Introduction to Liquid Biopsies

Introduction

This module reviews key aspects of research and clinical applications of liquid biopsies in cancer genomics, including the use of both circulating tumor cells (CTCs) and cell-free DNA (cfDNA). Topics include the clinical implications of liquid biopsies on screening, detection of mutations for therapy selection, disease prognosis and drug response and resistance. The module also describes the risks vs. benefits of applying liquid biopsies in cancer genomics, screening and treatment.

What is a Liquid Biopsy?

Blood contains two types of cancer-derived materials that are susceptible to detailed molecular analysis: intact circulating tumor cells (CTCs) and cell-free circulating tumor DNA (cfDNA; also referred to as circulating tumor DNA, or ctDNA). As tumors increase in volume, the capacity of phagocytes to eliminate and clear apoptotic and necrotic fragments can be exceeded, leading to passive release of cfDNA into the bloodstream. Physicians can use the blood drawn from a patient's arm to analyze DNA that tumors typically shed into the bloodstream. Depending on the tumor size and vascularity, the amount of cfDNA released in the circulation can vary from 0.01% to 90% of all DNA present in plasma. Therefore, liquid biopsies provide a noninvasive approach to tumor molecular profiling without having to obtain tumor tissue.

The evolution of sensitive CTC and cfDNA detection technologies has enabled the development of liquid biopsies with many clinical applications, including:

• screening for presence of disease;
• patient stratification and therapy selection (companion diagnostics);
• monitoring treatment response and drug resistance; and
• detection of minimal residual disease after surgery/recurrence.

The molecular analyses enabled by isolation of CTCs and cfDNA in liquid biopsies may be applied to guide different treatment strategies at different events in the initial diagnosis and treatment of patients with cancer (Figure).

Enlarge 

Risk Versus Benefit

Although liquid biopsies hold the promise of overcoming many of the drawbacks associated with tissue biopsies, it is likely that the latter will remain the gold standard for years to come. Until better technologies are available for testing liquid biopsies, tumor tissue will allow for a more thorough analysis, including the identification of more mutations than what is possible with a blood sample. However, having the option of a blood draw to learn about the genomics of a particular cancer will be valuable for many applications described earlier in this review. Advantages and disadvantages to liquid biopsy analysis are summarized below.

Advantages and Benefits

• Non-invasive method for identification of tumor markers, either as an alternative for patients whose tissue is unable to be biopsied or as an adjunct to evaluate drug response.
• Possibly less costly than tumor biopsy and analysis.
• Provides an accurate snapshot of the genomic landscape of the tumor, bypassing issues such as intratumor heterogeneity, as it is speculated that CTCs and cfDNA carry the driver mutations causing metastases.
• Able to obtain serial samples during treatment to assess for drug resistance and tumor progression. This is not picked up with a tumor biopsy given that tumor biopsies are generally only done prior to treatment, therefore mutations indicating resistance would not be picked up as these generally arise after starting therapy.
• Unlike tissue biopsy where the tumor DNA is preserved in formalin-fixed paraffin embedded (FFPE) blocks, DNA cross linking does not occur with liquid biopsy; thereby facilitating tumor DNA sequencing.

Liquid Biopsies' Greatest Benefits

Video 1-6

Video 1-6: Wafik El-Deiry MD, discusses the benefits that liquid biopsies can offer patients.

Disadvantages and Challenges

• Potentially miss biomarkers expressed in the tumor
• Test variability and assay sensitivity and specificity
• CTCs are rare, fragile and heterogenous
• Lack of consensus in technical approaches of choice

Reliability of Liquid Biopsy Results

Video 1-7

Video 1-7: Wafik El-Deiry MD, discusses whether liquid biopsy results are less reliable than traditional tissue biopsies.

Approaches to Liquid Biopsy Analysis: CTCs and cfDNA

The concept of using liquid biopsies to examine the genetic profile of tumors was first described in 1948 by Mandel and Metais. However, this topic did not receive much attention until recently due to the advances in molecular techniques coupled with the advent of molecularly targeted therapies for treating various types of cancers. Although the mechanism(s) through which tumor cells shed their genetic material into the systemic circulation is not fully understood, this process can be broadly categorized into passive and active mechanisms. Without delving into the intricacies of each process, passive mechanisms generally involve the release of tumor DNA into the circulation after cell necrosis or apoptosis, whereas in the latter, the tumor spontaneously sheds fragments of its DNA into the blood stream to affect the transformation of susceptible cells at distant sites.

The two main approaches that are currently pursued to detect tumor DNA in the blood include analyzing cfDNA and detecting CTCs. Of note, recent studies suggest that tumors eject small vesicles also referred to as exosomes, which may also be used to detect tumor DNA; however, this section primarily focuses on the clinical applications of CTCs and cfDNA.

Circulating tumor cells are intact tumor cells that are dislodged into the systemic circulation. These cells serve as seeds for subsequent growth of additional tumors (metastasis) at distant sites. Circulating tumor cells are primarily detected in various metastatic carcinomas such as breast, prostate, lung and colorectal cancers. Given the fact that most CTC isolation platforms necessitate that the blood sample be processed within 96 hours after collection, one of the major pitfalls of this approach is the inability to biobank the collected intact cells. On the other hand, cfDNA are fragments of tumor DNA, around 160 to 180 base pairs in length with a higher prevalence of mutations in the smaller fragments. One of the main advantages of cfDNA is that it can be analyzed from biobanked biological fluids (including frozen plasma or serum).

Technologies for Isolation and Detection of CTCs and cfDNA

One of the main challenges with isolating CTCs is their low abundance relative to the normal circulating blood cells. Even in metastatic settings, patients generally have fewer than 10 CTCs/mL of blood. Hence, most isolation techniques start with an enrichment step in which the concentration of the CTCs is increased by several log units to enable or facilitate their detection. Numerous CTC detection methods have emerged over the years. Nonetheless, they all take advantage of the physical properties of the CTCs such as their large size in comparison to circulating leukocytes (median diameter 15 µm vs. 10 µm), or the unique expression of cell surface markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratins that are heavily expressed on the surface of epithelial cancer cells. Of note, CTCs can undergo epithelial to mesenchymal transformation, and thereby lose their expression of EpCAM as well as other surface markers via this process.

Detection of cancer by monitoring cfDNA has also garnered much enthusiasm because tumor specific alterations in cfDNA are unique to the tumor and not present in normal or noncancerous cells. Hence, cfDNA potentially offers a sensitive and specific strategy for cancer detection. Similar to CTCs, one of the greatest technical challenges is the identification of very low amounts of tumor cfDNA where it accounts for less than 1% of total circulating free DNA in the blood. Thus, standard sequencing techniques, such as Sanger sequencing or pyrosequencing, can detect cfDNA only among patients with heavy tumor burden. However, the introduction of digital polymerase chain reaction; beads, emulsification, amplification and magnetics; or pyrophosphorolysis-activated polymerization have enabled the detection of cfDNA derived from tumors in a considerably consistent manner.

Liquid Biopsy Applications

The use of tumor biopsies for diagnosis, prognosis and therapy selection will remain the mainstay in cancer genomics for many years; however, the addition of liquid biopsies as an alternative in patients who are unable to undergo an invasive tumor biopsy allows for many clinical applications, including screening, detection of mutations for therapy selection, disease prognosis and drug response and resistance. The ability to perform serial blood sampling provides a unique mechanism to monitor the course of disease without having to obtain tissue at frequent intervals. This section discusses various clinical studies and data demonstrating the potential applications liquid biopsies may have on clinical practice in the near future.

Video 1-8

Video 1-8: Liquid Biopsies vs. Tissue Biopsies. Wafik El-Deiry MD, discusses the pros and cons of both tissue and liquid biopsies.

Screening

The concept of using a blood test to screen for and monitor cancer is not new (eg, PSA test for prostate cancer, carcinoembryonic antigen test for colorectal cancer, carbohydrate antigen 19-9 test for pancreatic cancer or cancer antigen 125 test for ovarian cancer). However, these previous methods can be extremely nonspecific and result in high rates of false positives. Given that tumors shed CTCs and cfDNA into the blood stream, it is possible to use liquid biopsies for cancer screening. In fact, labs around the world are currently competing by developing screening tests based on a simple blood draw.

To be able to use liquid biopsies for cancer screening, liquid biopsy analyses would have to be sensitive enough to detect when the tumor is actually releasing cancer cells. For example, the process of cancer development from in situ to invasive disease could be associated with the activation of innate immunity, and such changes might be detected in the blood before they show up on an imaging scan. Another possibility of cancer detection could be the identification of products of tumor metabolism related to the cancer.

For example, studies have reported on the potential utility of microRNAs (miRNAs) to diagnose non–small cell lung cancer (NSCLC). Shen and colleagues determined that a panel of four miRNAs in plasma (miRNA-21, miRNA-126, miRNA-210 and miRNA-486-5p) were significantly elevated in patients with NSCLC (n = 58) compared with healthy individuals (n = 29), with 86% sensitivity and 97% specificity.

The use of liquid biopsies to screen for and diagnose cancers is still many years away. However, significant progress has been made in detection of actionable mutations for therapy selection, patient stratification, drug resistance and disease monitoring.

Detection of Actionable Mutations for Therapy Selection

More data are accumulating to suggest that cfDNA is a broadly applicable, sensitive and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer. In the same method that tumor molecular profiling is used to detect actionable mutations for therapy selection, liquid biopsies may offer an alternative method for mutation detection to guide therapy selection in patients unable to undergo a tissue biopsy (or as an adjunct to a tissue biopsy).

• A study by Bettegowda and colleagues (n = 640 patients with various cancers) demonstrated that cfDNA was detectable in more than 75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular and head and neck cancers, but it was detectable in less than 50% of patients with primary brain, renal, prostate or thyroid cancers. In patients with localized tumors, cfDNA was detected in 73%, 57%, 48% and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer and breast adenocarcinoma, respectively. In a subset of 206 patients with metastatic colorectal cancer, the sensitivity of cfDNA for detection of KRAS mutations was 87% and specificity was 99%. In another subset of 24 patients who objectively responded to epidermal growth factor receptor (EGFR) inhibitor therapy but subsequently relapsed, 96% developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway.
• In one of the largest studies investigating cancer mutations in liquid biopsies, Zill and colleagues demonstrated that a liquid biopsy identified cancer mutations in 85% of all advanced tumors. In 49% of the cases, these biomarkers were associated with an approved targeted drug. Investigators used the commercially available Guardant360 assay (Guardant Health), a highly sensitive next-generation sequencing technique, to look for patterns of genetic changes (approximately 70 actionable tumor mutations) in 17,628 blood specimens from 15,191 patients. The results showed that cfDNA mutation patterns were highly consistent with distribution in tumor tissue by the publicly available The Cancer Genome Atlas (TCGA). The investigators report that correlations with TCGA ranged from 0.92 to 0.99. Furthermore, the EGFR T790Mresistant mutation was seen in the blood but not the original tumor biopsies, because it emerges after treatment with EGFR inhibitors, which were given after tumor biopsy. Taking into account FDA-approved agents and eligibility for clinical trials, the cfDNA assay identified a possible treatment option for 63.6% of all patients. The overall accuracy of cfDNA sequencing in comparison with matched tissue tests in a subset of 386 patients was 87%. The accuracy increased to 98% when blood and tumor were collected less than 6 months apart.
• Levy and colleagues reviewed the clinical applications of liquid biopsy technologies, including CTCs, proteomics, miRNA and cfDNA for NSCLC, and provided insight into the diagnostic and therapeutic implications and challenges of these platforms. One study found the concordance rate between EGFR mutations in serum and tissue was 92.9%, with a sensitivity of 85.7%. Another study determined the mutation concordance rate between 652 matched tumor and plasma samples before treatment was 94.3%, with a sensitivity of 65.7% and specificity of 99.8%. However, rates of concordance across all studies varies (27.5%-100%) with low to high sensitivities (17.1%-100%) and consistently high specificities (71.4%-100%). The disparities in sensitivities depend heavily on technology and platforms. Two meta-analyses assessing the diagnostic accuracy of cfDNA EGFR mutations demonstrated a pooled sensitivity of 61% and 67.4% and specificity of 90% and 93.5%, respectively.
• In a phase 4 trial of gefitinib (Iressa, AstraZeneca) in patients with NSCLC, those with EGFR mutation–positive cfDNA, regardless of mutation subtype, had a similar overall response rate as that of patients with tissue EGFR mutation–positive tumors (76.9% and 69.8%, respectively), suggesting that the blood-based EGFR test might be as predictive to tyrosine kinase inhibitor treatment as tissue. In fact, response rates were higher in patients with matched samples who harbored mutations in both blood and tissue (76.9%; 95% CI, 65.4-85.5) compared with patients with only mutation-positive tumor tissue (59.5%; 95% CI, 43.5-73.7). Similar results were seen in an exploratory analysis of patients treated in the IPASS study. Given the number of studies that have demonstrated EGFR mutations in cfDNA are reliable predictive biomarkers of treatment with erlotinib (Tarceva; Genentech, Astellas) or gefitinib, the FDA approved the cobas EGFR Mutation Test v2 (Roche Molecular Systems), a blood-based companion diagnostic for erlotinib and the first FDA-approved liquid biopsy assay.
• Schwaederle and colleagues published their pilot experience in 168 patients with diverse cancers who underwent digital next-generation sequencing of plasma for 54 cancer-related genes. Fifty-eight percent of patients had at least one cfDNA alteration, 71.4% of whom had at least one alteration potentially actionable by an FDA-approved drug. The overall concordance rates for tissue and cfDNA were 70.3% for TP53 and EGFR, 88.1% for PIK3CA, and 93.1% for ERBB2 alterations. Importantly, there was a significant correlation between the cases with one or more than one alteration with cfDNA greater than or equal to 5% and shorter survival (median OS 4.03 months vs. not reached at median follow-up of 6.1 months; P < .001). Approximately 42% of patients matched to a targeted therapy achieved stable disease greater than or equal to 6 months compared with 7.1% for the unmatched patients (P = .02).

Disease Prognosis

As mentioned previously, detection of CTCs and cfDNA can also be used to assess disease prognosis. Studies have demonstrated that, in addition to the detection of mutations in cfDNA, the number of CTCs at baseline and throughout treatment can be predictive of clinical outcomes.

• In a prospective, multicenter study, Cristofanilli and colleagues tested 177 patients with metastatic breast cancer for levels of CTCs both before the patients were to start a new line of treatment and at the first follow-up visit. Patients in a training set with CTC levels greater than or equal to 5 per 7.5 mL of whole blood, as compared with the group with fewer than 5 CTCs per 7.5 mL, had a shorter median PFS (2.7 months vs. 7.0 months, P < .001) and shorter OS (10.1 months vs. >18 months, P < .001). A multivariate analysis demonstrated that, of all the variables in the statistical model, the levels of CTCs at baseline and at the first follow-up visit were the most significant predictors of PFS and OS.
• Sausen and colleagues performed whole-exome sequencing on 24 tumors, targeted genomic analysis on 77 tumors, and cfDNA analysis to examine tumor-specific mutations in the circulation of these patients. Liquid biopsy analyses demonstrated that 43% of patients with localized disease had detectable cfDNA at diagnosis. Detection of cfDNA after resection predicted relapse and poor outcome, with recurrence by cfDNA detected 6.5 months earlier than with radiologic imaging.
• The Spanish Lung Cancer Group demonstrated EGFR status on cfDNA was prognostic of clinical outcomes in patients with NSCLC. A prespecified analysis of 76 patients with identifiable cfDNA EGFR mutations demonstrated a shorter median OS in patients with the L858R mutation than in those with the exon 19 deletion (13.7 months; 95% CI, 7.1-17.7; vs. 30.0 months; 95% CI, 9.3-37.7; P = .001). Among the 41 patients with the L858R mutation in tissue, those in whom the L858R mutation was also detected in cfDNA had notably shorter median survival than those in whom the mutation was not detected in cfDNA (13.7 vs. 27.7 months; HR = 2.22; P = .03), suggesting a prognostic value of plasma cfDNA L858R mutations.

Drug Response and Resistance

Last, and perhaps most importantly, detection of CTCs and cfDNA can be used to predict drug response and drug resistance in patients initiating a targeted therapy. One major advantage of liquid biopsies is the ability to obtain serial blood samples for cfDNA analysis, which provides the opportunity to examine molecular changes during therapy, with the hope of detecting drug resistance before seeing radiographic evidence of disease progression.

• A study by Mok and colleagues used the EGFR cobas test to evaluate plasma EGFR mutations in patients treated with gemcitabine/platinum plus sequential erlotinib or placebo. For patients treated in the erlotinib arm who tested positive for EGFR cfDNA at baseline, the disappearance of cfDNA at cycle 3 was significantly associated with improved PFS (HR = 0.38; P = .0083) and a trend toward longer OS (HR = 0.45; P = .0831) compared with patients who continued to express EGFR in cfDNA.
• Tseng and colleagues prospectively evaluated matched cfDNA and tissue samples from 62 patients with EGFR-mutated NSCLC. The study demonstrated that failure to clear cfDNA EGFR mutations was an independent predictor of lower disease control rate (OR = 5.26; 95% CI, 1.13-24.44; P = .034), shorter PFS (HR = 1.97; 95% CI, 1.33-2.91; P = .001) and decreased OS (HR = 1.82; 95% CI, 1.04-3.18; P = .036).
• In another study, Marchetti and colleagues performed polymerase chain reaction and deep sequencing on serial plasma samples from 520 patients with NSCLC with known tissue and plasma EGFR mutations before treatment. Patients who had a greater than or equal to 50% reduction in plasma EGFR copy number at 2 weeks had a greater mean percentage of tumor shrinkage than patients who had less than 50% reduction in plasma EGFR copy number (70% vs. 30%; P = .0001).
• Oxnard and colleagues studied whether genotyping of cfDNA was a useful biomarker for prediction of outcome from a third-generation EGFR tyrosine kinase inhibitor, osimertinib. Patients who were included had acquired drug resistance and evidence of a common EGFR-sensitizing mutation, T790M. Sensitivity of cfDNA genotyping for detection of EGFR T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 31%. The response rate (63%) and median PFS (9.7 months) were similar in patients positive for T790M in cfDNA. Although patients with T790M-negative plasma still had an overall response rate of 46% and median PFS of 8.2 months, tissue genotyping distinguished a subset of patients with T790M-positive tumors who had better outcomes (69% response rate and 16.5 months median PFS) and a subset of patients with T790M-negative tumors with poor outcomes (25% response rate and 2.8 months median PFS).
• Wang and colleagues investigated the presence of the T790M resistance mutation in pre- and post-treatment cfDNA in 135 patients with advanced NSCLC who had clinical benefit of 6 months or more with first- or second-line TKI treatment. In the cohort of 83 patients with known EGFR mutations by tissue, the patients were categorized into three groups according to the quanity of T790M in pretreatment plasma samples (high, >5%; low, 0%-5%; nil, 0%). The median PFS was 7.1, 9.5 and 12.8 months (P = .001) and the median OS was 18.2, 21.2 and 32.5 months (P = .005) for the high, low and nil groups, respectively, suggesting that a high pretreatment T790M mutational load might define patients less likely to benefit from TKI therapy.

Concordance Between Tumor and Liquid Biopsies for Mutational Analysis

Summary of Tumor and Liquid Biopsy Concordance

One important issue to consider is whether tumor DNA obtained from liquid biopsy serves as a reliable proxy for mutations identified in tissues. The Table below highlights the results from several studies that sought to interrogate the concordance rates between mutations identified in tissues and liquid biopsies. The results from these studies lend further support to the use of liquid biopsy as an alternative or adjunct to tissue biopsy.

Enlarge 

Video 1-9

Video 1-9: Cancer Types for Liquid Biopsies. Wafik El-Deiry MD, discusses what cancer types are most appropriate for liquid biopsies.

Select Commercially Available Liquid Biopsy Assays

Liquid biopsies are beginning to fill a major niche in molecular diagnostics and the number of commercial companies offering liquid biopsy analysis is continuously increasing. Experts estimate that using DNA blood tests for cancer screening will be a $10-20 billion a year market by 2020. Companies are investing hundreds of millions of dollars to develop the innovative technologies required to test for liquid biopsies. Ultimately, a liquid biopsy may provide a less-expensive and less-invasive way to monitor patients throughout treatment, and large molecular diagnostic companies see this investment as high risk and even higher reward. As clinicians, it will be important to weigh the risks and benefits discussed earlier in this review, and compare and contrast the available liquid biopsy assays available. A non-inclusive list of commercially available liquid biopsy assays is listed here:

• Guardant360 (Guardant Health)
• FoundationACT^TM (Foundation Medicine)
• Grail (Grail Bio, an Illumina spinoff)
• Cobas EGFR Mutation Test v2 (Roche)
• Cancerintercept (Pathway Genomics)
• PlasmaSelect-R^TM (Personal Genome Diagnostics)
• myRisk (Myriad Genetics)
• Oncotype SEQ (Genomic Health)
• OncocEE (Biocept.com)

Video 1-10

Video 1-10: Liquid Biopsy Testing Systems. Wafik El-Deiry MD, discusses the emerging technology of liquid biopsy testing systems.