The current status of the clinical utility of liquid biopsies in cancer

Abstract

Introduction:

Liquid biopsies have attracted considerable attention as potential diagnostic, prognostic, predictive, and screening assays in oncology. The term liquid biopsies include circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in the blood. While many liquid biopsy technologies are under active investigation, relatively few liquid biopsy assays have been proven to serve as a diagnostic surrogate for biopsies of metastatic disease as predictive biomarkers to guide the selection of therapy in the clinic.

Areas Covered:

The objective of this review is to highlight the status of liquid biopsies in solid tumors in the oncology literature with attention to proven utility as diagnostic surrogates for macrometastases.

Expert Opinion:

Carefully designed clinical-translational studies are needed to establish the diagnostic accuracy and clinical utility of liquid biopsy biomarkers in oncology. Investigators must fully consider relevant pre-analytical variables, assay sensitivity, bioinformatics considerations as well as the clinical utility of rare event profiling in the context of the normal blood background. Future liquid biopsy research should address the concern that not all DNA mutations are expressed and should provide the means to discover potential therapeutic targets in metastatic patients via a minimally invasive blood draw.

1. Introduction

In the last two decades, oncology research has exponentially grown with the advent of cancer genomic sequencing (Table 1). The vast amount of data produced from these efforts highlights that cancer is extremely heterogeneous, and a ‘one size fits all’ treatment regimen does not hold. Therefore, the forefront of cancer research has focused its efforts on tailoring treatments guided by the genetic makeup of a patient’s malignancy.

Table 1.

Glossary of commonly used study terms, abbreviations, and definitions

Key Term	Abbreviation	Definition
Circulating tumor cell	CTC	Cells in the bloodstream that originated from an original tumor site. These cells traveling throughout the vasculature have the potential to cause tumor metastasis.
Cell free DNA	cfDNA	Circulating non-encapsulated DNA fragments present in the bloodstream. Produced by cellular lysis, which may be caused by events such as apoptosis or necrosis. The cfDNA in the bloodstream may originate from any cell type, including tumor cells.
Circulating tumor microemboli	CTM	Clusters of CTCs (usually 3 or more cells)
Circulating tumor DNA	ctDNA	Cell free DNA fragments originating from tumor cells. ctDNA is thought to make up < 1% of total circulating cfDNA in the bloodstream of cancer patients. Due to their origin, they have genomic variations associated with the tumor cell from which they originate. They are currently being used and tested as biomarkers for cancer detection, diagnosis, and treatment selection.
Next Generation Sequencing	NGS	A variety of tests that involve parallel sequencing of numerous DNA fragments and comparing these fragments with the human reference genome in order to find variations, including differences in bases, exons, genes, translocations, and inversions
Liquid biopsy		A type of blood test used to detect circulating tumor cells or circulating tumor cell DNA in cancer patients. Due to its non-invasive nature, it is being studied as a surrogate for or addition to traditional tissue biopsies with some liquid biopsies now FDA approved for use. Further research continues to study the clinical validity and utility of these types of tests.

1.1. Incorporating molecular pathology in the clinic

The field of oncology continues to rely heavily on anatomic pathological staging and morphology phenotyping that provides relatively limited data on pathobiology. However, this stance is quickly changing. The American Joint Committee on Cancer (AJCC) 8^th edition staging guidelines for breast cancer, released in 2016, includes estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), tumor grade, and in some cases Oncotype Dx (Genomic Health, Redwood, CA, USA) recurrence score to assign a prognostic stage [1]. In one study, it was found that over a third of women with stage I to III breast cancer that were staged with the 7^th edition criteria had a change in staging when the AJCC 8^th edition criteria were applied [2]. Given the rapid pace of scientific research, the staging guidelines acknowledge that biomarkers may be included in the AJCC staging going forward. Thus, there is a tremendous need to develop biomarkers that can help predict prognosis and guide treatment.

1.2. Advantages of liquid biopsies

Liquid biopsies such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) are emerging technologies that have strong potential to serve as future surrogate biomarkers, potentially directing treatment options in the clinic (Table 2). Ideally, biomarkers could be used for diagnosis, prognosis, prediction of disease progression, or as a surrogate marker of response to treatment (Table 3). Furthermore, if these biomarkers are validated in clinical trials to improve therapeutic efficacy, they bring several advantages over tissue biopsies: 1) They are noninvasive. 2) They can provide temporal data on tumor heterogeneity throughout treatment. 3) They can provide detailed information on genetic mutations and molecular drivers. 4) Over time it is likely the methods used to analyze liquid biopsies will be cost-effective and provide rapid results. However, very few of these assays have been cleared by the U.S. Food and Drug Administration [3].

Table 2.

Comparison chart of ctDNA, Tissue Assay, and CTCs

Metrics	ctDNA	Tissue Assay	Circulating Tumor Cells
Risks	Minimally invasive test Blood draw risks	Invasive test Biopsy risks	Minimally invasive test Blood draw risks
Factors that affect role as a tumor marker	Global sample of tumors Well suited to capture intratumor genetic heterogeneity Variability of ctDNA amount is dependent on cfDNA in sample, which can make it difficult to obtain enough sample, issues with minimal allele frequency required to detect, false positive and false negative tests	Regional sample of tumor based on location of biopsy site mean that heterogeneity within the tumor itself may be missed	Global sample of tumor Well suited to capture intratumor genetic heterogeneity Very low concentrations in blood leading to low capture rates Misses low-EpCAM CTC populations if based on affinity marker testing. Issues with potential low purity if isolated based on size or negative selection.
Role as a screening test	Currently being studied as a screening test for multiple cancers.	Current practice for diagnosis of a cancer type. Not a screening test.	Not cost effective as a screening test currently. Not all cancer patients have detectable CTCs.
Selecting targeted therapy	If targets are known or prevalent, ddPCR or targeted panels for ctDNA may be favorable	Evidence exists for treatment selection based on tissue biopsy results in early and advanced cancers	If potentially actionable treatment targets are unknown, CTCs may be preferable to discover the full spectrum of available targeted therapy options
Advantages in monitoring therapy effectiveness	Possible ability to gather temporal data on tumor heterogeneity throughout treatment, but no evidence of clinical validity. More studies needed.	More difficult to re-biopsy to show evolution of metastases or response to treatment	Possible ability to gather temporal data on tumor heterogeneity throughout treatment. However, more studies and research needed.
Clinical Utility	Limited evidence of clinical utility for treatment selection, and no evidence for other uses	Evidence of utility in treatment selection for early and advanced cancers	Limited evidence of clinical utility for treatment selection
Clinical Validity	Insufficient evidence for most assays.	Current standard practice for treatment selection for early and advanced cancers.	Insufficient evidence for most assays.

ctDNA: circulating tumor DNA

CTC: circulating tumor cells

Table 3.

Markers detectable via liquid biopsy

Field of study	Cells being detected	Clinical Utility
Oncology	Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA) Circulating Tumor Microemboli (CTM)	CTCs, CTM and ctDNA are currently being studied as biomarkers in oncological screening, diagnosis, and treatment selection.
Cardiology	Circulating Endothelial Cells (CECs)	CECs are currently being studied as diagnostic biomarkers for endothelial damage. Studies have found CECs to be elevated in myocardial infarction, diabetic nephropathy, heart failure with preserved ejection fraction, and arterial hypertension.
Obstetrics	Cell-free Fetal DNA (cffDNA)	cffDNA from maternal blood has been studied to detect fetal aneuploidies, Rhesus-D status, fetal sex, single gene disorders, and pre-eclampsia.

1.3. FDA approved assays

The three U.S. Food and Drug Administration (FDA) approved assays are the CellSearch CTC assay (Menarini Silicon Biosystems Inc., Huntington Valley, PA, USA), the cobas EGFR Mutation Test v2 (Roche Molecular Systems Inc., Pleasanton, CA, USA), and the therascreen PIK3CA RGQ PCR kit (QIAGEN, Germantown, MD, USA) (Table 4). CellSearch is an EpCAM and cytokeratin based assay meant to capture CTCs for analysis and is currently approved in metastatic breast [4,5], prostate [6], and colorectal [7] cancers. Cobas EGFR Mutation Test v2 is a real-time PCR test that identifies 42 mutations in exons 18, 19, 20 and 21 of the epidermal growth factor receptor (EGFR) gene, including the T790M resistant mutation. Interestingly, cfDNA EGFR captured from non-small-cell lung cancer (NSCLC) patients using the cobas EGFR assay could predict prognosis in those treated with erlotinib. Specifically, those with cfDNA EGFR mut⁺ had worse progression-free survival (PFS) and overall survival [8]. The therascreen PIK3CA assay detects 11 mutations in the PIK3CA gene, which are estimated to be present in 40% of metastatic estrogen receptor positive breast cancer [9]. The SOLAR trial demonstrated that treatment with alpelisib plus fulvestrant had improved progression-free survival in advanced breast cancer[10], particularly for patients who had genomic testing of tumor tissue or ctDNA [9]. The cobas EGFR and therascreen PIK3CA assays are examples of predictive diagnostic biomarkers that could help direct treatment. It is quite probable that additional liquid biopsy assays will be reported in the near future to add to this current list of FDA approved tests. Despite this, the American Society of Clinical Oncology and College of American Pathologists recently convened to review the literature on ctDNA assays in solid tumors and concluded that there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer [11], despite much excitement in the field.

Table 4.

FDA Approved Liquid Biopsies

Name of Liquid Biopsy	Targets	Types of Cancer Used For	Manufacturer
CELLSEARCH® Circulating Tumor Cell Assay	Detects circulating tumor cells (CTC) of epithelial origin (CD45−, EpCAM+, and cytokeratins 8, 18+, and/or 19+)	Metastatic breast, prostate, and colorectal cancer	Menarini Silicon Biosystems
cobas® EGFR Mutation Test v2	Detects 42 mutations in exons 18, 19, 20 and 21 of the epidermal growth factor receptor (EGFR) gene, including the T790M resistant mutation	Non-small cell lung cancer (NSCLC)	Roche Molecular Systems
therascreen® PIK3CA RGQ PCR kit.	Detects 11 mutations in exons 7, 9, and 20 of the PIK3CA gene	PIK3CA mutated HR+/HER2− advanced breast cancer	QIAGEN

1.4. Tumor heterogeneity

It is well established that there is incredible spatial and temporal intratumoral genetic heterogeneity within metastatic disease [12], which is thought to lead to treatment resistance and failure [13,14]. There is evidence from studies performing multiregional sampling that within a primary tumor there are separate yet distinct subpopulations of cells [15,16]. Studies have demonstrated that there is considerable discordance between primary and metastatic tumors in colorectal cancer [17], lung cancer [18], melanoma [19], renal cancer [20], and breast cancer [21,22]. With this understanding, there has been a paradigm shift that cancer is not a stagnant but rather a dynamic process. Therefore, an essential aspect of a liquid biopsy is the ability to capture tumor heterogeneity.

2. Next Generation Sequencing

Next-generation sequencing (NGS) has brought about a ‘golden era’ for cancer genomics. For decades, before NGS adoption, Sanger sequencing was the only method available for gene sequencing; however, it was limited by its capillary-based technology to analyzing a single DNA fragment at a time. NGS technology allowed for massive parallel sequencing thus simultaneous sequencing of millions of DNA fragments per run [23]. Several methods of NGS have been used to analyze CTCs such as targeted sequencing [24], whole-exome sequencing (WES) [25], whole-genome sequencing (WGS) [26], and RNA-seq [27]. There is currently not one method that is superior for clinical application in the field of liquid biopsies. While there is an urgent unmet need to translate liquid biopsy assays to the clinic, further analytical and clinical validation is required to move the field towards clinical utility for cancer patients. Molecular profiling of advanced cancers has excellent potential to improve care via precision medicine directed at oncogenic driver mutations. Liquid biopsy offers the prospect of a real-time assessment of tumor biology under the selection pressures of treatment, better delineating tumor targets and eradicating or controlling disease.

A significant question remains for the optimal molecular analysis of liquid biopsy biomarkers such as CTCs and ctDNA. NGS has revolutionized the way scientists have been able to study cancers by allowing examination of cancer genomes, exomes and transcriptomes across all types of malignancy. However, CTCs and ctDNA require PCR or amplification in order to detect their rare nucleic acids. Given that there is more RNA compared to DNA in cellular composition [28], issues related to mutant allele frequencies and depth of coverage are important considerations for NGS of liquid biopsies, particularly for ctDNA profiling. For this reason, many groups have preferred targeted deep sequencing of panels rather than more shallow, but broader high throughput sequencing approaches [29]. As discussed above, Torga et al.’s findings of discordance between the Guardant360 and PlasmaSelect assays for paired samples from the same patients likely reflects different amplification and library preparation strategies as well as differences in the specific sequences being assayed [30].

Shi et al. recently reported that genuine intratumoral genetic heterogeneity is very difficult to distinguish from sequencing artifacts and that the majority of somatic variants contributing to heterogeneity are technical noise [31]. Furthermore, cancer-only WES pipelines were found to be unreliable with 69% of somatic mutations proven to be false positives [31]. Hedegaard reported a success rate of 29.5% in generating DNA exome libraries from formalin-fixed paraffin-embedded (FFPE), which was much more challenging than generating RNA Seq libraries from FFPE [32]. Similarly, Li et al. demonstrated that RNA Seq of FFPE provided excellent but not perfect correlation compared to fresh, frozen tumor [33].

At present, the ability of liquid biopsies to benchmark against RNA and DNA based orthogonal validation of tumors is incompletely explored. Parsons et al. reported that their study aimed at comparing the FoundationOne ctDNA NGS platform to orthogonal FFPE specimens met the pre-specified futility threshold due to inability to acquire the FFPE block from pathology and render results within 28 days for clinical decision making at a molecular tumor board [34]. Confounding factors such as tissue fixation or time to assay or preservation tube strategies aimed at maintaining RNA stability in intact cells are incompletely explored when considering the adaptation of these NGS technologies in liquid biopsies for clinical validation against orthogonal reference material.

CTCs in both early and metastatic breast cancer have considerable heterogeneity in their gene expression by whole transcriptome RNA Seq [35], suggesting that limited panels evaluating the RNA or DNA of liquid biopsy specimens would be unlikely to show clinically relevant positive test results. In addition, it would be improbable to select targets for qPCR or ddPCR without prior knowledge of gene expression. Therefore, if targets are known or prevalent, ddPCR or targeted panels for ctDNA may be favorable, however, if potentially actionable treatment targets are unknown, CTCs may be preferable to discover the full spectrum of available targeted therapy options. In particular, since ctDNA may be derived from the responding rather than the resistant tumor fraction, viable CTCs may theoretically be more useful to profile in heterogeneous tumor types where targets may vary. This is particularly a concern since not all DNA mutations are expressed and RNA is a functional indication of tumor biology.

As it is not yet possible to directly sequence RNA molecules without amplification [36], we have previously reported the assessment of the high fidelity of the amplification techniques that we have selected for whole transcriptomic studies [37,38]. We utilized a rapid processing strategy for gene expression profiling of CTCs given prior studies demonstrated that pre-analytical variable of time resulted in changes in gene expression if specimens were processed more than 2–3 hours after blood draw [39,40]. It is clear that further evaluation is needed to determine the relationship between CTCs and ctDNA in the context of orthogonal reference validation studies to better understand the underlying tumor biology of both types of liquid biopsies in the context of various types of malignancies.

3. Circulating Tumor DNA

Cell-free DNA (cfDNA) is composed of nucleic acid fragments that are released from apoptotic or necrotic cancer cells into the bloodstream [41], of which the total concentration of ctDNA is usually <1% of total cfDNA [42]. Presently, there are no techniques available to isolate ctDNA from other circulating DNA in the blood. Thus, analysis of tumor DNA fragments is affected by the background of normal DNA fragments found in the blood. The quantity of ctDNA increases in correlation to tumor burden [43]. The half-life of ctDNA in the circulatory system ranges from 15 minutes to several hours [44]. Currently, ctDNA assays have been demonstrated to be prognostic in breast cancer [45], ovarian cancer [46], NSCLC [47], gastrointestinal cancers [48,49], and melanoma [50].

3.1. ctDNA Detection

Identification of ctDNA is based on the detection of tumor-specific mutations, which can limit analysis as it requires detailed knowledge of the tumor before analysis [51]. For example, digital droplet PCR (ddPCR) as a ctDNA assay requires a priori knowledge of the genes of interest and only three genes (TP53, PIK3CA, and GATA3) have somatic mutations in primary breast cancer at a 10% incidence or greater [52]. Hence, when a priori knowledge of tumor mutations is absent untargeted approaches with next-generation sequencing (NGS) of ctDNA is required.

The most significant advantage of NGS is the ability to identify novel tumor mutations and track changes temporally with treatments. However, these techniques require a large amount of ctDNA, ≥10% in a blood sample, to reconstruct tumor-specific copy numbers consistently [53]. Low ctDNA concentrations are problematic as it was mentioned earlier that the total concentration of ctDNA obtained in a sample is generally <1% of total cfDNA. Another method of detection is Cancer Personalized Profiling by deep Sequencing (CAPP-Seq), which has been successfully applied in NSCLC [54] but may be applied to any cancer type with a known pattern of genetic mutations. Instead of targeting recurrent point mutations that may or may not be present in all patients, CAPP-Seq designs a library based on databases such as The Cancer Genome Atlas (TCGA) that targets reoccurring mutated regions in the cancer of interest [55].

Several commercial tests for ctDNA sequencing are available and can cover hundreds of mutational hotspots and genes. One of the first liquid biopsy tests to be developed was Guardant360. Guardant360 identifies single nucleotide variants (SNVs) in 73 clinically relevant genes, indels in 23 genes, copy number amplifications (CNAs) in 18 genes, and fusions in six genes [56]. Guardant360 can accomplish high confidence testing by limiting sequenced regions to short hotspots, which in turn might decrease the false positive rate. In contrast, traditional NGS assays, which sequence larger regions, have high false positive rates when there are low concentrations of tumor DNA [57]. Although a plethora of commercial tests are available, some which are commonly used include FoundationOne Liquid (Foundation Medicine Inc., Cambridge, MA, 02141) and PlasmaSelect (Personal Genome Diagnostics, Baltimore, MD, USA). FoundationOne Liquid uses a 62-gene panel to analyze specific cancer mutations, while PlasmaSelect implements a 64-gene panel using NGS.

3.2. ctDNA Clinical Applications

Garcia-Murillas et al. used targeted capture sequencing of primary tumors to develop custom ddPCR assays to monitor early stage breast cancer patients for minimal residual disease based on a priori knowledge of primary tumor sequencing [58]. Murtaza et al. used deep sequencing of ctDNA and multiple tumor deposits from a single patient to demonstrate that private, subclonal mutations could be detected in ctDNA liquid biopsies, which may represent the heterogeneity of metastatic sites [59]. Also, several studies have demonstrated that ctDNA mutations recapitulate those found in tissue biopsies and can monitor the evolution of metastatic disease [60–62]. Despite these remarkably innovative study designs, oncologists treating patients in the clinic are faced with a lack of clinically validated liquid biopsies approaches that can serve as a minimally invasive indicator of potential treatment opportunities based on a blood draw alone.

Clinical trials have already begun to incorporate ctDNA assays as exploratory, correlative biomarkers in breast cancer. Chandarlapaty reported that the presence of specific ESR1 mutations in ctDNA predicted that the addition of everolimus to exemestane improved PFS in metastatic breast cancer [63]. Campone et al. showed that buparlisib plus fulvestrant had improved PFS in patients with PIK3CA mutations in their ctDNA [64]. Leighl et al. found that the ctDNA Guardant360 assay detected guideline-recommend biomarkers just as effectively as tissue genotyping in patients with untreated newly diagnosed metastatic NSCLC [65]. However, Torga et al. demonstrated that there was very low concordance between blood samples drawn from the same person when comparing NGS assays Guardant360 and PlasmaSELECT [30]. Also, Bettegowda et al. recently reported that ctDNA was detectable in >75% of patients with advanced malignancies of several types, but present in <50% of patients with brain, renal, prostate or thyroid cancers, suggesting that the specific biomarkers studied are not universal across all cancers [43]. Recently, Cohen et al. published the CancerSeek assay based on 61 ctDNA amplicon and protein features used to detect early-stage malignancies as a potential screening tool [66]. The assay performed well in detecting many cancers but only had a sensitivity of 35% for breast cancer [66].

Currently, there has been considerable research surrounding the use of immunotherapy and stimulating a patients’ immune system to help fight their cancer. Several studies have found that tumor mutation burden (TMB) could be used as a surrogate for neoantigen levels [67], and thus possibly determine likelihood of response to immunotherapies. This prompted a study by Gandra et al., who reported that tumor mutational burden derived from ctDNA in blood samples predicted PFS of NSCLC patients treated with PD-L1 inhibitor atezolizumab [68]. However, further studies need to be carried out to confirm these findings.

3.3. ctDNA Disadvantages

Additional clinical trials incorporating CTC or ctDNA correlative biomarkers in their design are urgently needed; however, concerns exist about the analytical validity and clinical usefulness of these assays based on current data [11]. Many different ctDNA assays claim analytical validity, but few independent or cross-platform validation studies have been done. As mentioned earlier, a very poor ctDNA correlation was found between liquid biopsy platforms Guardant360 and PlasmaSELECT when examining 42 genes that overlapped between the two platforms [30]. Although this was a small study involving 40 metastatic prostate cancer patients, 25 of 40 had alterations in overlapping genes, but only three had complete congruence with ≥1 alteration, six had partial congruence, and sixteen had no congruence. The remaining 15 patients had no reportable alterations [30]. The findings of the study were concerning as Guardant360 and PlasmaSELECT both report high clinical sensitivity and specificity. Besides, somatic mutations are very heterogeneous within [69] and across different cancer types [70], thus platforms that rely on small gene panels will inevitably miss important actionable sequencing regions. NGS has a random error rate of 0.1% to 1% [71], making accurate detection of mutations difficult when allelic fractions are less than 1% [72]. In some cases, fresh tissue is available as orthogonal reference material, but in most cases, either formalin-fixed paraffin-embedded (FFPE) tissue or worse yet, no tissue is available as an orthogonal validation for liquid biopsies [3,73]. Currently, the only ctDNA assays that have been proven to be predictive biomarkers are single marker assays, not NGS multi-gene panel testing involving hundreds of genes.

Until procedures isolating ctDNA are standardized, reproducible and demonstrate clinical validity in clinical trials with therapeutic interventions directed by liquid biopsy predictions, it is doubtful that guidelines will support the routine use of ctDNA in cancer treatment. There is no dispute that DNA is more stable as a biomarker compared to RNA and that circulating cell-free DNA is more abundant in the plasma than CTCs, but these factors alone do not constitute analytical validity. Currently, there is no method to differentiate ctDNA derived from responding, resistant, or apoptotic tumor cells - it is simply assumed that ctDNA originates from some source of dead tumor cells. If the source of ctDNA is held in question, it makes it challenging to base clinical decisions upon the genomic findings in the absence of showing clinical validity via prospective trials. Finally, ctDNA of defined targets may be useful in clinical trials, but when resistance emerges, it may be necessary to utilize CTCs as a discovery tool and functional assay.

4. Circulating Tumor Cells

CTCs are intact whole cancer cells that have shed from a primary or secondary tumor and disseminate through hematogenous means to a distant site. CTC’s are extremely rare, and the estimated concentration of CTCs in a patient with cancer is one CTC per billion normal blood cells [74]. CTCs are found to be prognostic in lung [75], pancreatic [76], colon [77], and prostate [78]. Also, CTCs have been found in all stages of breast cancer, and their quantity related to tumor burden [4,45,79,80]. In addition, CTCs could be detected in 76% of all breast cancer patient regardless of their cancer subtype [81]. CTC counts are prognostic in PFS and OS in those with metastatic breast, whereas carcinoembryonic antigen and cancer antigen 15–3 serum tumor markers were not prognostic [82]. CTCs have also been found to have sub-populations with distinct genomic signatures that can direct treatment. One study discovered, there are CTCs sub-populations associated with breast cancer brain metastasis that could predict those at risk for brain metastasis [83]. Furthermore, a specific cell population, VIM⁺/CD45⁻, in prostate cancer was associated with poor prognosis and higher disease burden. Sparano et al. demonstrated in a secondary analysis of a clinical trial that even a single CTC was associated with late recurrence of estrogen receptor-positive breast cancer [84]. Finally, CTCs are well suited to demonstrating tumor heterogeneity, as many studies report a high concordance rate when comparing mutations between CTCs and tumor biopsies [85–87].

4.1. Circulating Tumor Microemboli

Clusters of CTCs are known as circulating tumor microemboli (CTM). These clusters contain an aggregation of a heterogeneous population of 3 or more cancer cells [27]. It is thought that CTMs shed from a primary tumor as whole unit cells rather than single CTCs aggregating together [88,89]. Of note, CTMs are rarer than CTCs and estimated to be 2–5% of any given CTC population. Interestingly, CTMs may have a survival advantage over CTCs due to their evasion of anoikis [90] and have been shown to have increased metastatic potential compared to single-cell CTCs [88,91]. Isolating CTMs has been challenging due to their rarity and the fact that most isolation techniques have been honed to capture CTCs. Also, based on a study by Aceto et al., CTMs have very short half-lives compared to CTCs and were cleared from circulation three times faster than CTCs [88]. Several clinical studies have demonstrated that CTC clusters have prognostic value and higher numbers are related to shorter PFS and OS in breast [92], lung [90] melanoma [93], ovarian cancer [94], gastric [95], colorectal [96], liver [97], and pancreatic ductal adenocarcinoma [98].

4.2. CTC Detection

Due to the rarity of CTCs circulating within the body [99], multiple methods have been created to isolate CTC populations. The two broad categories of isolation techniques are based up physical properties (i.e., size, density, deformability, and electrical charge) [43,100–103] and affinity-based selection [5,99,104–106]. Currently, immunomagnetic separation remains the most widely adopted technique. This process employs antibody-coated magnetic cells to positively enrich for CTCs through epithelial cell adhesion molecules (EpCAM) or negatively enrich for CTCs through CD45. Our group has previously reviewed the various assays in the liquid biopsy field [107]. Despite a multitude of enrichment methods, the anti-EpCAM antibody-based CellSearch assay remains the only Food and Drug Administration (FDA) approved platform. However, one of the issues with EpCAM based isolation methods is it potentially underestimates CTC numbers. Notably, Hyun et al. found that breast cancer CTCs, which undergo epithelial-mesenchymal transition, have low expression patterns of EpCAM [108]. Thus, approaches that rely on EpCAM isolation techniques miss low-EpCAM CTC populations, which is significant given that EMT is thought to play a role in tumor progression in metastasis [109,110].

Interestingly, several studies have noted a weak correlation between the detection of specific ctDNA transcripts and first-generation CTC assays, such as CellSearch, which permeabilizes CTCs, degrading their RNA [43,45]. Epitope-based selection is not logistically compatible with pursuing clinical validation studies both due to the unavailability of blood preservation strategies that do not compromise CTC viability and their repertoire of RNA transcripts. New CTC strategies have emerged that avoid some of these limitations, such as epitope independent filtration based approaches (i.e., ANGLE Parsortix) [111–115] or high definition imaging/proteomics platforms (i.e., Epic Biosciences) that image all of the blood cells. It has also been found that the type of blood collection tube a sample is collected in can affect CTC numbers. Specifically, blood collection in cell-free DNA tubes improves CTC detection numbers and has less degradation compared to EDTA, citrate, and heparin tubes [116]. Another study found that recovery of spiked prostate cancer cells was not affected by blood tube types (EDTA, citrate, and preservative-containing blood tubes) for up to 48hrs; however, tumor-cell RNA could not be recovered in tubes containing preservatives, while mRNA in EDTA tubes could be recovered for up to 48 hours after collection [117]. Our group has previously shown a rapid decline in cell recovery rate based on time elapsed; therefore, the RNA profiled after 48 hours is likely quite distinct from the RNA collected immediately after blood draw [38].

4.3. CTC Clinical Applications

CTCs have been demonstrated to be predictive biomarkers. Kuboki et al. found that CTC counts before receiving a cetuximab-containing regiment as third-line treatment were prognostic for OS but not PFS in advanced colorectal cancer [118]. Messarikakis et al. demonstrated that a higher number of CTCs prior to treatment and at disease progression were associated with decreased PFS in small cell lung cancer patients [119]. Given the evidence for CTC numbers and its association with outcomes, there has been a push to examine sub-populations of CTCs that can further guide treatments and help improve cancer survival predictions. Schneck et al. were able to isolate breast cancer CTCs with PIK3CA mutations [120], which is vital as drug resistance to HER2-targeted therapy have been associated with alterations in PIK3CA [121]. Pailler et al. showed that CTCs could predict crizotinib efficacy. Specifically, their group found an association between increased PFS and decreased CTC sub-populations with anaplastic lymphoma kinase gene copy number gain (ALK-CNG) after receiving crizotinib in NSCLC [122]. Bulfoni et al. examined sub-populations of CD45^neg CTCs and found those expressing epithelial and mesenchymal markers were associated with poor PFS and OS, while those with no expression of epithelial and mesenchymal markers were associated with secondary brain lesions [123]. Based upon a CTC 4-gene signature (EPCAM, ERBB2, MUC1, and KRT19), Bredemeier et al. demonstrated that CTCs with one of these expression genes were more likely to be overall non-responders to treatment for metastatic breast cancer [124]. In some cases, CTCs may provide more information on clinically actionable genes compared to a primary tumor biopsy. For instance, CTCs in breast cancer can have different HER2 and ER expression when compared to the primary tumor [81]. Findings such as this could have enormous implications for treatment if these differences are found to have effects on therapy responses.

The CirCe01 phase III trial was a non-randomized run-in phase trial that found patients with ≥5 CTC/7.5 ml at baseline had better PFS if CTC concentrations decreased by 70% of baseline or to <5 CTC/7.5 ml [125]. The STIC CTC phase III non-inferiority trial randomized ER+ HER2− metastatic breast cancer patients to either receive hormone therapy (HT) or chemotherapy (CT) based on a clinical decision versus receiving HT if CTC counts were <5 CTC/7.5ml or CT if ≥5 CTC/7.5 ml. They found that CTC counts could help in either escalating to CT when CTC counts were high or de-escalating to HT when CTC counts were low [126]. The Treat CTC trial discovered that trastuzumab did not decrease CTC counts in women with high risk, HER2 nonamplified, early breast cancer, which was fascinating as trastuzumab generally improves outcomes in women with HER2 positive breast cancer. The GeparQuattro clinical trial found that CTC detection prior to neoadjuvant therapy in locally advanced breast cancer was found to have lower disease-free survival (DFS) and OS [127]. The phase III JO21095 trial randomized HER2 negative metastatic breast cancer patients to concurrent capecitabine plus docetaxel or sequential docetaxel followed by capecitabine at progression and measured CTCs at baseline, prior to the start of a new cycle, and at progression. The results of the study found baseline positive CTC ≥2 CTC/7.5 ml were strongly associated with poorer PFS and OS. Interestingly, those with ≥2 CTC/7.5 ml CTC at baseline also had worse OS despite the fact that CTC counts decreased at the second cycle of therapy [128]. Finally, a phase II trial found that aspirin could decrease CTC numbers in metastatic colon cancer but not in metastatic breast cancer [129]. Although this is a small sample of clinical trials involving CTCs, they highlight the promising prognostic value of CTCs and how CTCs can add to the clinical picture; however, further studies need to validate the findings in these trials.

5. CTC versus ctDNA

There have been limited numbers of studies that compared CTCs and ctDNA within the same study. Germano et al. evaluated CTCs and ctDNA in metastatic colorectal cancer and noted ctDNA could be collected in all samples, but CTCs were only recovered in a third of the patient samples. They found an 84.6% concordance rate in ctDNA samples when compared to tissue biopsies. However, genotyping and concordance studies could not be performed on CTCs due to a low capture rate [130]. It must be noted that the low capture rate could be attributed to their CTC capturing method given newer CTC assays seem to have higher recovery rates of cells. Yanagita et al. found that NSCLC patients with high levels of baseline cfDNA were associated with poor PFS compared to lower levels of cfDNA. However, no difference in PFS could be found in those with high versus low CTC levels. In addition, both CTC and ctDNA provided information on mutations on actionable genes that were not present on the tissue biopsy, thus complementing each other [131]. Kidess-Sigal et al. reported high concordance for KRAS, BRAF and PIK3CA mutations for matched CTC and ctDNA samples, however, in several cases CTCs detected a mutation not found in ctDNA and vice versa [132]. It may be found that the two biomarkers complement each other in directing clinical decision making, but it is clear that there is much improvement to be made in the detection, isolation, and analysis of both CTCs and ctDNA.

6. Expert Opinion

A clinical-translational perspective

Carefully designed clinical-translational studies are needed to establish the diagnostic accuracy and clinical utility of liquid biopsy biomarkers in oncology. Investigators must fully consider relevant pre-analytical variables, assay sensitivity, bioinformatics considerations as well as the clinical utility of rare event profiling in the context of a normal blood background. Future liquid biopsy research should address the concern that not all DNA mutations are expressed and should provide the means to discover potential therapeutic targets in metastatic patients via a minimally invasive blood draw. Given this concern, our group has focused on gene expression profiling of CTCs to evaluate for potentially clinically actionable genes using well-validated biomarkers specific for our disease of interest. Other investigators have focused on transcriptomic profiling of CTCs as well, given this concern and the more functional role of RNA as a determinant of subsequent protein expression [133–136]. This approach allows us to compare CTCs and tumor biopsies across a wide range of potentially clinically actionable targets permitting the discovery of which genes are expressed in both types of specimens, as well as which genes are unique to CTCs. Our group has focused on whole transcriptome RNA Seq of CTCs, a powerful approach that permits pathway analysis, analysis of single nucleotide variants for mutation calling, and discovery of novel therapeutic strategies based on CTC biology.

Companion diagnostics in trials:

Future research on liquid biopsies will involve multi-institutional clinical trials in which treatment decision making may be made based on liquid biopsy results. There are numerous examples in which ctDNA was used as a companion diagnostic in clinical trials evaluating novel therapeutic agents prospectively. However, there are relatively few studies in which CTCs were incorporated as companion diagnostic testing that resulted in therapeutic decision-making based on either CTC enumeration or specific molecular biology features. Research in the field of CTCs will need to move beyond enumeration to looking at potential treatment targets based on the idea that CTCs reflect the real-time status of tumor biology under the selection pressure of treatment. The incorporation of gene expression profiling of CTCs in multi-institutional trials will require further refinements in blood preservation tubes to ensure that pre-analytical variables such as time since blood draw, temperature, and cell viability do not confound assay results. Currently, most clinical trials incorporating CTCs have used fixation methods such as CellSave tubes, which permeabilize the CTCs, thus limiting the ability to perform many relevant molecular biology assays of interest for characterization.

It is critical that liquid biopsy studies utilize appropriate negative controls, demonstrating that the targets of interest are not found in healthy controls. Commercially available reference standards derived from human cells and prepared as fragments of human genomic DNA rather than serial dilutions are preferable for demonstrating the lower limits of detection of ctDNA assays. CTC mimics using cancer cell lines spiked into peripheral blood should also be used to test the limits of detection for CTC assays. However, despite these efforts to establish suitable positive controls, profiling the blood of actual cancer patients with liquid biopsies is far more challenging than working with contrived specimens due to the rarity of signal to noise from the background peripheral blood. Liquid biopsies will need to demonstrate sensitivity, specificity, accuracy, and reproducibility to be accepted into clinical trial testing. Ultimately, liquid biopsy strategies must be tested prospectively in clinical trials in order to demonstrate clinical validity and utility by helping to select the appropriate population for treatment based on these biomarkers. There have already been a few successful liquid biopsy assays that have demonstrated clinical validity and earned FDA approval. Within the next decade, the goal for liquid biopsies should be the capacity to control cancer through the profiling of patients serially over time, identifying disease progression and informing clinicians of options for single or combination therapies that will successfully control the disease. Although treatment resistance and disease progression will emerge in the absence of highly effective treatments, the Holy Grail for liquid biopsies is to discover new opportunities for the clinician to intervene in order to successfully eradicate disease. The success of liquid biopsy biomarkers is intimately tied to the success of available therapeutic strategies – liquid biopsies cannot be useful unless therapeutic agents are effective for particular cancers. Researchers in the liquid biopsy field will need to focus on the role of CTCs, ctDNA and other circulating entities as companion diagnostic tests in order to transition from basic discovery to actual clinical utility.

Article highlights:

• Circulating tumor cells and circulating tumor DNA have great potential as liquid biopsies in cancer to guide treatment for patients with metastatic disease.

• Only 3 liquid biopsy assays have sufficient clinical evidence to achieve FDA approval for use in the clinic as either a companion diagnostic or prognostic marker.

• There is currently insufficient data demonstrating clinical validity and clinical utility for the majority of liquid biopsy assays

• The ability of liquid biopsies to accurately characterize potential treatment targets is critically important, particularly for heterogenous tumors

• Orthogonal tissue validation is required to determine if a liquid biopsy may serve as a diagnostic surrogate for metastatic disease.