Are Liquid Biopsies Ready for Prime Time of Clinical Applications? Shuhang Wang, MD, MS,a Jie Wang, MD, PhDb,*
Molecular targeted therapy represented by EGFR tyrosine kinase inhibitors (TKIs) based on genotyping has become a paradigm for precision management of NSCLC. To realize detection and monitoring of EGFR activating and resistance mutations during EGFR TKI therapy, it is important to access tumor samples at different time points. However, this is extremely challenging in patients with lung cancer. Even with access to tumor biopsy specimens, the spatial and temporal heterogeneity of EGFR mutations in NSCLC complicate interpretation of test results. Liquid biopsy has played a prominent role in dynamic and quantitative detection by conquering the difﬁculty of repeat tissue biopsies and intratumor genetic heterogeneity.1,2 However, is current evidence sufﬁcient to support using EGFR mutation detection assays based on liquid biopsy in routine clinical practice? Throughout serial studies on liquid biopsy in recent years, detecting EGFR activating mutations by using circulating tumor DNA (ctDNA) in peripheral blood has been demonstrated to be feasible and reliable for patients with advanced NSCLC, with consistency rates ranging from 78% to 95%, sensitivity rates from 63% to 82%, speciﬁcity rates from 92% to 100%, and comparable predictive power to predict EGFR TKIs responses comparable to that of tissue-based detection.3–6 On the basis of this evidence, the European and Chinese instructions for geﬁtinib use have been amended to “plasma ctDNA may be used for EGFR mutation detection when tumor tissues are unavailable.” However, most evidence provided in literature has been from singlecenter or multicenter controlled clinical studies, which raises the issue of whether such an approach is applicable in routine clinical practice. In the current issue of the Journal of Thoracic Oncology, Reck et al. reported a large, multicenter (in Europe and Japan), noninterventional diagnostic study intended to investigate the utility of plasma ctDNA EGFR mutation testing in a real-world diagnostic setting (the ASSESS study).7 They observed that the concordance of mutation status is 89% (sensitivity 46%, speciﬁcity 97%, positive predictive value [PV] 78%, and negative PV 90%) in 1162 matched plasma ctDNA and tissue
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samples. Compared with the results of previous studies on ctDNA-based EGFR mutation detection,3–6 this study presented lower sensitivity, lower PV, and a higher falsenegative rate. It is possible that the use of multiple detecting methods, including the QIAGNE therascreen EGFR mutation test (Qiagen, Hilden, Germany), Roche cobas EGFR mutation test (Roche Molecular Systems, Pleasanton, CA), and peptide nucleic acid–locked nucleic acid for EGFR mutation analysis, in this study might have contributed to the inferior performance because these technologies have different quality control systems and diverse detection sensitivities. Other factors such as the methods used to obtain tumor samples (surgical resection, biopsy, or cytologic examination), the location of tumors (primary versus metastatic site), the time point of sample collection (fresh versus stored samples), and disease staging (advanced versus locally advanced NSCLC) could all affect the results. In addition, this study was limited by the fact that it was merely a molecular epidemiological investigation and did not include information on the treatment efﬁcacy of EGFR TKIs, so that EGFR mutation status in ctDNA could not be correlated with the clinical outcome of EGFR TKIs. However, the large sample size helped to demonstrate the challenges of plasma ctDNA detection in the real world, suggesting that the standardization and optimization of sample collection, storage, DNA isolation, and mutation detection are factors vital to ensuring high sensitivity, speciﬁcity, and predictive accuracy of ctDNA detection for EGFR mutation. *Corresponding author. a
Beijing Cancer Hospital, Beijing, People’s Republic of China, and Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China. b
Disclosure: The authors declare no conﬂict of interest. Address for correspondence: Jie Wang, MD, PhD, Cancer Hospital Chinese Academy of Medical Sciences, Chaoyang District, Panjiayuan Nanli No. 17, Beijing, People’s Republic of China. E-mail: [email protected]
yahoo.com ª 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 http://dx.doi.org/10.1016/j.jtho.2016.08.125
T790M mutation is the most common mechanism of resistance to EGFR TKIs,8 and multiple studies have demonstrated that T790M mutation detection in plasma ctDNA possessed excellent sensitivity, speciﬁcity, and predictive accuracy in relation to third-generation TKIs, such as AZD9291 and CO1686, compared with tissue samples from repeat biopsies.9,10 These results impelled the U.S. Food and Drug Administration’s recent approval of the cobas EGFR Mutation Test v 2 (Roche Molecular Systems), which is speciﬁcally designed to detect T790M mutations in the blood of patients with NSCLC, as a companion diagnostic test, which is a testament to the application of liquid biopsies in the clinical setting. Besides plasma ctDNA, urine may also be used as a biological specimen for genetic testing. In this issue of the Journal of Thoracic Oncology, Reckamp et al. reported for the ﬁrst time that T790M mutation in patients with metastatic lung adenocarcinoma treated by EGFR TKIs could be detected in urine. In this study, EGFR activating and T790M mutations in paired tumor tissues and ctDNA from blood and urine samples were analyzed through the hypersensitive next-generation sequencing technique, and the high sensitivity, speciﬁcity, and predictive accuracy of T790M mutation in urine ctDNA to third-generation TKIs were presented, especially when urine volume reached more than 90 mL. This is clinically signiﬁcant because testing of urine ctDNA provides a truly noninvasive and easy to self-manage alternative to tissue biopsy for detection of the T790M mutation status of patients with advanced NSCLC.11 In addition, the longitudinally dynamic alteration of the T790M signal in urine correlated with tumor shrinkage was observed in a subset of nine patients receiving treatment with rociletinib, and similar results have been reported in studies involving plasma ctDNA detection. These results suggest that urine-based genetic analysis may be used to monitor patients’ response and foresee the appearance of novel resistance genes before clinical or radiological progression. If the cutoff value of “molecular resistance” in urine or plasma can be determined in further study, the antiresistance strategy will switch from treatment to prevention, which may bring patients a greater survival beneﬁt. The other strength of this study is that 12 T790Mpositive patients who were not detected through tumor tissue samples were identiﬁed by using urine and plasma samples, supporting the evidence that liquid biopsy has advantages for overcoming the heterogeneity of repeat biopsy tissue assays. This also raises the question of whether liquid biopsy is more “precise” than tumor biopsies, especially when liquid biopsy is used for monitoring of resistance related genes change. Further studies will be needed to support the hypothesis.
Liquid Biopsies and Clinical Applications
The detection and monitoring of relapse and metastasis of early-stage NSCLC after radical resection at the cytological and molecular genetic levels have become hot issues in recent years. As another vector in peripheral blood, the circulating tumor cell (CTC) appears to be an excellent model to investigate tumor recurrence and metastasis.12 Previous research focused mainly on determining the relationship between alteration of CTC count after treatment and patients’ outcome in advanced cancers such as breast cancer and prostate cancer.13,14 Recent studies of single–driver gene or genome sequencing in a single CTC or bulk CTCs extends the realm of liquid biopsy–based research.15–17 However, highly efﬁcient acquisition of CTCs is a cornerstone for subsequent analysis of cancer-speciﬁc genomic alteration. Crosbie et al.18 analyzed the correlation between tumor recurrence and metastasis and CTC enumeration (a single cell versus a cluster) in tumordraining pulmonary veins and peripheral blood in early lung cancer. They found that pulmonary vein CTC count was signiﬁcantly higher when compared with that in matched peripheral vein samples. Pulmonary vein and peripheral vein CTC counts were both signiﬁcant risk factors for tumor recurrence. Pulmonary vein circulating tumor microemboli or two or more CTCs per 7.5 mL of peripheral blood were related to high-risk for both tumor recurrence and death. However, it is too early to draw conclusions on account of the limited sample size. Furthermore, the results of this study were affected by the fact that it involved only the analysis of CTC quantity and did not examine the role that hotspot genetic alteration may play in recurrence and metastasis of lung cancer. Notwithstanding, Crosbie et al. provide the evidence for conducting additional large-scale, multicenter studies to ascertain whether CTCs or CTM in pulmonary veins represent the cluster of cells with the highest capacity for invasion and metastasis. In summary, after nearly a decade of exploration and development, detection of EGFR activating and resistance mutations on the basis of plasma ctDNA has gradually been applied in clinical practice of NSCLC, ﬁnally becoming the prerequisite and basis for accomplishing precision management of lung cancer. Urinary DNA– and CTC-based genetic detections will enrich the utility of liquid biopsy after undergoing large-sample and multicenter studies. The three studies published in this issue of Journal of Thoracic Oncology have added evidence from different perspectives for clinical utilization of liquid biopsy.
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