid biopsies, the highly multidisciplinary ULTRAPLACAD project relies on two advanced plasmonic biosensor technologies: i) Plasmon-enhanced fluorescence spectroscopy imaging (PEFSI); and ii) nanostructure-enhanced surface plasmon resonance imaging (NESPRI). Plasmonic methods have already been shown to be useful for biomedical applications for the analysis of nucleic acid analytes in real samples (including human blood) with a limit of detection (LOD) from low femtomolar to attomolar concentrations (162,205,206). In the ULTRAPLACAD project, novel plasmonic structures will be delivered based on surface plasmon modes supported by continuous metallic films and arrays of metallic nanoparticles. Those structures hold the potential for a breakthrough enhancement of sensitivity. Moreover, this method will be implemented in an innovative compact biosensor device based on an integration of key optical elements for the excitation and collecting of fluorescence light directly on the sensor chip. The SPRI approach is based on the detection of target analytes through biomolecular binding-induced refractive index changes. In the ULTRAPLACAD project, an SPRI platform based on the diffraction coupling of light into surface plasmons will be developed, in order to increase sensitivity and limit the signal-to-noise ratios. With respect to the proposed diagnostic model systems, KRAS and BRAF mutations are considered, since they are routinely assessed in genomic DNAs from surgically removed lesions of colorectal carcinoma, breast cancer, melanoma and other tumors. As far as miRNA target sequences are concerned, miR-221, miR-222, miR-141, miR-155, miR-21, let-7a and miR-16 are considered, since these miRNA molecules are available in circulating exosomes, and could mark tumor aggressiveness. ULTRAPLACAD is the only project in which ctDNAs, miRNAs and proteins, e.g., the three major analyte classes, are combined in a single detection platform (www.ultraplacad.eu).
9. Conclusions
The liquid biopsy of cancer is mainly based on the analysis of CTCs and/or cell-free nucleic acids in the peripheral blood of cancer patients, as well as in other body fluids suitable for diagnostic assessment. Among these, cerebrospinal fluid for tumors of the central nervous system, saliva for tumors affecting the head and neck, pleural effusion in the case of respiratory tract cancers and urine for urinary tract cancers. At present, liquid biopsy should be considered one of the most advanced non-invasive diagnostic systems suitable for performing key clinically relevant actions possibly leading to precision medicine. Historically, the applications of liquid biopsy for the characterization of cancer patients have been focused on CTCs. More recently, this analysis has been extended to cfDNA and miRNAs, demonstrated to be associated with cancer, with potential applications in early diagnosis, staging, prognosis, prediction of therapeutic responses, therapeutic outcome, and follow-up during therapeutic intervention. Liquid biopsy measures alterations in gene structure, regulation and expression that are the hallmarks of cancer. These analytes include nucleotide variants, promoter methylation, copy number variations of specific genes, chromosomal rearrangements, mutations affecting transcription, splicing and RNA maturation, translational efficiency and differences in miRNA signatures. The analysis of all these parameters can be approached by a variety of technological platforms. Moreover, the great interest of liquid biopsy is that this approach avoids certain key issues associated with invasive surgical biopsy. These include, but are not limited to: i) A static representation of the tumor pathology strictly limited to the tumor tissue sampling; ii) ethical and practical issues preventing repeated tissue biopsy; iii) tumor heterogeneity, particularly during progression and metastatic dissemination (making multiple sampling necessary); iv) easier patient monitoring by non-invasive analytical procedures (i.e., liquid biopsy). Therefore, despite the fact that the liquid biopsy approach suffers from important drawbacks (fragmentation of cfDNA, instability of RNA, low yield of isolated samples to be analyzed and variable presence of normal DNA and RNA) this approach is generally deemed of great interest for future applications, patent development and clinical trials. These will ultimately verify the potential of liquid biopsy in cancer. It is not expected that liquid biopsy will replace surgical biopsy; however, it will probably complement within a few years the information routinely obtained by excisional, incisional, surgical and needle biopsies, becoming a tool of choice for the dynamic monitoring of patients during clinical treatment and during the long-term surveillance of their health status.
Acknowledgments
Not applicable.
Funding
This study was supported by the European Union (EU) Horizon 2020 Research and Innovation Programme (GA #633937, project ULTRAsensitive PLAsmonic devices for early CAncer Diagnosis (ULTRAPLACAD), and by Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG #13575 to RG, IG# 14204 and IG #19052 to PG). MA is the recipient of an AIRC, Nuvenia Fellowship id. 19503.
Availability of data and materials
Not applicable.
Authors' contributions
All authors were involved in the conception of the study, and revised and approved the final manuscript. All authors take the responsibility for publishing this review paper. AF, PG, GS and RG performed the literature search, wrote the manuscript, and critically analyzed the existing knowledge; AF and RG designed the figures; AF, MA, JG and RG have ideated and drew the tables; AF, JG and RG were involved in the acquisition of data and in the analysis of patents and clinical trials; JG and MA significantly contributed to editing the manuscript; PG, DAS, GS and RG significantly contributed to the analyses of international networks focusing on liquid biopsy. AF, PG, DAS, GS and RG were significantly involved in the drafting of the manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article.
Abbreviations:
|
CRC |
colorectal cancer |
|
HCC |
hepatocellular carcinoma |
|
CTCs |
circulating tumor cells |
|
cfDNA |
circulating free DNA |
|
cfRNA |
circulating free RNA |
|
miRNA or miR |
microRNA |
|
UTR |
untranslated region |
|
CDS |
coding sequence |
|
RISC |
RNA-induced silencing complex |
|
PCR |
polymerase-chain reaction |
|
RT |
reverse transcription |
|
qPCR |
quantitative PCR |
|
ddPCR |
droplet digital PCR |
|
WGS |
whole genome sequencing |
|
NGS |
next generation sequencing |
|
SPR-I |
surface plasmon resonance imaging |
|
SNPs |
single nucleotide polymorphisms |
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