Chest CT is a fundamental imaging tool for the evaluation of lung cancer and is most commonly used as a noninvasive modality for the screening and staging of lung cancer. It is especially useful to define the size, location, and characterization of lung lesions, which are surrounded by air-filled lung tissue. Additionally, mediastinal and hilar lymphadenopathy, pleural effusion, metastasis in the liver, adrenal glands, bone, and other parts of the thoracic cavity can be assessed by chest CT. Since September 2019, low-dose chest CT (LDCT) has been adopted? for the screening of lung cancer in high-risk individuals in Korea. Screening with LDCT rather than conventional chest radiography is efficient to detect small lung nodules, leads to early intervention, and improves prognosis of lung cancer patients. The U.S. National Lung Cancer Screening Trial demonstrated that LDCT screening leads to a 20% reduction in lung cancer-associated mortality in high-risk populations compared to that seen with chest X-ray. Patients with positive results after LDCT need to be further evaluated using other diagnostic procedures [9].

Positron emission tomography (PET), using uptake of the radiolabeled glucose analogue [18F]-fluoro-2-doxyglucose by metabolically active cells, is a useful test for staging and thus deciding a treatment modality. It is also useful for the detection of extrathoracic disease. However, a false negative result may occur in cases of small lesions less than 1 cm in size and in tumors with low metabolic activity such as in case of carcinoid tumors or bronchioloalveolar cell carcinoma. In contrast, false positives can also occur when inflammatory conditions, such as pneumonia or granulomatous disease, are present. According to a report by Lee et al. [10], the false positive rates for PET-CT in the identification of mediastinal lymph node (L/N) metastasis in Korea can be as high as 87% when using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) as a gold standard and significantly higher in patients older than 65 years with squamous cell carcinoma or pneumoconiosis. However, the same study reported that current or past history of tuberculosis did not significantly affect the false positive rate, unlike previous reports indicating that the usefulness of PET-CT decreased in areas with a high prevalence of tuberculosis [10].

Lung cancer, along with breast and malignant melanoma, are common causes of brain metastases, with lung cancer being the most common. Brain metastases can be detected as an early symptom in approximately 10% of diagnosed patients and associated with significant morbidity and limited survival [11]. The presence of symptomatic brain metastases is an important consideration in the selection of therapeutic agents and treatment modality. As the survival of lung cancer patients increases, improvement of drug permeability of the bloodbrain barrier has become an important consideration for pharmaceutical companies when developing anti-cancer drugs. HIRA guidelines recommend assessment of brain metastases for initial staging and preoperative evaluation of lung cancer [5]. However, with the recent increase in the early diagnosis of lung cancer, the need for brain magnetic resonance imaging (MRI) for staging tumors less than 2 cm in size has been questioned [12].

Approximately 30% to 40% of non-small cell lung cancer (NSCLC) patients develop bone metastases that lead to poorer prognosis and worsen quality of life during the remaining life. Whole body bone scintigraphy (WBBS) is useful for the detection of bone metastases, when used in conjunction with other relevant clinical findings such as bone pain, elevated alkaline phosphatase, and hypercalcemia. It also has a high false positive rate due to trauma, inflammation and degenerative change of skeletal system and thus, the routine use of WBBS is not recommended by the National Comprehensive Cancer Network guidelines [13]. Moreover, implementation of WBBS for lung cancer is on the decline in Korea as the HIRA indicator does not include bone scans as imaging tools conducted for lung cancer bone metastasis [5].

The bronchial pathway to the target lesion and location where the sampling to be performed using forceps and brushes should be determined by careful review of the patient’s anatomy before fiberoptic bronchoscopy (FBS) procedure. The diameter of commercial FBS is around 6 mm and lesions locating up to subsegmental bronchus can be detected and biopsied using FBS. The diagnostic yield for FBS is low, ranging from 20% to 60% [14]. The size and location, and visibility of lesion are important factors that influence the diagnostic yield. Lung lesions less than 2 cm in diameter located in peripheral lung areas typically lead to low yields. In the case of endobronchial lesions that are located in central regions, three or more biopsies are recommended to achieve high diagnostic yields. Transbronchial biopsy, bronchial washing, and bronchial brushing can also improve the diagnostic yield for the lesions that are not directly visible by FBS. For peripheral lung lesions that cannot be reached by routine FBS, fluoroscopy guided transbronchial lung biopsy can increase the accuracy of the diagnosis. Recently, other fusion techniques, including EBUS and navigation bronchoscopy, have been developed and used to improve diagnostic yield.

Using a radial EBUS probe, it is possible to detect peripheral lung lesions surrounding the tip of the probe. The image of radial-type EBUS can be obtained using a guide sheath (GS). Biopsy forceps and bronchial brushes can be placed using GS. This method is superior to FBS or EBUS accessing small peripheral lung lesions [14]. The accessible airways closest to the lesion are marked as targets. The radial probe is then inserted into the target lesion through the bronchoscope. After retraction of the radial probe, forceps or brushes can be placed to further biopsy target lesions. Until recently, the diagnostic yield and safety of EBUS-GS have not been fully evaluated. Most studies investigating this issue were based on single center case reports or were meta-analyses. However, a larger number of studies have now shown the good diagnostic yield of EBUSGS and confirmed that it is a well-tolerated procedure [14,19,20].

Navigation bronchoscopy is generally adopted for small peripheral lung lesions to increase diagnostic accuracy. This technique includes three stages. In the first stage, or the planning stage, a CT scan is reconstructed and a virtual road map to the target is created. In the second stage, or registration stage, an electromagnetic field is created around the body using sensors to overlap the virtual and the actual body. The last stage is the navigation stage where the above information is used for navigation to guide the probe to the target site [21]. The diagnostic yield of this method has been found to range from 33% to 97% [22]. Incidence of pneumothorax is the most frequent complication for this method and occurs in approximately 3% of all cases [23]. The technician’s expertise; the volume, location, and characteristics of the lung lesions; concurrent EBUS; and the use of catheter aspiration are important factors that can influence the diagnostic yield of this method.

CT-needle aspiration biopsy (NAB) is one of the most commonly used tools to secure lung tissue, and its diagnostic yield is dependent on the size of the target lung lesion. Typically, lung nodules over 1 to 2 cm in size are good candidates for CT-NAB. Previous studies have shown that the diagnostic yield for CT-NAB ranged from 77% to 94%. It is an invasive procedure and can provide an alternative to surgery for the confirmation of lung cancer diagnosis. However, this technique is associated with a risk of complications. A recent study revealed that the associated rates of pneumothorax and bleeding were 20% and 3%, respectively [24]. Another study showed that pneumothorax requiring intervention occurred in 4.3% of patients, and that major complications developed in 4.4% of those patients [25]. Larger needle diameters, increased transverse lung parenchyma, and smaller lesion sizes are risk factors for the development of major complications in this procedure [25]. Contraindications to the use of CT-NAB are bleeding diathesis, low platelet count, prolonged prothrombin time or partial thromboplastin time and international normalized ratio, pulmonary hypertension, emphysematous disease and chronic obstructive pulmonary disease, large bullae in the biopsy path, and patients with challenges such as intractable cough or those using mechanical ventilation. The ability of patients to hold their breath and maintain their position is important for this procedure. A careful selection of patients is needed in order to attain better yields and reduce complication rates. Recently, improvements have been made to this technique including the addition of laser guidance to improve its accuracy and reduce the risk of complications [26].

Various types of needles are available for CT-NAB and the choice of needle is dependent on the size, location, and characteristics of the target lesions, and the technician’s expertise and preference. There are mainly three types of needles used: aspiration needles, cutting needles, and automated core biopsy needles. Aspiration needles are thin-walled and flexible, and cellular material for microscopic or cytological diagnosis can be obtained using these needles [27]. Cutting needles, which are modified aspiration needles with a side cutting tip, are used to obtain histologic specimens. Lastly, core biopsies can be performed using a biopsy gun without causing crushing injuries. Specimens obtained from core biopsies increase the rate of definite benign disease and allow for the characterization of different cell types. The diagnostic yield of core biopsy is superior to that of CT-NAB particularly in benign disease and specimen obtained from core biopsy is more suitable for immunochemical and genetic variation test compared to that from fine needle aspiration. On the other hand, it has an increased associated risk of complications. The reported overall complication rate and major complication rate are 38% and 6%, respectively, for this technique. In a previous study, pneumothorax and pulmonary hemorrhage developed in 25% and 18% of patients, respectively [25]. The choice of biopsy needle for transthoracic needle biopsies should be considered on a case-by-case basis for the improvement of the diagnostic yield, purpose of biopsy, and the reduction in the rate of complications.

Polymerase chain reaction (PCR) is a revolutionary method invented in 1983 that allows for large amounts of DNA to be amplified in vitro using two primers. PCRbased assays have been continually amended to make it easier to find driver mutations such as EGFR in the medical practice. Dideoxynucleotide sequencing, developed by Sanger et al. [29], of PCR-amplified DNA is the standard method for the detection of genomic mutations; however, it shows suboptimal sensitivity, is labor- intensive, and has long turnover times. Other modified methods, such as PCR-single-strand conformation polymorphism, TaqMan PCR, Cycleave PCR, PCR-restriction length polymorphism, peptide nucleic acid-locked nucleic acid PCR clamp, and mutant-enriched PCR, have been developed and showed improved sensitivity when compared to conventional PCR methods. Kim et al. [30] compared the PNA-mediated PCR clamping method and the direct-sequencing method using the tissues of 112 lung cancer patients. They demonstrated that mutants were identified in 45 samples using the PNA-mediated clamping method, 10-fold more than that when using direct-sequencing, and indicated that this method can be useful for detecting driver mutations [31]. Now, PNA-mediated clamping method is one of the most commonly used methods to detect driver mutations in cancer tissue specimen in Korea.

Next generation sequencing (NGS) is a methodology that quickly decodes a large amount of genome information by breaking down a genome into numerous fragments, reading each fragment simultaneously, and finally combining the data obtained using bioinformatics techniques [32]. Hybrid capture sequencing is used when whole genome, whole exome, or large targeted panels are assessed, whereas amplicon sequencing is used when in-depth reading is required and assay sensitivity is being evaluated. Targeted NGS panels have been validated in several previous studies. Targeted NGS panels, including those for EGFR, KRAS, BRAF, and MET, are widely used in lung cancer [33]. Wider analyses can take up to a week to complete. From these large NGS datasets, in addition to EGFR, KRAS, and BRAF mutations, 36% of lung cancer patients can be found to harbor other potential driver mutations (FGFR, ERBB2, AKT, and MAP2K1, STK11) [34-36]. Many NGS panels that include more diverse genes, tumor mutational burden, microsatellite instability status detection, and fusions are currently under development and will provide more detailed information on lung cancer genetics. Since 2017, the NGS technology base genetic panel test has been designated as an object for national health insurance benefits in Korean lung cancer patients. When applying NGS in lung cancer in Korea and claiming insurance, the essential requirements of the genetic panel should include following 14 genes; HER2, EGFR, ALK, KRAS, NRAS, BRAF, BRCA1, BRCA2, KIT, PDGFRA, IDH1, IDH2, MYC, MYCN [37]. However, NGS can yield false negatives or positives, and therefore, additional tests such as fluorescence in situ hybridization or immunohistochemistry (IHC) for protein overexpression may improve the patients’ diagnosis.

There are two types of commonly used real-time PCR methods: real-time PCR using a TaqMan probe and real-time PCR using SYBR Green. Although tissue biopsies are the gold standard for detecting driver mutations, they are invasive and sometimes challenging to obtain due to the patient’s condition and tumor location or size. Contrary to tissue biopsy, a liquid biopsy from plasma, pleural effusion, or bronchoalveolar lavage fluid is typically less invasive [38]. Previous studies have shown promising data using liquid biopsies and real-time PCR [39]. Shin et al. [39] reported a 100% sensitivity and concordance rate of 98.7% with real-time PCR for EGFR using pleural effusions when compared to Sanger sequencing and PNA-mediated PCR clamping.

In transcriptome analysis, analysis is performed using microarray, an RNA sequencing method that separates mRNA, converts it into cDNA, and analyses its sequence using NGS. Whole transcriptome profiles can easily be obtained from Gene Expression Omnibus (GEO) databases. Lim et al. [40] integrated robust datasets into the bioinformatics pipeline using statistical methods and presented normalized datasets in lung cancer. Bang et al. [41] conducted transcriptome analyses for 10 NSCLC patients and reported that genes related to the cell cycle were highly upregulated in lung cancer. They validated these results using public data available in GEO and The Cancer Genome Atlas (TCGA). MFAP4 and AGER genes were significantly downregulated and the SPP1 gene was upregulated in NSCLC, and these genes were significantly associated with poorer prognoses.

IHC is used in the differential diagnosis of adenocarcinoma and squamous carcinoma (SqCC); neuroendocrine marker identification; driver mutation assessment, including that for EGFR/ALK/ROS1 and PD-L1/PD-1 expression; and the differential diagnosis of lung cancer and mesothelioma [7]. Thyroid transcription factor-1 and napsin A are used as adenocarcinoma markers, and p40, CK5/6, and TP63 can be used as SqCC markers [44]. According to World Health Organization classification, assessment of both neuroendocrine morphology and immunohistochemical expression (chromogranin A, synaptophysin, or CD56) is required for the diagnosis of large cell neuroendocrine carcinoma. For the detection of driver mutations, the U.S. Food and Drug Adminstration (FDA)-approved ALK D5F antibody is used for ALK-rearranged NSCLC as a companion diagnostic tool [45]. However, IHC analysis is not used in EGFR mutation screening due to its low sensitivity. IHC methods for PD-L1/PD-1 varies between providers. The U.S. FDA approved the 22C3 antibody for pembrolizumab, the 28-8 antibody for nivolumab, and SP142 for atezolizumab [46]. Many studies have reported that PD-L1/PD-1 levels correlate to treatment response and prognosis; however, further research will be needed to fully understand these pathways in cancer [47]. Calretinin, BAP1, podoplanin, CK5, CK5/6, and WT1 can be potentially used for the diagnosis of mesothelioma [48].