According to World Health Organization (WHO) data, globally, lung cancer accounts for 18% of annual cancer deaths in both sexes at all ages [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 87% of all lung cancer patients [2]. Among these, 40% are adenocarcinoma, 25–30% are squamous carcinoma and 10–15% are large cell carcinomas [3].

In the era of precision medicine, NSCLC has become an eminent example of how therapeutic decision making is based on the identification of specific biomarkers, called oncogenic drivers, designed by the National Comprehensive Cancer Network (NCCN) guidelines, such as the epidermal growth factor (EGFR), rearrangements of anaplastic lymphoma kinase (ALK), ROS proto-oncogene1 (ROS1), serine/threonine-protein kinase B-Raf (BRAF), ERBB2, MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping, RET proto-oncogene (RET) and PDL-1 (programmed death ligand 1) ^[4][5][4,5] present in approximately 30% of NSCLC patients ^[3][6][7][3,6,7]. More recently, the G12C missense mutation of the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been added to these and new therapeutic agents (i.e., adagrasib, sotorasib) have been developed [8]. These advances in cancer genetics are leading to the birth of mutational oncology.

Unfortunately, over the years, it has been observed that the number of diagnoses in advanced stages of the disease (stages III and IV) have increased [9], and even if low-dose spiral computed tomography (LDCT) is currently 4 times more sensitive ^[10][11][10,11] than X-ray imaging, it does not detect the small lesions of lung cancer at the early stage ^[12][13][12,13]. For this reason, to date, histopathological investigation still remains the diagnostic gold standard. Despite this, even with acceptable adverse events, tissue biopsies often need to be repeated, which means additional stress and pain for patients [14].

At the same time, international scientific entities such as the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) recommend the use of liquid biopsy to accompany tissue biopsy when the tissue sampling turns out to be inadequate or infeasible [15]. A liquid biopsy is defined as a test performed on a blood sample to search for circulating substrates coming from cancer cells in a non-invasive manner. Therefore, different types of liquid biopsy can be used for molecular diagnostic investigation through the collection of different body fluids, the most used of which is the peripheral blood sample, with minimally invasive methods. The different substrates obtained from body fluids, such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes and circulating miRNAs, are used for diagnosis, prognosis and disease monitoring following clinical treatment. Given the study of the main substrates, it is possible to capture tumor heterogeneity as well as evaluate the molecular changes longitudinally due to genomic instability and treatment resistance ^{[16][17][18][19]}[16,17,18,19].

Undoubtedly, one of the most studied liquid biopsy substrates is the circulating tumor cell (CTC), reported for the first time in 1869 by the Australian doctor Thomas Ashworth. CTC detection is widely used to predict the prognosis of various lung cancers, including small cell lung cancer, squamous cell lung cancer, lung adenocarcinoma and large cell lung cancer [20]. CTCs are released from the primary tumor and/or metastases by dispersing into the bloodstream and represent the intermediate stage between the primary tumor and metastases as determined by the ability of tumor cells to invade other organs leading to the formation of a metastatic lesion [21].

Basically, there are two methods for isolating CTCs: (i) label-dependent systems/methods based on the detection of specific surface markers of CTCs and (ii) label-independent systems/methods based on physical or biological properties of CTCs [22]. The first is dependent on the label, and is characterized by the use of specific markers such as the adhesion molecule of epithelial cells (Ep-CAM), the human epidermal growth factor receptor 2 (HER2) [23], mucin 1 (MUC1), cytokeratins, etc. ^[24][25][24,25]. However, due to their ability to undergo epithelial–mesenchymal transition (EMT), CTCs have different phenotypes that cannot express these markers, disabling this isolation method. To overcome this obstacle, several label-dependent methods using negative depletion of CD45-positive leukocytes have been introduced [26].

The amount of CTCs in the blood is not high: it is estimated from 1 to 10 cells per 10 mL of blood due to the process of EMT ^[27][31] closely related to the higher stage and poorer prognosis of small cell lung cancer ^[28][32]. For this reason, there are preliminary studies showing that CTC sampling gives greater detection success if it is performed at the pulmonary vein level instead of in the peripheral blood ^[29][30][33,34]. In order to have greater clinical usefulness information, the quantity of CTCs must also be considered for the evaluation of prognosis. It is demonstrated how the count of CTCs at baseline is used as a predictor of progression-free survival, PFS, and overall survival, OS, but it is also used for monitoring treatment and the progress of the disease towards clinical relapse or remission. Thus, the count of CTCs during therapy could serve as an indication to modify the chemotherapy drug ^[31][32][33][35,36,37]. Morphology and immunohistochemistry are other characteristics that can be studied for more complete information. Morphology is necessary to distinguish the CTCs from the rest of the surrounding cells by distinguishing the size and shape of the nucleus, the structure of the chromatin, the nucleoli and more. Immunocytochemistry, on the other hand, is studied to evaluate cancer antigens, e.g., the expression of PD-L1 in the CTCs of NSCLC patients as an indication for treatment using PD-1/PD-L1 immune checkpoint inhibitors ^[34][38].

With genomic analysis, mutations in the DNA of CTCs are studied, leading to a significant clinical value for the evaluation of the treatment ^[35][39]. However, it is also possible to proceed with the study of RNA, mainly mRNA, through transcriptomic analysis. In fact, gene expression is in agreement with the number of mRNA transcripts ^[36][40]. Again, the data of a high expression of genes associated with chemoresistance should lead to a re-evaluation of the incompatible treatment. Then, through mass spectrometry or Western blotting, it is possible to carry out the proteomic analysis of the CTCs ^[37][38][41,42]. The advantage in clinical practice in the use of CTCs lies in the relatively inexpensive and non-invasive tool for the study of cancer. Cancer cell counts allow for real-time assessment of disease progression, and an increase in the number of CTCs is interpreted as a relapse and a decrease as a sign of remission, even before these can be seen with obvious clinical signs ^[32][39][40][36,43,44]. Through gene profiling it is possible to evaluate the clones of resistance to therapy ^[32][36], thus being able to adopt the right treatments, and it is possible to screen and detect the CTCs in the blood in the early stages of the tumor ^[41][45].

2.2. Circulating Tumor DNA—ctDNA

Tumor-derived biomarkers used for liquid biopsy can arise from several biofluids including plasma, serum, urine, saliva or exhaled breath condensate (EBC), and pleural and cerebrospinal fluid. Among the various biomarkers that can be obtained, the most widely used is undoubtedly the cell-free tumor DNA (ctDNA), genotyped as a tumor marker 17 years after its discovery in cancer patients in 1977 ^[42][50]. The ctDNA is released by the neoplastic cells in the body fluids in variable amounts. In particular, the amount of ctDNA dispersion increases with the stage and metastatic sites ^[34][43][44][38,51,52]. The short half-life of ctDNA of ≃1 hr makes it very suitable for measuring tumor burden in real-time in response to therapy. The challenge remains the detection of ctDNA in plasma versus the rest of the much more abundant wild-type cfDNA, released by non-tumoral cells ^[34][38]. The ctDNA detection is further influenced by the clinical condition of the patient, the timing of sampling collection and the site of metastases. Therefore, the analysis of ctDNA is influenced by several factors such as (i) sampling in ethylenediaminetetraacetic acid (EDTA) tubes or specific cfDNA storage tubes such as Streck (cell-free DNA BCT) ^[45][53]. The collection in the first type of tubes implies rapid processing of the sampling, while the second one that preserves the quality of small fragments of DNA, allows the holding of the blood sample as it lasts for several days at room temperature without the need for on-site processing; (ii) storage and transport of the blood sample [15]; (iii) timing between blood sampling and plasma extraction; and (iv) the amount of blood drawn from the patient, which is commonly established as 20 mL but is still not standardized ^[46][54]. The need for predictive biomarkers in NSCLC has therefore pushed research in implementing liquid biopsy through the study of ctDNA for the treatment of NSCLC. A meta-analysis study in 1017 patients from 10 studies suggested that the reduction in early ctDNA was associated with improved progression-free survival (PFS), overall survival (OS) and objective response rate (ORR) in ICI-treated patients with advanced NSCLC ^[47][62]. However, not all patients are able to benefit from this type of treatment due to drug resistance ^[48][63], disease progression ^[49][64] or adverse immune-related events (irAE) ^[50][65].

2.3. microRNA-miRNA

miRNAs, also called microRNAs, are a class of small, single endogenous RNAs with a length of about 20–22 molecules that do not code for any protein. They act as post-transcriptional gene regulators by binding to the complementary 3′- untranslated regions (3′-UTR) of target mRNAs and causing translational inhibition or mRNA degradation ^[51][69]. The first discovery of miRNAs dates back to 1993 by Ambros and colleagues from Caenorhabditis Elegans ^[52][70]. Since, they have been increasingly studied, and in 2002, Dr. Croce’s group provided the first evidence of miRNA’s involvement in the pathogenesis of human cancer. There is growing evidence on the role of miRNAs in the development, progression and metastasis of various cancers ^[53][71] and in a particular way in NSCLC ^[54][55][56][72,73,74]. Indeed, in 2008, the discovery of circulating miRNAs in plasma and serum meant that these began to be studied as biomarkers for the diagnosis and treatment of NSCLC ^[57][58][75,76]. In NSCLC, miRNAs act as tumor suppressors or oncogenes to regulate progression and metastasis by modulating their target genes. There are several families of miRNAs that act as tumor suppressor genes such as the Let-7 family, which effectively induces cell cycle arrest and cell death in murine lung cancer cells expressing KRAS (G12D) ^[59][77]; miR-34 is a direct proapoptotic transcriptional target of p53. Down-regulated miR-34a expression is often observed in NSCLC, contributing to tumorigenesis by attenuating p53-dependent apoptosis ^[60][78]; miR-486 is considered an ideal biomarker in cancer diagnosis ^[61][79] targeting components related to insulin growth factor (IGF) signaling and functioning as a tumor suppressor in NSCLC ^[54][72]. miR-218, which targets the EMT, Slug and ZEB2 regulators ^[62][80], and miR-200 inhibit EMT by targeting ZEB1 and ZEB2. Given the controversial function of miRNAs, there are data in the literature that support their role also as oncogenes in NSCLC including miR-196b, miR-221/222, miR-17/92, miR-21 and miR-224 ^[63][81]. MiRNAs have been shown to be involved in genetic alterations, epigenetic changes and transcriptional control with a key role in NSCLC. Some miRNAs are inherently involved in NSCLC metastasis processing, so a deep understanding of the miRNA signaling network will help identify therapeutic targets. In addition, miRNAs contribute to the drug resistance of NSCLC. Zhao et al. ^[64][82] demonstrated how changes in the nature and amount of miRNA in exosomes are associated with the resistance of NSCLC cells to chemotherapy drugs. Other studies argue that miRNAs are differentially expressed in the exosomes of cisplatin CDDP-resistant NSCLC cells ^[65][83].

2.4. Exosomes in NSCLC

The exosomes are nano-sized vesicles with a diameter of about 30–200 nm. They are released during the fusion processes of plasma membranes through active processes of exocytosis ^[66][67][86,87]. A cancer cell can actively release more than 20,000 exosomal vesicles in 48 h ^[68][88], which contain RNA, DNA and proteins that represent the information biological load of cancer cells ^[69][70][71][89,90,91] contrary to the release of ctDNA and miRNA derived from “dying” cells in necrosis or apoptosis ^[72][92]. These differences in secretion point out that the information on exosomes comes from living cancer cells, suggesting a possibility of diagnosing lesions earlier. There is evidence showing that exosomes are closely related to lung carcinogenesis by playing a key role in the growth and progression of lung malignancies through tumor angiogenesis and EMT ^[73][93].

2.5. Liquid Biopsy in Pleural Effusion

For the purpose of promising precision medicine, confining the study of cfDNA or CTCs and extracellular vesicles to the bloodstream can be limiting. Indeed, malignant pleural effusion (MPE) is rich in tumor cells for patients with advanced lung cancer. The extracellular vesicles and the cfDNA are the two main targets currently explored using MPE. The MPE is an accumulation of extra fluid in the pleural space between the lungs and chest wall and its use may have great clinical significance ^[74][98]. The MPE is common in patients with metastatic lung cancer with a frequency ranging from 8% to 15% and with a higher incidence for lung adenocarcinoma due to its peripheral location in the lung, being able to invade the pleural space more easily ^[75][99]. As a blood sampling, the MPE is also easy to collect and it is more informative for mutation rates than tissue biopsy samples ^[76][100].