Liquid biopsy for the detection of EGFR mutation can play many roles in cancer diagnostics [12-14,16,17]. Patients diagnosed with lung adenocarcinoma harboring EGFR mutation will be the first candidates when they develop resistance against first-line TKIs. Especially, when the tumor is too small or located in a challenging region to be sampled, liquid biopsy can be a good alternative [14-18]. Patients with poor performance status can also benefit from this technique.

Sample collection and processing is a critical step in liquid biopsy. Since ctDNA is rapidly degraded by the nuclease in blood and contaminated by genomic DNA from blood cells, it is essential to separate plasma from the sample [13,14]. The routine venipuncture technique will be sufficient to collect blood from the patients. The sample collection tube should be chosen considering each institution’s setting. Conventional ethyldiaminetetraaceticacid (EDTA) tube can be used if the samples are processed without delay [19,20]. Recently, specialized tubes for delaying degradation of ctDNA are commercially available [19,20]. The tube from Streck (Omaha, NE, USA) has been the most widely used collection tube. Roche diagnostics and Qiagen have also marketed specialized tubes. According to a study [19], conventional EDTA tube and Streck tube do not show much difference in their performance when samples are processed within 6 hours. When incubated longer in EDTA tube, cell-free DNA may be released from the blood cells, and EDTA will hinder the polymerase chain reaction (PCR) [20]. Tubes from Roche and Qiagen showed similar performance, and they are slightly better than Streck tube [20]. Specialized tubes can sustain sample quality for several days at room temperature before processing further (Table 1).

Before ctDNA extraction, blood should be processed into plasma through double centrifugation. Plasma samples are better than serum samples, which can be contaminated by DNA released from immune cells [13]. Since a small amount of ctDNA is present in plasma, isolation is a critical step in the process for saving tumor DNA. Several commercial kits for isolation are available in the market (Table 2) [21,22]. These are manual, semiautomatic, and fully automatic. Manual protocol uses column-based method while semi-automatic instrument works with magnetic beads. Previous studies showed variable results depending on the extraction kits, though they all had similar performances [21,22]. The technician’s skill and protocol optimization may be one of the critical factors for yielding better ctDNA. Table 1 summarizes commercial ctDNA extraction kits.

High sensitivity detection methods are required to detect EGFR mutations from liquid samples. Kits for detecting mutations have been developed and are commercially available [23-25]. Each kit requires different quality and amount of DNA (Table 3). They depend on real time PCR technology with their own variations. Roche Cobas uses real time PCR with Taqman like probe and Qiagen has released ARMS based kits, Therascreen EGFR RGQ. Another PCR based technique uses peptide nucleic acid clamping and Panamutyper (Panagene, Daejeon, Korea). The Roche and Qiagen systems use their own PCR machine from Roche and Qiagen while Panamutyper can run on any qualified PCR machines. The number of mutations these kits can detect are different; however, together they include exon 19 deletion, T790M and L858R. Currently, only Roche kit has acquired FDA approval. The most important element of these kits is how sensitively and specifically they can detect mutations in liquid samples. There are certain studies to evaluate their performance and report sensitivities ranging from 62% to 67.5% and specificities ranging from 88% to 97% [26-29]. In the ASSESS study, these three kits showed high specificity, however, sensitivity was equal to or less than 75% [25]. For T790M, sensitivity was 41% and 29% for Cobas and Therascreen, respectively, and specificity was 100% for both kits from the patients enrolled in AURA trial [10]. Therefore, deciding the best kit will depend on the laboratory’s choice with consideration of their requirements. Features of these products are summarized in Table 3. Other platforms using digital PCR and next generation sequencing are still far from widespread use in clinical setting [24].

Once liquid biopsy for detecting T790M mutation is done, the reports should contain the following information: pathologic number, age, sex, hospital unit number, sample source, requesting physician, requesting department, adequacy for testing (amount of DNA extracted), receipt day, report day, storage tube, methodology used, exons tested and associated range of detectable mutations, mutation status, comments, testing technician, and corresponding pathologist. Since the patients already have sensitizing EGFR mutation, it is recommended to include the type of original EGFR alteration and previous histologic diagnosis.

Since liquid biopsy technique has not been validated yet, vigorous quality assurance is necessary. Although there is no recommended program for external quality assessment (EQA), one pilot trial for EGFR testing in blood is ongoing in Germany [30]. Another program for BRAF and KRAS is also being conducted [31]. Since patient derived standard sample is difficult to store and distribute, artificial sample mimicking the real one can be used instead [30,31]. We are in the process of developing Korean EQA program.

Performance and interpretation of liquid biopsy require broad knowledge in lung cancer pathology. Pathologists have an important role in the diagnosis and management of cancer and thus can interpret liquid biopsy results in conjunction with the histologic diagnosis, previous status of EGFR-activating mutation, and clinical situation. The liquid biopsy in lung cancer is usually performed in patients whose previous EGFR mutation status has been known. The sole purpose of this technique is to detect a T790M mutation responsible for TKI resistance. Unlike tissue specimens, in which the pathologists can determine the percentage of tumor cells, it is extremely difficult to estimate whether the blood sample contains a sufficient amount of tumor DNA. If the sample is adequate, the test generally finds the original EGFR-activating mutation, which may act as an internal control for the presence of ctDNA [13]. When it has not detected any EGFR-activating mutation including previously existing one or reported mutations other than the preexisting ones or T790M, pathologists should be able to interpret the result. In the former, test should be repeated because the samples might have been degraded and contain insufficient ctDNA. In the latter, the newly emerged mutation, in the presence of newly developed lesion, may indicate a metachronous primary tumor. The communication between pathologists, clinicians, and radiologists is important for further diagnosis and management of cancer. Moreover, lung adenocarcinoma undergoes frequent transformation into small cell carcinoma when it is treated with TKI, while maintaining the original EGFR mutation [7,8,32]. Recommended interpretation is suggested in Table 4.

EGFR mutation testing performed with blood or other liquid sample is a non-invasive method, which can be more widely adopted. Laboratories must get familiar with liquid samples and develop their own protocols to handle these specimens. They can choose appropriate sample tubes, extraction kits, detection methods, and other instruments. They should select the most suitable combination in accordance with their requirements, unless the detection kits indicate specific methods and instruments [31]. Although sensitivity of tissue biopsy is higher than liquid biopsy, both are far from perfection and T790M mutation can be detected only in one of the two methods. Reportedly, allele fraction of T790M mutation tends to correlate with treatment efficacy of osimertinib [33]. Therefore, absence of T790M in tumor tissue while it is detected in plasma might reflect low allele frequency and lead to poor response. Therefore, the two methods are complementary to each other and should be selected according to each patient’s condition (Fig. 1).

Liquid biopsy is a promising method, which is safe and convenient. Before more experiences and data are accumulated, liquid biopsy should be performed with great caution. There are a few steps in liquid biopsy, which can produce false negative or false positive results. Interpretation requires profound knowledge of lung cancer including diagnosis, treatment, and prognosis. However, in debatable cases, discussion between pathologists, physicians, and radiologists is critical. This method will soon play a major role in early diagnosis, monitoring of treatment, and detection of minimal residual disease. Currently, it cannot replace the conventional pretreatment tissue diagnosis [14]. It is important to validate and improve the performance of this technique before it is widely used in clinical practice. Liquid biopsy performed in EGFR has provided a platform for determining gene mutations in KRAS, ALK, PI3CA, and BRAF as well.