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.
Wafik El-Deiry MD, discusses the pros and cons of both tissue and liquid biopsies.
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 T790M resistant 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).
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.
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