Liquid Biopsy in Non-Small Cell Lung Cancer

Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as “standard” biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.

occasionally intervene and fixation, embedding, or staining are equally infrequent. In addition to the "biopsy" half, the "liquid" half in the term "liquid biopsy" can also be misleading. The materials originated in the tumor that are to be detected in such "biopsies" are never liquid. Some of them are cells or fragments of cells, such as circulating tumor cells (CTCs), exosomes, or tumoreducated platelets (TEPs); others are nucleic acids dissolved in the blood, such as circulating tumor DNA or RNA (ctDNA, ctRNA). Each of these materials offers unique opportunities to test different biomarkers and analyze particular characteristics of the tumors ( Table 1).
The differences between a "real" and a "liquid" biopsy-or "liquid sample, " as the pathologists would probably prefer to call them-explain the advantages of the latter. "Liquid" biopsies will never replace real biopsies, which are irreplaceable sources of information that cannot be obtained by any other means, such as tumor type and histology. However, they offer all sorts of additional data that cannot be obtained in any other way. In patients who cannot be biopsied, or where biopsies do not have enough tissue, "liquid biopsy" is the only alternative to perform genetic testing for targeted therapy. Also, in patients with advanced disease, it is not feasible to obtain biopsies of every metastasic site. But blood reaches both the primary tumor and the metastases, and materials coming from all can be found in a "liquid biopsy. " Finally, unlike "real" biopsies, blood can be repeatedly obtained without the risk of comorbidities and used to monitor the course of the disease, including early detection of response and relapse or emergence of resistance to a particular therapy.

CiRCULATinG TUMOR DnA
Circulating free DNA (cfDNA) can be found dissolved in plasma and serum, at variable amounts. In the case of cancer patients, a fraction of the cfDNA is tumor derived, and ctDNA represents from less than 0.1% to more than 10% of the total cfDNA. This percentage has been shown to depend on stage, tumor burden, vascularization of the tumor, biological features like apoptotic rate and metastatic potential of the cancer cells, and factors affecting the blood volume of the patient (1,2). In addition, variations on the relative abundance of ctDNA correlate with response to therapy (3)(4)(5). ctDNA is released by passive mechanisms, such as lysis of apoptotic and necrotic cells or digestion of tumor cells by macrophages, and also by active mechanisms. In this respect, cfDNA shows and enrichment in 150-180 bp fragments typical of the nucleosomal pattern of DNA fragmentation during apoptosis (6)(7)(8)(9). The ctDNA carries the same somatic alterations as the tumor itself and can be used to detect clinically relevant mutations such as those in the epidermal growth factor (EGFR) or KRAS genes. This is particularly useful when no biopsy is available for genetic analyses and, in this setting, the European Medicine Agency recommends EGFR testing in liquid biopsies to select patients for tyrosine kinase inhibitor (TKI) therapy (10). However, many standard techniques for mutation detection are not useful for ctDNA analyses due to an insufficient sensitivity. Since ctDNA often represents a small percentage of the total cfDNA, somatic mutations coming from the tumor can be present at allele fractions as low as 0.01%. Highly sensitive methodologies, or variations of preexisting methodologies, have been developed in order to detect low abundance mutations in cfDNA (6,11).
Modified real-time PCR techniques have been widely used to identify genetic alterations in the cfDNA of cancer patients. They include amplification-refractory mutation system [ARMS (12),], Scorpion-ARMS (13), and peptide nucleic acid (PNA) or locked nucleic acid (LNA) mutant-enriched PCR (14)(15)(16)(17). The diagnostic sensitivity of these techniques, when compared to tumor tissue, ranges from 43 to more than 90%, while the specificity is usually close to 100%; and the two commercially available methods to determine EGFR mutations in the cfDNA of cancer patients (Therascreen Plasma from Qiagen and COBAS Blood from Roche Diagnostics) are based on them. In our group, we have developed a quantitative PCR technique in the presence of PNA to detect EGFR, KRAS, and BRAF mutations in the cfDNA of advanced lung, colon, and cancer patients that achieves 75-80% sensitivity with 100% specificity (18,19). Digital PCR, droplet digital PCR, and beads, emulsion, amplification, and magnetics (BEAMing) system constitute further refinements of the PCR-based techniques and have also been used to determine mutations in cfDNA (14,(20)(21)(22)(23)(24)(25)(26) (Table 2).
Most modified PCR techniques are easy, comparatively unexpensive, and have a quick turnaround time (19), but have the disadvantage that can only detect mutations in a limited number of loci, usually within a single gene. Next-generation sequencing methodologies can overcome these limitations but, while tissue-based NGS genotyping is already well established, the application of NGS technologies to liquid biopsies is challenging and an ultra-deep sequencing approach is commonly used in order to improve sensitivity. In this approach, the gene panels are limited so that each read is sequenced thousands of times (39, 50, 51). However, this requirement of a high sensitivity may easily lead to false-positive results and requires a careful validation of the whole testing process. Examples of NGS protocols specifically developed for ctDNA analysis include TAm-Seq (tagged-amplicon deep sequencing), which combines site-specific primers with universal tails (52, 53); Safe-SeqS (Safe-Sequencing System) (54), and CAPP-seq (capture based sequencing), which relies on hybridization-based capture of target regions followed by amplification (38, 55) ( Table 2).
The detection of mutations in cfDNA by modified PCR or NGS techniques is not only useful in lung cancer patients at presentation. It has also been successfully employed for patient monitoring, including early evaluation of response and relapse, which are associated with changes in the EGFR or KRAS mutational burden in cfDNA; and for early detection of acquired resistance to EGFR TKIs, associated in many patients with the emergence of the p.T790M mutation in blood (26,56). In this respect, p.T790M testing in cfDNA has been recently recommended in patients eligible for osimertinib treatment, in order to avoid unnecessary rebiopsies (33, 36, 56). CiRCULATinG TUMOR RnA Similar to ctDNA, RNA derived from tumor cells (ctRNA) is present in the plasma of cancer patients and can be used for detection of the clinically relevant ALK, ROS1, and RET fusion genes and METΔ14 splicing variant. However, genetic analyses in cfRNA present specific challenges and have not been widely used. Unlike cfDNA, cfRNA degrades very quickly and needs to be purified rapidly after blood extraction. The alternative is adding a preservative such as Trizol and freezing the sample at −80°C, but this procedure is not easily accessible to many clinical sites. Despite these limitations, our group has a 5-year experience in detection of EML4-ALK fusion transcripts in plasma cfRNA by retrotranscription PCR (RT-PCR) (49) and, using improved processing and purification methods, we have demonstrated that the sensitivity of the technique can be significantly improved.

TUMOR-eDUCATeD PLATeLeTS
Platelets have been recently demonstrated to sequester tumor RNA by a microvesicle dependent mechanism, and the socalled TEPs (57, 58) can be used as a source of tumor RNA for genetic analysis. Platelets can be isolated from blood by simple centrifugation steps, and its RNA content easily purified and used for the detection of gene fusions and splicing variants. Using a RT-PCR approach, our group has detected EML4-ALK fusion transcripts in TEP RNA from advanced lung cancer patients with 65% sensitivity and 100% specificity (49). In addition, we have demonstrated that the disappearance of fusion transcripts in platelets correlates with response to crizotinib treatment. Regarding splicing variants, the clinical relevance of METΔ14 in lung cancer was only described in 2015 (59-61), and there are no reports in the literature about detection of METΔ14 transcripts in liquid biopsy. However, we have recently detected the alteration in the TEP RNA of a NSCLC patient positive in tumor tissue, who attained a partial response to crizotinib (unpublished data). Platelet RNA can also be analyzed by multiplexing techniques, and a recent report has demonstrated the diagnostic potential of this approach. Using mRNA sequencing and surrogate TEP RNA profiles of 283 samples, 228 cancer patients of six different origins were discriminated from 55 healthy individuals with 96% accuracy. Tumors with specific genetic alterations, such as KRAS or EGFR mutations, were also distinguished and the location of the primary tumor identified with 71% accuracy (58).

eXOSOMeS
Exosomes are small vesicles present in blood and other body fluids (62)(63)(64). With a 30-100 nm diameter, they are constitutively released through exocytosis by many cells, including tumor cells, in physiological and pathological conditions. Exosomes contain lipids, proteins, mRNA, several types of non-coding RNAs, and double-stranded DNA; and their composition partly reflects that of the parental cells (65). In addition, being generated by the cell secretion pathway, all exosomes carry some common proteins independent of their origin, such as ALIX, CD63, or TSG-101 (66). Exosomes are generally isolated by sucrose gradient ultracentrifugation or immune-bead isolation techniques (such as magnetic activated cell sorting), and there are commercial kits available. Once isolated, exosomes are characterized by transmission electron microscopy, Western blot, FACS, or other methodologies (67).
Although being more difficult to purify than other materials, the lipid bilayer of exosomes makes their cargo particularly stable, theoretically allowing the identification of the tumor of origin, genetic alterations or resistances to treatments. In this respect, EML4-ALK fusion transcripts have been recently identified in the exosomal RNA of NSCLC patients (68). In addition, some studies indicate that micro RNA (miRNA) analysis of exosomes might be useful for the diagnosis of lung adenocarcinoma (69)(70)(71) and that particular miRNAs can offer prognostic information in advanced NSCLC. For example, downregulation of miRNA-373 and miRNA-512 has been associated with a poor prognosis (72), miR-208a and miR-1246 with resistance to radiotherapy (73,74), and miR-221-3p and 222-3p with good response to osimertinib in EGFR mutated patients (75).

CiRCULATinG TUMOR CeLLS
Together with ctDNA, CTCs are the most widely investigated material in liquid biopsies of cancer patients. First observed in 1869 (76), they are cancer cells detached from the solid tumor mass that circulate in the blood and lymphatic system (77) as single cells or as aggregates, the so-called circulating tumor microemboli (78)(79)(80). In advanced NSCLC patients, CTCs are relatively rare, 1-10 per mL against a background of 10 6 -10 7 peripheral blood mononuclear cells. This low abundance poses formidable challenges for the development of robust and sensitive enrichment protocols (81).
Some CTC capture methods are label dependent, based on specific epithelial cell surface markers, such as epithelial cell adhesion molecule (EpCAM) for positive selection or CD45 for negative depletion. One of such techniques, the CellSearch ® system (Veridex), has been approved by the FDA for monitoring some type of tumors (82-84), but not lung cancer. In advanced NSCLC, CellSearch ® has shown a limited detection efficiency, with CTCs detectable in only 20-40% of patients (85)(86)(87). Label-dependent methods do not select CTCs that have undergone epithelial to mesenchymal transition (88) or those with stem cell characteristics that have not started epithelial differentiation. Label-independent techniques, which are based on physical characteristics such as size, can overcome this limitation. Isolation by Size of Epithelial Tumor cells (ISET ® , Rarecells), based on filtration and cytological characterization, has shown an increased sensitivity in NSCLC (89)(90)(91)(92) with an 80% detection rate of CTCs in blood from 40 stage IIIA-IV patients compared with 23% using CellSearch ® (85). Another technology based on size, ScreenCell ® , allows not only the detection but also the isolation of CTCs, which can be subjected to further morphological studies and used for genetic testing. Isolated CTCs can be cultured or injected into mice to generate xenografts (93)(94)(95)(96) and CTC-derived tumor cells can thus be obtained in enough numbers for molecular and pharmacological profiling. CTC counts have been extensively researched as a prognostic factor in NSCLC (97). In early-stage patients, the decrease or disappearance of CTCs after surgery has been reported to correlate with better clinical outcomes (98,99), while its persistence was associated with shorter progression-free survival (PFS) (100). Regarding advanced NSCLC, some studies have reported that a higher CTC count at presentation correlates with advanced stage and shorter PFS and overall survival (85,101). Also, the decrease or disappearance of CTCs after chemotherapy or targeted therapy has been consistently associated with better outcomes (102)(103)(104).
Finally, CTCs have also been investigated as a tool to identify clinically relevant genetic alterations in NSCLC ( Table 2). Using NGS and modified PCR techniques, EGFR-sensitizing mutations and the p.T790M resistance mutation have been detected in the CTCs of EGFR-positive patients at presentation and after progression to TKI treatments, respectively (28,41,42,105). The sensitivities reported range from 47 to 100%. However, unlike cfDNA, CTCs are not used for EGFR testing in the routine clinical practice. EML4-ALK fusions have been identified by fluorescence in situ hybridization (FISH) and immunochemistry in CTCs isolated using ISET (44) or other enrichment methodologies (47, 48). In some cases, filter-adapted FISH was employed, a methodology that has also been demonstrated to successfully identify ROS1 rearrangements in CTCs isolated by ISET (46).

AUTHOR COnTRiBUTiOnS
All the authors contributed to the writing and critical revision of the review.