EBUS-TBNA samples undergoing immunohistochemical analysis in addition to conventional histomorphological evaluations have led to higher rates of identifying the histological subtypes of non-small cell lung cancer (NSCLC) specimens [38]. However, the quantity of material that can be obtained by needle biopsy is small, and therefore attempts have been made to improve the processing methods used for to EBUS-TBNA samples to obtain a pathological diagnosis [39]. A needle biopsy specimen is fundamentally cytological material, and “proper preconditioning” is important for maximizing the information derived from a very small sample. However, although the effectiveness of rapid on-site evaluation (ROSE) for improving the diagnostic yield of EBUS-TBNA is still controversial, ROSE may aid in deciding how to process the sample for additional evaluations [40]. In addition, ROSE was found to increase the chances of performing successful lung cancer genotyping from small numbers of needle biopsy samples that were obtained by EBUS-TBNA [41]. The recent guidelines on the techniques of EBUS-TBNA recommended obtaining samples for histopathological diagnosis and additional samples for molecular testing [18]. The cell block method [42] and the “tissue coagulation clot” method [43] have allowed histological evaluations and have been reported to improve the diagnostic yield of EBUS-TBNA. These “core” building techniques may facilitate biomarker testing in lung cancer [44]. The World Association for Bronchology and Interventional Pulmonology recently published guidelines on the acquisition and preparation of needle aspiration samples. The guidelines also encourage bronchoscopists to have discussions with their pathologist colleagues about the most suitable methods for processing specimens [45] (Fig. 5.6).

Specimens obtained by EBUS-TBNA can be processed for multidirectional analysis [39]. Gefitinib, the first epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), was introduced for the treatment of lung cancer in 2002 [46]. It marked the beginning of molecularly targeted therapy for lung cancer. The association between EGFR gene mutations and sensitivity to gefitinib was reported in 2004 [47, 48]. Since then, clinical molecular testing for the detection of molecular targets has increasingly been required. Because the majority of the lung cancer patients have advanced disease at the time of diagnosis, the development of molecular testing techniques on a very small biopsy specimen was warranted. The initial attempt to detect EGFR mutations in specimens provided by EBUS-TBNA was reported in 2007 [49]. Following improvements in molecular analysis, the detection sensitivity improved, and other investigators reported similar attempts at detecting EGFR mutations in EBUS-TBNA specimens [50, 51]. Multiplex mutation testing [52, 53] and mutation testing of the solution used to rinse the TBNA needle are used currently to increase the sensitivity of mutation detection [53]. In addition to EGFR gene mutations, the anaplastic lymphoma kinase (ALK) fusion gene was found to be a very strong oncogenic driver in 2007 [54]. The detection of the aberrant fusion gene will initially require immunohistochemical techniques and reverse transcription polymerase chain reaction (RT-PCR) or FISH. Sakairi et al. was able to detect the EML4-ALK fusion gene in an EBUS-TBNA sample using RT-PCR, with the result confirmed by FISH [55]. Biomarker testing of biopsy material is important for identifying which molecularly targeted therapeutic agents are useful for the patient with NSCLC. Testing should be performed prior to the start of treatment [56]. Patients with lung cancer who received the appropriate targeted therapy were reported to obtain improved survival compared with patients not receiving targeted therapy or with no oncogenic driver targets [57]. The use of cell block preparations of EBUS-TBNA specimens for molecular testing has obtained excellent results for lung adenocarcinoma; EBUS-TBNA specimens from 93 % of patients were sufficient for at least one round of molecular testing for EGFR mutations, the ALK fusion gene, and KRAS mutations [58].

The repeatable nature of EBUS-TBNA should be a powerful tool for identifying the appropriate molecularly targeted therapeutic agents. Gefitinib initially showed dramatic effects in patients with lung cancers harboring sensitive EGFR mutations; however, patients develop resistance to EGFR-TKIs. The well-known secondary changes which result in resistance to EGFR-TKIs include the substitution of methionine for threonine at position 790 (T790 M) in exon 20, which was reported in 2005 [59] and focal amplification of the MET proto-oncogene, which was reported in 2007 [60]. The secondary changes leading to resistance to ALK-TKIs include a mutation in the ATP-binding domain (L1196 M) and a mutation in the non-ATP-binding domain (C1156Y), which were reported in 2010 [61]. In addition, many other mechanisms have been reported to be involved in the development of ALK-TKI resistance [62]. Second- and third-generation TKIs have been developed to overcome various types of resistance [62]. Actually, the new-generation drugs have shown dramatic benefits for the patients with acquired resistance to initial TKI treatment [66, 64]. A previous analysis of tumor specimens taken at the time of acquired resistance to EGFR-TKI found secondary mutations, aberrant gene amplification, and transformation to small cell lung cancer [65]. The European Society for Medical Oncology (ESMO) guidelines have stated that the emergence of molecular resistance suggests that a repeat biopsy should be performed at the time of tumor progression [66]. Rebiopsy by EBUS-TBNA has been thought to be feasible [67] (Fig. 5.7); however, rebiopsy itself has so far not been a standard clinical practice because of patient factors (tolerability), physician preference, and limited resources [68].

Multidirectional analysis of samples obtained by EBUS-TBNA has allowed assessment of oncogenic drivers in clinics, as well as the identification of genetic signatures for lung cancer research. We can extract DNA, RNA, and protein from EBUS-TBNA samples, and these materials can be used for transcriptome and proteome analysis. Analysis of specimens with aberrant DNA methylation can be used for assessment of chemosensitivity [69] and also used for making higher sensitivity for the detection of nodal metastasis by EBUS-TBNA [70]. Expression of the unique vascular endothelial growth factor-c (VEGF-C) mRNA was reported from EBUS-TBNA samples of metastatic lymph nodes. The higher expression of VEGF-C was observed in metastatic lymph nodes with high vascularity features using Doppler mode image [71]. High-quality EBUS-TBNA samples can be used for comprehensive mRNA and microRNA expression analysis using microarray technology [72]. Primary xenograft technology using EBUS-TBNA samples has been evaluated for overcoming the limitations in sample size [73, 74].

We often encounter difficulties obtaining tissue samples, since many patients have advanced disease at the time of first presentation and are not eligible for surgery. EBUS-TBNA can solve that problem, because it can obtain tumor samples from patients with advanced disease who are not candidates for surgery. Because it can easily and safely obtain samples that are evaluable by today’s technology, EBUS-TBNA may greatly expand the knowledge base that supports lung cancer research.