Bronchology – A Well Branched Tree

Lung cancer is responsible for 1.3 million deaths worldwide annually, and it is the most common cause of cancer-related death in men and the second most common in women. Lung cancer staging is the assessment of the degree to which a lung cancer has spread from its original source. As with most cancers, for lung cancer staging is of paramount impor‐ tance for the treatment planning process and prognosis. Two primary methods of lung can‐ cer staging are available: clinical staging and pathologic staging. In clinical staging, information is provided by noninvasive or minimally invasive techniques, such as physical examination, radiologic examination, endoscopic ultrasound, bronchoscopy, mediastinosco‐ py, and thoracoscopy. In pathologic staging, information obtained from clinical staging is combined with findings from both the invasive surgical procedure and the pathologic evalu‐ ation of excised tissue. Clinical staging is important and can help to determine the next ap‐ propriate step in therapy, such as the decision to proceed with pathologic staging, which remains the reference standard because the overall level of agreement between the two sys‐ tems only ranges from 35% to 55% [1].

ku (Chiba), Robert Rintoul (Cambridge) and Armin Ernst (Boston) that published in the Journal of Bronchology a description of how to do an EBUS-TBNA thus offering a detailed description of local lymph node position and orientation within the mediastinum [12]. EBUS EBUS-TBNA was proven in 2007 by Wong and Yasufuku et al. on 65 patients to be a safe method allowing a high yield also for the diagnosis of sarcoidosis [13].
It soon became widely accepted that EBUS-TBNA is a reliable diagnostic tool for enlarged lymph nodes in patients with NSCLC and that lymph nodes below the one centimeter range could also be sampled. This led to a study with 100 patients published in 2006 in European Respiratory Journal [14] that showed that every sixth patient with no evidence of mediastinal disease on CT was diagnosed positive using EBUS-TBNA. Thus, EBUS-TBNA showed potential to avoid unnecessary exploratory thoracoscopies. Following the same idea, in 2008 Hwangbo et al. showed that in cases with both CT-and PET-negative and -positive scans, EBUS-TBNA is an excellent tool for detecting mediastinal metastasis, thereby confirming that EBUS-TBNA is an effective invasive method following CT and PET scanning [15].
In the same year, Armin Ernst et al. showed in Journal of Thoracic Oncology on 66 patients that EBUS-TBNA can have a superior yield compared to cervical mediastinoscopy, which intuitively suggested that mediastinoscopy is not necessarily of additional diagnostic benefit in evaluating negative EBUS-TBNA staged lymph nodes [16]. However, mediastinoscopy has an important role especially in operable patients with non-enlarged lymph nodes for assessing local mediastinal invasion and the exclusion of metastatic disease.
Herth et al. evaluated EBUS-TBNA for re-staging in 124 patients with tissue-proven IIIA-N2 disease after induction chemotherapy (Journal of Clinical Oncology 2008) and concluded that EBUS-TBNA is a valuable and practical tool for re-staging with a sensitivity of 76%, specificity of 100%, PPV of 100%, NPV of 20% and diagnostic accuracy of 77% [17]. These results imply that a negative EBUS-TBNA for re-staging should be surgically re-staged.
In 2006, the compatibility between EBUS-TBNA and the Aloka Prosound alpha5 ultrasound processor made additional Doppler modes available, and this led to a consecutive study by Herth et al. on 89 patients that described changes in flow resistance parameters (resistance index by Pourcelot) in malignant lymph nodes [18].
Yasufuku et al. from Chiba University have shown great dedication to evaluating the benefits of EBUS-TBNA samples for immunohistochemical analysis and reported encouraging results with cell cycle-related proteins in chemotherapy patients in Thorax 2008, and a year earlier in Chest the same group showed that epidermal growth factor receptor (EGFR) mutation can be easily detected in metastatic lymph node samples for EBUS-TBNA and so the samples gained by EBUS-TBNA allowed genetic evaluations of tumour cells from lymph nodes [19,20].
In 2009 Tournoy et al. provided a detailed analysis of endosonographic landmarks (where available), describing the anatomic borders of the lymph node stations as defined in the 7th edition of the IASLC's TNM-staging nomenclature, which is relevant for correctly staging patients with lung cancer [21].
Building on the strong results of combined EUS-FNA and EBUS-TBNA procedures -a study published by Vilman et al. in 2005 had already indicated their complementary nature -Annema et al. challenged in 2010 the pre-dominant surgical staging algorithm by comparing the combined EBUS-TBNA and EUS-TNA with surgical staging alone [22] and showed that combining endosonographic and surgical staging resulted in greater sensitivity for mediastinal nodal metastases and fewer unnecessary thoracotomies. These results indicated that the combination of both procedures may be able to replace surgical stages as the primary staging method for patients with lung cancer.
Instead of using different scopes for EBUS-TBNA and EUS-TNA, two separate studies published in Chest in 2010 by Hwangbo et al. and Herth et al. [23] used only one bronchoscope for both procedures, starting via the trachea and continuing via the oesophageal route. They came to the conclusion that EBUS-TBNA and EUS-TNA are complementary methods and showed that both procedures can be performed with a single EBUS echoendoscope in one sitting by one operator. A further study in 2011, "Nonsurgical staging of the mediastinum: EBUS and EUS" conducted by Herth, stated that the combination of both procedures achieves a complete and accurate mediastinal staging. Therefore it can be expected that the implementation of combined EBUS-TBNA and EUS-FNA will reduce the need for surgical staging of lung cancer significantly [24].
The summary of scientific studies on EBUS-TBNA provided about clearly shows the procedure's power in helping to improve mediastinal staging of lung cancer during the past 10 years. The technological development of less invasive staging and sampling devices continues endoscopists using endosonography, we can expect further exciting developments in clinical practice in the years to come.
Endobronchial ultrasound with real-time-guided transbronchial fine-needle aspiration (EBUS-TBNA) is a minimally invasive outpatient procedure by which mediastinal [25] and hilar lymph nodes [26] as well as centrally located primary lung lesions [27] can be visualised and sampled under ultrasound guidance. In a systematic review, EBUS-TBNA has shown a pooled sensitivity of 93% in the staging of non-small-cell lung cancer. In a direct comparison with surgical staging, EBUS-TBNA even showed to be superior [28]. Therefore, EBUS-TBNA has been adopted in the most recent lung cancer staging guidelines as a minimally invasive alternative to surgical staging [29] EBUS-TBNA is an accurate, minimally invasive and safe staging procedure and can be considered the procedure of choice for patients with extrathoracic malignancies in whom hilar or mediastinal lesions are observed. In patients with (concurrent or previously treated) extrathoracic malignancy, EBUS-TBNA has a sensitivity of 85% to demonstrate metastatic spread. Implementation of EBUS-TBNA in these patients obviates invasive surgical diagnostic procedures in 61%. [30,31] The development and introduction of the new convex probe endobronchial ultrasound (CP-EBUS) that performs endobronchial ultrasound-guided transbronchial needle aspiration has changed the practice of bronchoscopic biopsy of the mediastinum in respiratory diseases. In particular, the role of EBUS-TBNA in the diagnosis and mediastinal lymph node staging of lung cancer, the leading cause of death from malignant disease worldwide [32], is becoming an interest to pulmonologists as well as thoracic surgeons. The newest CP-EBUS now being used in clinical practice is a hybrid bronchofibervideoscope which features a unique optical system that exploits both video and fiber-optic technologies (BFUC160F-OL8, Olympus, Tokyo, Japan). This CP-EBUS is a linear curved array transducer that scans parallel to the insertion direction of the bronchoscope. Images can be obtained by directly contacting the probe or by attaching a balloon on the tip and inflating with saline ( Figure 1). The outer diameter of the insertion tube of the CP-EBUS is 6.2 mm, and that of the tip is 6.9 mm. The angle of view is 80º and the direction of view is 35º forward oblique [33]. The outer diameter of the insertion tube of the flexible bronchoscope is 6.2 mm. CP-EBUS has a linear curved array ultrasonic transducer of 7.5 MHz. The balloon attached to the tip of the bronchoscope is inflated with normal saline (A). The dedicated TBNA needle is inserted through the working channel (B) [33].
The built-in CCD in the control section allows sharp images similar to those of regular videoscopes and allows a slimmer insertion tube of 6.2 mm. The ultrasound images can be frozen and the size of lesions can be measured in two dimensions by the placement of cursors. It is also equipped with the Color Power Doppler mode. The display range covers 2-24 cm ( Figure 2).
The dedicated 22-gauge needle is used for EBUS-TBNA ( Figure 3). The needle is a single use aspiration needle with echogenic dimpled tip design to improve visibility on ultrasound images. This needle has various adjuster knobs which work as a safety device to prevent damage to the channel. The maximum extruding stroke is 40 mm and to prevent excessive protrusion, a safety mechanism stops the needle at the stroke of 20 mm. The needle is attached onto the working channel of the bronchoscope which allows the operator to actually perform EBUS-TBNA. The needle is also equipped with an internal sheath which is withdrawn after passing the bronchial wall, avoiding contamination during TBNA. This internal sheath is also used to clear out the tip of the needle after passing the bronchial wall. The use of this sheath has significantly increased the yield of EBUS-TBNA. The exit of the needle is at 20°w ith respect to the outer covering of the insertion tube. The needle can be visualized through the optics and on the ultrasound image [33]. Mediastinal staging can be divided into noninvasive staging (imaging) and invasive (sampling) staging. Computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and PET-CT are used for noninvasive imaging [34]. Other imaging modalities include the use of transesophageal ultrasonography (EUS) and endobronchial ultrasound (EBUS) using a radial probe for detecting even small mediastinal lymph nodes [35][36]. Mediastinoscopy is still the gold standard for mediastinal lymph node staging [37]. However, it requires general anesthesia and complications cannot be ignored.

Changes to T descriptors [38-39]
Rami-Porta R et al. evaluated in 2007 for the 7th Edition of the TNM Classification of Malignant Tumors only patients without metastasis and, although there was information about different aspects of the T component, they only could analyze in detail tumor size, existence of accompanying nodules and pleural dissemination.
The prognostic value of the tumor size was studied in patients with completely resected pathological T1 and T2 N0 M0 tumors who had not received adjuvant therapy . The statisti-cal calculations determined three cut-points at 2, 5 and 7cm, which, in addition to the 3cm,  the border between T1 and T2, gave rise to 5 groups of tumors with significantly worse survival with larger tumor diameters. The groups and their 5-year survival rates were: T1≤2cm,  77%; T1>2cm and ≤3cm, 71%; T2>3cm and ≤5cm, 58%; T2>5cm and ≤7cm, 49%, and T2>7cm,  35%. This prognostic gradation was maintained when less selective patient populations were evaluated: clinical staging, incomplete resection and different lymph node affectation. With such arguments, it was decided to subdivide the T1 tumors into T1a (≤2cm) and T1b (>2cm and ≤3cm), and T2 tumors into T2a (>3cm and ≤5cm) and T2b (>5cm and ≤7cm). Likewise, the 5-year survival was compared between patients with T2>7cm tumors and T3 tumors. Similar results were found in the different populations, except in the N0 cases with complete resection, in which it was verified that the survival was even higher in the T3 (41%) than in the T2>7cm (35%), therefore it was decided to reclassify the latter as T3. When they analyzed the tumors that, with pathological staging, presented additional nodules, it was observed that: (a) the 5-year survival of the T3 (31%) was similar to the T4 classified as such due to the existence of an additional nodule or nodules in the same lobe as the primary tumor (28%); (b) the T4 due to other factors had the same survival as those classified as M1 due to an additional nodule(s) in a different homolateral lobe than the primary tumor (22%); and (c) the T4 due to pleural dissemination had a clearly worse prognosis (11% 5-year survival). For the new classification, it was therefore recommended to consider as T3 those tumors with additional nodule(s) in the same lobe as the primary tumor, to consider as T4 those tumors with additional nodule(s) in a homolateral lobe other than that of the primary tumor, and to include in the M category those tumors with pleural dissemination (Table 1). -T2 > 7 cm became T3.

Multicentric tumors of similar histology:
-Separate nodule(s) in the primary lobe: became T3 from T4.
In the AJCC Cancer Staging Manual and UICC TNM Classification of Malignant Tumors the T factor is divided into four descriptors (T1-4) depending on size, site, number, and local extent of the primary tumor: the size and non-size based descriptors [40].
The tumor size threshold of 3 cm was set-up on the 2 nd edition of the TNM classification of malignant tumors in 1974. [46], and despite the advances in surgical procedures, adjuvant treatment, and mostly in imaging technology, this measure remained unchanged for 35 years. The 7 th edition of the TNM for lung cancer confers more importance to the size-based T descriptors and divides them as seen earlier. These modifications have been validated by recent studies that showed better survival stratification and prognosis estimation with the new T definitions [47][48][49]. The existence of multiple primary cancers (MPC) was initially reported by Warren and Gates in 1932 [54], but in spite of these past 80 years, to date accurate diagnosis of MPC is not yet clearly established due to a lack of consensus on definition and diagnostic criteria [55] In 1975, Martini and Melamed were the first to propose clinical and histopathologic criteria for the differential diagnosis of second lung cancers [56]. MPLCs are defined synchronous, if detected simultaneously, or metachronous, if tumors are separated in time [57][58]. Synchronous nodules may represent a MPLC (second primary), a metastasis, or an extension from the primary (satellite nodule) [59] (  The concept of satellite nodules was not considered in NSCLC staging system until 1992 by the AJCC and in 1993 by the UICC [63][64].
Prior to this, all nodules were classified as M1.
The T4 descriptor includes diverse tumors with different evolution and prognosis: • invasion of the mediastinum, heart, great vessels, trachea, esophagus, vertebral body, and carina; • tumor with a malignant pleural or pericardial effusion, or with satellite tumor nodule(s) within the ipsilateral primary-tumor lobe of the lung.
The IASLC lung cancer staging project committee has acknowledged the multiple reports showing better survival for primary tumors with satellite nodules than other T4 tumors and this is the reason why they downstaged them accordingly [38,65].
Later studies found that the new T descriptor for satellite nodules proposed by the IASLC reflects better the outcomes of that group of patients, which showed superior survival rates, and now these patients are to be considered for surgery [66][67].

Pleural dissemination and pericardial effusion
According to the TNM staging manual, pleural dissemination is defined as the presence of ipsilateral malignant pleural effusion (MPE) or pleural nodules [68]. Pleural nodules are defined as pleural tumor foci separated from direct pleural invasion by the primary tumor, classified as T4 [69]. These pleural tumors must be differentiated from direct tumor invasion to the visceral (T2) or parietal pleura (T3) ( Introduction of a new accurate definition of visceral pleural invasion (VPI); VPI is a pT2 descriptor ( Table 4). The abbreviation PL is used instead of P which is also used for designation of pTNM in distinction from cTNM. The IASLC also recommends the use of elastic stains to distinguish between PL0 and PL1 when hematoxylin and eosin (H&E) sections are not helpful [71].
In TNM staging, either pleural fluid cytology or clinical judgment are valid to establish the diagnosis of a MPE and consider it as a T4 factor [72], in the absence of relevant conventional or guided pleural biopsy. Malignant pericardial effusions are classified according to the same rules. Pleural dissemination and pericardial effusion are T4 descriptors, grouped into stage IIIB in absence of distant metastasis. The MPE is considered to be a sign of advanced disease, and almost every cancer can involve the pleura [73][74]. However, most MPE's are due to NSCLC's and encompass the worst prognosis [75][76][77] Ipsilateral MPE is a locally advanced disease that precludes surgical treatment in lung cancer [82][83]. Unlike other malignant effusions, those caused by NSCLC have low sensitivity to chemo-and radiotherapy [84][85][86]; therefore these patients are candidates for palliative therapy. In a similar way, NSCLC is the most frequent cause of malignant pericardial effusion, which has a grim prognosis too [87][88][89].
The IASLC reclassified pleural dissemination from T4 to M1a based on the magnitude of evidence demonstrating that postoperative survival rates of patients with stage IIIB due to MPE are no different from those with stage IV disease and significantly lower than in patients with no pleural effusion and even with non-malignant pleural effusion [90][91][92][93][94][95].

Changes to N descriptors [97-98]
The accurate assessment of lymph node involvement is an important part of the management of lung cancer. Lymph node "maps" have been used to describe the location of nodal metastases. However, there are discrepancies in nomenclature among maps used by Asian and Western countries. The IASLC proposed a new lymph node map that reduces these differences among currently used maps, and provides precise anatomic definitions for all lymph node stations. It has also been proposed a new method of grouping lymph node stations together into "zones" for the sake of future survival analyses [99].

Milestones:
1. No changes were made to N descriptors. Analysis from the international database of the IASLC showed that current N descriptors provide good survival stratification, and therefore considered appropriate to maintain them without modifications [100].

2.
New International Lymph Node Map: The IASLC has developed a new lymph node chart to resolve disagreements in nomenclature between Naruke's (The Japan Lung Cancer Society) and Mountain-Dresler's maps (American Thoracic Society). Although the nomenclature has changed, the general concept remains the same. Patients without nodal metastatic disease are designated as N0. Patients with N1 disease are defined as having metastatic involvement of lymph nodes in the ipsilateral peripheral or hilar zones. The N2 designation signifies metastatic extension to lymph nodes in the ipsilateral mediastinal (upper, aorticopulmonary, lower) or subcarinal lymph node zones. The N3 nodal designation includes metastatic involvement of any nodes in the supraclavicular lymph node zone or nodes in contralateral mediastinal, hilar-interlobar, or peripheral zone.

3.
New classification of lymph nodes by grouping stations into seven "Nodal Zones" for prognostic analysis: supraclavicular, upper, aorticopulmonary, subcarinal, lower, hilarinterlobar, and peripheral (Table 6 and Figure 4); this proposal needs to be validated with prospective studies and it is not yet effective in the new TNM system.
Survival differences were also calculated on the basis of the number of lymph node zones involved in any single nodal designation. For instance, in pathologically staged patients with any T and M0, those with nodal metastases to a single N1 zone had a median survival of 52 months whereas those with metastatic spread to nodes in multiple N1 zones had a median survival of only 31 months. Similar decreases in survival were also seen in patients with multiple N2 nodal zone involvement (median survival 19 months) compared with those with disease in a single N2 nodal zone (median survival 35 months) [65]. These results showed improved survival in patients with a single N2 zone involved compared with those with multiple N1 zones involved. These findings were validated by an external study [102], and raising the possibility of subdividing the N1 and N2 classifications into N1a (singlezone N1), N1b (multiple-zone N1), N2a (single-zone N2), and N2b (multiple-zone N2).

4.
Given the continuous ascension of endoscopic ultrasound techniques such as EBUS (endobronchial ultrasound) and EUS (endocopic ultrasound) guided transbronchial (TBNA) and transesophageal (FNA) fine needle aspiration for the sampling of mediastinal lymph nodes, the borders between lymph node stations have been reconsidered, limiting as much as possible the subjectivism and trying to better standardize and define nodal stations.
As Tournoy et al. showed in this graphic representation, the reach of EUS-FNA and EBUS-TBNA is partly overlapping and partly complementary. EBUS-TBNA follows the large airways, whereas EUS-FNA is excellent for the left, posterior and lower mediastinal and paraoesophageal stations. When both techniques are available, many of the mediastinal and hilar lymph nodes can be reached for fine-needle aspiration. In addition, with EUS-FNA, the left liver lobe, celiac trunk nodes, and the left adrenal gland can be assessed. With EUS, lymph nodes can be identified if they are located in the vicinity of the esophagus. The nodes to be identified with EUS are described in relation to vascular structures (including the aorta, the azygos vein, the left atrium, and the pulmonary artery) and the diaphragm [103][104].
With EBUS, the ultrasound window angle is much smaller, when compared with EUS (50-60 degree angle versus 150-180 degree angle), which makes the visualization and identification of large vessels or ultrasound landmarks easier with the latter ( Figure 5). EUS helps in the identification of structures and landmarks through movements of backward, forward and rotation of the scope. In contrast to EUS, EBUS has the advantage of having a real-time bronchoscopic view into the airways during procedures, which helps with a more accurate identification of the lymph node stations [12].
In general, the lymph nodes are characterized based on EBUS imaging as follows: a. Size (in short axis): less or more than 1 cm.
b. Shape: oval or round; when the ratio of short vs. long axis of lymph nodes is smaller than 1.5, the lymph node is defined as round; if the ratio is bigger than 1.5, it is oval.
c. Margin: indistinct or distinct; if the majority of the margin (>50%) is clearly visualized with a high echoic border, the lymph nodes are determined as distinct. If the margin is unclear, they are determined as indistinct. f. The presence or absence of coagulation necrosis sign (CNS). CNS is a hypoechoic area within the lymph node without blood flow. Typical coagulation necrosis sign represents a low echoic area within the lymph node and that sometimes occupy the majority of the lymph node. The presence of CNS had the highest specificity (92.6%) and the highest hazard ratio (5.6) for prediction of metastatic lymph nodes [106] Since we consider that in the light of the latest TNM classification the knowledge of lymph node stations and their borders is of absolute importance, we will debate about it on a larger scale in this chapter. This, according to our opinion, is going to help especially the EUS-EBUS practitioners, since we are going to correlate from literature the schematics with CT's and ultrasound in every lymph node station, in order to have a detailed mental image of the mediastinum. (Figure 6). Figure 7) are located caudal to the inferior margin of the cricoid but cranial to the incisura jugularis of the sternum and cranial to the clavicles bilaterally. Therefore, the supraclavicular nodes are also part of station 1. The latter can be felt by a clinical examination when enlarged; however, external ultrasound has shown to be useful for their localization, identification, and puncture.21-24 21 [107][108][109][110].

Station 1 lymph nodes (
Although the paratracheal part of station 1 can be reached by EBUS-TBNA (1R/L; bilateral-the midline of the trachea serves as the border) or EUS-FNA (1L; left), the proposed anatomic borders cannot be recognized with endoscopic ultrasound. Because these nodes are localized extrathoracic, an endoscopic approach is very unpractical. The endoscopes are not stable for these very proximal stations making interpretation and sampling technically difficult and uncomfortable for the patient. Therefore, endoscopic ultrasound is of limited value for identification, delineation, and sampling of the paratracheally located station 1 nodes [21]. Discriminating left and right-sided nodes has clinical implications (N2 versus N3). In most of the cases, there is no discussion about the position because the presence and the size of the nodes as seen and measured on the CT scan also help the endoscopist in the identification ( Figure 9). However, it can be that similarly enlarged nodes are found in this region and that attention is needed for making the difference between N2 and N3. As a general rule, it can be said that EUS-FNA can only reach the left paratracheal lymph node stations. With EBUS-TBNA, both stations can be approached while no clear endoscopic or ultrasonographic landmarks are available to discriminate left and right. Although the bronchoscopic image helps during EBUS-TBNA, the left side of the trachea never identifies as a straight plane. The large arteries (subclavian artery or the aortic arch) cannot help much because their position relative to that sagittal plane is variable. In addition, the smaller ultrasonography window of EBUS-TBNA also makes the visualization of the anatomic ultrasound landmarks is more limited [21].   . Station 2 node in front of the trachea, i.e. a 2R-node. There is also a small prevascular node, i.e. a station 3A node.

Endoscopy
Station 3 lymph nodes are defined with an anterior part being the prevascular nodes and a posterior part being the retrotracheal nodes. The craniocaudal extent goes for both from the sternal notch down to the main carina. Although the prevascular station 3A can be visualized with EBUS as lying ventrally from the large vessels, there are no anatomic ultrasound landmarks to recognize the superior border of this lymph node station. Sampling station 3A by endoscopic ultrasound is impossible because of the interposition of the large vessels. This contrasts with station 3P, which is situated in between the pars membranacea of the trachea and the vertebrae (Figure 11). It can be seen and sampled by both EUS-FNA and EBUS-TBNA. Although there is no ultrasound landmark for the superior margin of 3P either the inferior margin being the main carina level corresponds with the level of the main stem or left pulmonary artery during EUS and can be seen as an anatomic structure during EBUS. The margin between 3P and 2/4L is the left posterior tracheal corner, which is identifiable by EUS-FNA or EBUS-TBNA. The margin between 3P and 2/4R is the right posterior tracheal corner that can be identified during EBUS-TBNA.
Station 4 lymph nodes are located paratracheally but situated caudal to the transverse aortic arch plane ( Figure 12). The sagittal plane on the left side of the trachea is the margin between left and right, just like in station 2 nodes. By consequence, EUS-FNA in general cannot approach a right paratracheal node. The comments made to discriminate between 4R and 4L with EBUS-TBNA are identical to those for stations 2. However, what can be helpful for the endoscopist is that 4R nodes are situated posterior to the superior vena cava and/or ascending aorta, both presenting as large vessels with a vertical course, which can be readily visualized by EBUS-TBNA. The inferior margins of station 4 nodes have been redefined with important clinical implications, also for endoscopic ultrasound. The pleural reflection no longer serves as the border between stations 4 and 10. Station 4L has now an inferior border defined by the superior rim of the left main pulmonary artery and a lateral margin defined by the aortopulmonary ligament (Figures 12 -14). With both EUS-FNA and EBUS-TBNA, the cranial rim of the left main pulmonary artery can be visualized. The aortopulmonary ligament is invisible for ultrasound. Station 4R's inferior border has now been redefined as the inferior border of the azygos vein. This new definition is better because the anatomic margin being the pleural fold is invisible for conventional or endoscopic imaging, whereas the azygos vein is always visible. During EBUS-TBNA, it typically presents in the right tracheobronchial corner as a kidney-shaped vessel (Figures 12, 13 Station 5 (AP-window) nodes are located laterally to the ligamentum arteriosum [111].   Station 5 lymph nodes are situated laterally to station 4L nodes with the ligamentum arteriosum as anatomic border (Figures 11, 13). The inferior edge is similar for both, whereas the cranial edge is not. Although 4L nodes are situated caudal to the superior border of the aortic arch, station 5 nodes are located caudal to the inferior border of the aortic arch ( Figure 19).
Because the ligamentum arteriosum cannot be discerned by means of ultrasound, the differentiation between 4L and 5 can be difficult, especially when both stations contain suspect lymph nodes. Station 5 nodes can be identified by EUS-FNA and EBUS-TBNA although the latter is often more demanding. Because of the interposition of aortic arch or pulmonary artery, station 5 can only be punctured in selected patients with enlarged nodes.
Station 6 mediastinal lymph nodes are located lateral to the ascending aorta and aortic arch, in between the transverse planes at the superior and inferior border of the aortic arch. (Figures 13, 14) These nodes can most often be identified by means of EUS-FNA, whereas this is not always possible with EBUS-TBNA. The nodes in station 6 can only be punctured by a transaortal approach, but extended mediastinoscopy is advised instead [21,112]. Station 6. Para-aortic nodes: Para-aortic (ascending aorta or phrenic) nodes are located anteriorly and laterally to the ascending aorta and the aortic arch from the upper margin to the lower margin of the aortic arch [111].
Station 7 lymph nodes have an inferior border that is redefined. (Figure 15).On the left side, this is the superior border of the lower lobe bronchus, and on the right side, this is the inferior border of the intermediate bronchus. (Figure 16) The lymph nodes in this station can be seen and biopsied by both EUS-FNA and EBUS-TBNA. Formerly, an anterior and posterior part of this node was recognized. This was meaningful because a cervical mediastinoscopy cannot reach the posterior part of this station. With EUS-FNA and EBUS-TBNA, the entire subcarinal area can be approached. Although identification of the nodes in this station is easy for both EUS-FNA (the nodes lay just dorsally to the origin of the left pulmonary artery and cranial to the left atrium) and EBUS-TBNA (by means of the endoscopic view), the delineation of the inferior border by means of endoscopic ultrasound is, however, not easy. With EUS-FNA, the left atrium is generally seen as the anatomic border above which the subcarinal nodes are situated, although this is with the new definition probably too restrictive. The relation of the left atrium or pulmonary artery to the bronchus intermedius and the left lower lobe bronchus, the latter being the newly defined inferior borders of station 7, can be variable. With EBUS-TBNA, the delineation of the inferior border is possible because this investigation allows a simultaneous bronchoscopic view of the bronchial tree although there are no distinct corresponding ultrasound landmarks.

Stations 8 and 9
are the paraoesophageal and pulmonary ligament nodes, respectively, and are situated inferior to the inferior margins of station 7 lymph nodes. The superior border of these lymph nodes is as such defined by the inferior margin of the subcarinal area. Station 8 nodes are located along the left atrium (Figures 17, 18), whereas station 9 nodes are lying within the pulmonary ligament ( Figure 19). Although the latter is a structure that cannot be seen with endoscopic ultrasound, station 9 nodes are located just cranial to the diaphragm, which is readily identifiable with EUS-FNA. Stations 8 and 9 lymph nodes can be thus approached by means of EUS-FNA. Occasionally, station 8 nodes can be found by EBUS-TBNA. However, and as suggested above, one has to take into account the inferior stretch of the subcarinal nodes making this station in addition to station 9 becomes invisible for EBUS-TBNA. When performing EUS-FNA, one cannot confuse a lesion or lymph node with the esophagus. When performing EBUSTBNA, the esophagus can be seen as a multilayered structure with a hyperechogenic line in the middle corresponding with air not to be misinterpreted as a lymph node. The discrimination between the left-and right-sided nodes is the midline. Although no formal ultrasound characteristics for the midline are available, the relative position of the endoscope to the descending aorta can help.  Station 10 or hilar lymph nodes are situated immediately adjacent to the main stem bronchus but caudal to the inferior border of azygos vein on the right and superior rim pulmonary veins and artery on the left. These nodes can be seen and sampled by EBUS-TBNA (Figures 20, 21). The inferior margin of station 10R is the interlobar region. There is no unique ultrasound feature that defines that border, but because a bronchoscopic view is available during EBUS-TBNA, the secondary carina or the upper lobe split off can serve as surrogate here. EUS-FNA has been thought to be unable to see and sample hilar stations. However, there is no doubt that in certain cases, station 10 nodes located medially from the main stem bronchi can be assessed. Endoscopists should be aware of this because misinterpretation could lead to overstaging [114]. Once the secondary carina is reached, station 11 lymph nodes (or interlobar nodes) are encountered. From this station on, the nodes can only be approached by EBUS-TBNA and not by EUS-FNA. These nodes are located just underneath the mucosa of the secondary carina on the left. There is a division between 11s and 11i on the right side. The former indicate the nodes between upper lobe and intermediate bronchus, the latter are situated in between middle and lower lobe ultrasound landmarks are not available; however, the synchronous endoscopic view enables the identification of the relevant lobe split offs. In closing, the new IASLC map maintains the lymph node stations of the other maps, but it also groups those that are anatomically proximal in lymph node areas in order to make the lymph node classification easier, especially in patients who will not undergo surgery. In this map, all the lymph node stations are defined by anatomically precise limits that are easy to recognize with imaging techniques and inspection during invasive explorations or thoracotomy. The innovations of this lymph node map are: • The creation of a supraclavicular lymph node area that includes the supraclavicular, lower cervical (caudal on the lower edge of the cricoid cartilage) and the suprasternal fossa lymph nodes. If these lymph nodes are invaded by a tumor, they are classified as N3, regardless of the side of the tumor.
• The widening of the subcarinal lymph node station. It now includes all the lymph nodes from the tracheal bifurcation until the upper edge of the lower left lobar bronchus and the lower edge of the intermediary bronchus. If they are affected by tumors, these lymph nodes are classified as N2. This new subcarinal station includes lymph nodes that before, at least according to the Japanese map, were hilar (adjacent to the lower sides of the main bronchi), that could be classified as N1 or N3, depending on the side of the tumor. The larger size of this subcarinal station will mean an increase in N2 tumors in detriment of N1 and N3 tumors.
• The incorporation of precise limits for station number 10, the hilar station, which facilitates the prospective collection of data in order to clarify the prognostic role of this station, whose placement on other maps has always been controversial.
• The shift in the midline of the upper mediastinum from the tracheal anatomical midline to the left paratracheal margin exclusively affects the upper and lower right and left paratracheal stations. This modification implies that the affected lymph nodes that are to the left of the anatomical midline, but to the right of the new left paratracheal line, will be N2 for tumors of the right lung, but N3 for those of the left lung.

Changes to M descriptors [25, 39]
The patients with lung cancer studied for the 7th edition of the TNM Classification of Malignant Tumors presented the following survival rates at 1 and 5 years: T4 any N M0, 53% and 16%; pleural dissemination, 45% and 6%; contralateral pulmonary nodule(s), 46% and 3%, and distant metastasis, 22% and 1%; in this latter case, with significantly lower survival rates than previously cited [39]. With such references, it was decided to subdivide the M component into M1a (presence of pleural dissemination or contralateral pulmonary nodule(s)) and M1b (distant metastasis).
• Subclassify M1 by additional nodules in the contralateral lung as M1a.
• The MX and pM0 designation has been eliminated from the AJCC/UICC TNM system.

Additional changes
• Introduction of a new accurate definition of visceral pleural invasion (VPI); VPI is a pT2 descriptor (Table 8). To avoid confusion, the abbreviation PL is employed instead of P which is also used for designation of pTNM in distinction from cTNM. The IASLC also recommends the use of elastic stains to distinguish between PL0 and PL1 when hematoxylin and eosin (H&E) sections are not helpful [71].  [116].

PL category
• Pathologic TNM staging must be used for all SCLC cases [117]..The International Staging Committee of the IASLC has confirmed that the survival of patients with SCLC worsened as the T and N categories increased [116]. It was also observed that, except in stage IIA, which had only 55 patients for analysis, the 5-year survival worsened as the stage progressed: IA, 38%; IB, 21%; IIA, 38%; IIB, 18%; IIIA, 13%; IIIB, 9%, and IV, 1%. Based on this, the proposal to use the TNM system for staging SCLC was confirmed.
• Carcinoid tumors are now included within the TNM classification [118], a new staging system for neuroendocrine tumors. Lung carcinoids are staged in the same way as carcinomas.
Even though the 6th TNM classification specified that it was not applicable to carcinoid tumors, several studies have used it, finding prognostic differences among the stages. The IASLC has also confirmed that those classified as stage I lived significantly more than those in stage II, and these significantly more than those in stages III-IV; therefore, the new TNM classification of 2009 is recommended to describe the extension of these tumors [118].
Stage groupings were modified due to changes to the TNM descriptors (Table 5).
d. T4 tumors due to other factors, N0-1 are down-staged from IIIB to IIIA.
b. T2 tumors >7 cm, now T3, are up-staged: T3N0M0 from IB to IIB and T3N1M0 from IIB to IIIA.
c. Tumors with pleural nodules or malignant pleural (or pericardial) effusion were reclassified from T4 to M1a, therefore are up-staged from stage IIIB to IV [120].
The main limitations are derived from the retrospective character of some databases that were not designed to study the TNM classification and lack precise anatomical details about the tumor extension, the number and lymph node stations affected or the differences between the different forms of M1 disease. For this reason, the IASLC itself has initiated a prospective project aimed at once again updating the TNM classification in 2016, validating all the T, N and M descriptors, especially those who have not been until now. Thus, a large international database is being constituted that, correcting the geographical omissions and disproportions in the therapeutic modalities, includes patients with non-small-cell tumors, small-cell tumors and their neuroendocrine subtypes.
In conclusion, the IASLC staging classification is unquestionably a major advance. The size of the database, the broad international spectrum, the careful and detailed analysis, as well as the internal and external validation, are tremendous achievements and relatively unique among types of cancer [121].
So, the main advantages of the new classification would be: • The size of the database, the largest ever collected for any cancer type.
• For the first time, data was collected from different countries.

Endoscopy
• The timeframe of 10 years allowed for 5 years follow-up.
• Cases included all treatment modalities.
• Pathologic and clinical staging where considered in survival analysis.
• The statistical analysis included meticulous internal and external validation.
• Changes to the TNM descriptors and stage groups were derived strictly from the outcome measure of overall survival.
The new TNM system is less intuitive and more complex than the 6th edition, and will be more difficult to learn. The oncology community needs to overcome the higher learning curve in order to offer patients the most appropriate treatment choices based on more accurate prognostic information [120].

Electromagnetic navigation bronchoscopy
It is known that diagnostic yield of flexible bronchoscopy is limited by its inability to guide biopsy instruments directly to the lesion. As it was expected, diagnosis success rate is dependent on the size and location of the lesion. The diagnostic yield of flexible bronchoscopy is expected to be between 20 and 84%. However, current nonsurgical techniques available to diagnose small peripheral lung lesions (SPLL) are limited either by low accuracy [122][123][124][125] or by potential complications [126][127][128][129].
For lesions less than 2 cm in diameter, the diagnostic yield of flexible bronchoscopy is 14% for peripheral lesions in the outer third of the chest and as high as 31% if in the proximal two-thirds [130]. The diagnostic yield of flexible bronchoscopy for mediastinal lymph nodes using transbronchial needle aspiration (TBNA) is reported to be between 15 and 83% [131]. Also, diagnostic yield of TBNA in staging of bronchogenic carcinoma is reported to be between 50 and 60% [132].
Within the past several years, electromagnetic navigation guided bronchoscopy (EGB) has proven very effective at assessing pulmonary nodules accurately with very low complication rates. EGB consists of four elements:

1.
Computer software that utilizes thin-slice CT to create a three-dimensional rendering of the lung and tracheobronchial tree, which can then be used for virtual bronchoscopy.

2.
A sensor probe that fits through the small working/suction channel of a bronchoscope with a steering mechanism. Because the sensor probe is recognizable within the electromagnetic field, it can be navigated through the small airways of the lung toward peripheral lesions not reachable by conventional bronchoscopy (Figure 22).
3. An electromagnetic field encompassing the patient's thorax, so that the real anatomy can be merged with the computer generated (virtual) anatomy by use of standard "registration points" (e.g., carina, takeoff of the right upper lobe bronchus, bifurcation between the left upper and lower lobes).

4.
A hollow, extended working channel (EWC) that can be secured in a small peripheral airway and used to pass diagnostic instruments such as brushes, aspirating needles and biopsy forceps [133].
The ideal patient for EGB should be one with peripheral pulmonary lesion (solid or fatty solid nodule located beyond the visible range of flexible bronchoscopy) detected by chest radiography and CT or presenting with suspicion of cancer by CT morphology or positive positron emission tomography (PET) scan, as well as with a nondiagnostic conventional bronchoscopy in most of these cases; he should have absence of other metastatic lesions accessible for biopsy, a negative TTNA or contraindication for TTNA (severe pulmonary impairment, bleeding diathesis, lesions not accessible by TTNA as judged by a radiologist panel) and contraindication for straightforward curative surgery; in case of associated mediastinal lymph nodes should have a negative transbronchial needle aspiration (TBNA) or difficult to reach with TBNA lymph nodes.
Those patients who should not undergo EGB are those with contraindication to short-acting anesthetic agents, bleeding diathesis, presence of concomitant endobronchial lesion, presence of a pacemaker/defibrillator or a diagnosis by other means (sputum cytology, microbiology) that offers a reliable and easy to control course of treatment.
Navigation aim is to closely approach the target lesion (distance between sensor tip and lesion centre ≤15 mm) and take as many biopsies as possible for each lesion. At every three attempts is advised that the forceps should be withdrawn and the position of the sensor probe in relation to the target lesion checked.
Makris D et al. also recommends that all patients should undertake a CT scan prior to EGB with the following technical criteria: slice thickness 2-3.5 mm, interval between slices (with overlap of 1 mm) 1-2.5 mm, image size 512x512 pixels and dicom format [134].
As for anesthesia, Gildea TR et al. considers that all procedures (flexible bronchoscopy, bronchial washings, bronchoalveolar lavage and the actual EGB) can be safely performed using conscious sedation with intravenous 2 mg boluses of both midazolam and morphine with topical lidocaine [135].
In an emblematic study from Gildea TR et al. in 2006, the role of electromagnetic navigation bronchoscopy using super-Dimension/Bronchus System as a novel method to increase diagnostic yield of peripheral and mediastinal lung lesions was investigated. The superDimension/bronchus system is an image-guided localization system, which is designed to guide bronchoscopic tools to predetermined points within the bronchial tree. The device uses three separate technologies that are combined to enable navigation of dedicated tools within the lung in real time.
The first component is the planning software, which converts digital imaging and communications in medicine standards (DICOM) images from a computed tomography (CT) scan into multiplanar images with three-dimensional reconstruction and virtual bronchoscopy of the airways.
The second component is a steerable probe that contains a position sensor attached to an eightway steerable instrument that has the ability to navigate turns in the endobronchial tree [135]. The third component is an electromagnetic (EM) board, which is a field generator connected to a computer containing the planning data. The exact position of the steerable probe when placed within the EM field is depicted on the system monitor.
Registration is the process by which the computer links the five to six virtual fiducial markers to the actual position in the patient. Upon registration completion, the average fiducial target registration error (AFTRE) score was given in millimetres (mm). The AFTRE is the radius of expected difference of the location of the tip of the steerable probe in the actual patient compared with where it is expected to be in the virtual patient [135].
After registration, navigation is performed with simultaneous advancement of the steerable probe toward the target and directing steerable probe to the lesion. The closest distance between the steerable probe tip and the lesion centre is recorded. When navigation is completed, the steerable probe is removed, leaving the extendable working channel through which brushings and TBBXs or TBNA were performed [135].
In this study, biopsies were performed using a C-arm fluoroscopy unit. All instruments were visualized under fluoroscopy only after navigation was completed to confirm proper function and position of the bronchoscopic tools relative to the lesion and the pleura. Brush biopsies involved two to three passes, and four pieces of tissue were obtained by TBBX. TBNA usually was done with 2 to 4 passes of a combination of 19-G and 22-G needles depending on physician choice [130,[135][136][137][138][139].
Eberhardt et al. [18] reported in 2007 92 peripheral lung lesions biopsies from 89 consecutive subjects. The diagnostic yield of EGB was 67%, which was independent of lesion size. Total procedure time ranged from 16.3 to 45.0 min (mean [± SD] procedure time, 26.9 ± 6.5 min). The mean navigation error was 9 ± 6 mm(range, 1 to 31 mm). They reported two incidences of pneumothorax for which no intervention was required. When analyzed by lobar distribution, there was a trend toward a higher ENB yield in diagnosing lesions in the right middle lobe (88%). They concluded that EGB can be used as an independent bronchoscopic technique without the need for fluoroscopy when compared with other available studies. There was no increased risk of pneumothorax (2 of 89 patients; 2%). The upper lobes tend to have sharper angles in the bronchial tree that may be challenging to navigate even with a steerable sensor probe. The EWC ends close to the tip of the sensor probe and makes it less flexible. This reduces the range of deflection and, consequently, the ability to navigate. It can also make the probe flip into a different position when negotiating some tight angles in the bronchi. Navigation in the lower lobes is more affected by diaphragmatic movement during breathing and could result in larger errors than recorded. This is because the planning data are based on CT scan images acquired in a single breathhold [140,141].
Eberhardt et al. also showed that the improved yield of EGB compared to conventional transbronchial lung biopsy in small lesions (diameter ≤ 2 cm) can be attributed to the improved precision in navigation. This study has shown the yield, safety, and timesaving with use of the EGB system without the need for fluoroscopy. This system eliminates radiation exposure and could reduce procedure costs. The diagnostic utility of the use of EGB in the biopsy of peripheral lung lesions appears to be equivalent to other advanced techniques like endobronchial ultrasound [142][143][144].
Apparently, these techniques have pushed bronchoscopic biopsy yields closer to those achieved by CT scan-guided transthoracic needle biopsy and surgery. Given the relative comfort [145] and safety [146] of flexible bronchoscopy, and the recognized risks of both CT scan-guided [147][148][149] and surgical biopsies [150], there is growing need to develop and refine these techniques. The expanded role of lung cancer screening, in which the vast majority of lesions is benign, makes this all the more important [151]. Multimodality diagnosis by combining ENB with other bronchoscopic and imaging techniques may further enhance the diagnostic yield [140].

Autoflorescence Bronchoscopy (AFB)
Visible light perceived by the human eye comprises the whole wavelength range, between 400-700 nm. Conventional bronchoscopy illuminates mucosal structures of the airwaves with the full wave spectrum; therefore, light gets reflected, backscattered or absorbed by the structures it encounters, thus providing the human eye with an image [152]. Tissues show a natural autoflorescence when excited in the 200-460 nm range; however, only the visible part of this range is used in medical applications [153].
However, the light source attached to the bronchoscope can emit only blue light, as a proportion of the blue spectrum excites the cellular chromophores, such as collagen, elastin or keratin, contained in the layers of the submucosa, especially within the connective tissue comprised in the elastic fiber bundles, or on the exterior surface of the cartilages (perichondrium). When excited by blue-violet light in the 400-450 nm spectrum, the excited chromophores of normal cellular lines found in the mucosa display a green tint. Another aspect of the autoflorescence theory refers to the thickness and cellular morphology of the examined tissue. Tumors and premalignant lesions have an increased mucosal thickness and therefore absorb more of the excitation occurring from fluorescence light, in what is called "the architectural effect" [153,154]. Tissues that underwent a morphological change due to various pathological conditions, predominantly premalignant dysplasia or metaplasia-like phenomenon, are colored differently as the green fluorescence is affected by either alterations of cellular chemistry, morphology or epithelial thickness [155][156][157]. A wide range of AFB devices are currently available on the market: the Storz D-light system [158], Pentax SAFE-1000 [159], the Xilix LIFE system [158] or the DAFE system by Richard Wolf [160].
The main advantage of AFB is that progressive dysplasia of the mucosal layers also results in a progressive transition from normal green autoflorescence to a red-brown color, specific to precancerous or malignant lesions. This makes early premalignant lesions far easier to spot during bronchoscopy as compared to regular white-light based techniques. Inflammatory reactions, granulomas, scars, dysplasia/metaplasia and early malignant lesions which are hard to spot due to their submucosal confinement are therefore easily spotted due to their dark red-brown appearance, surrounded by normal green tissue [155][156][157].
The addition of AFB to conventional endoscopy can significantly increase diagnostic rate of early malignant or premalignant lesion, without the need of tumor sensitizers and therefore with no additional complications to standard bronchoscopy techniques. The detection rate of high-grade dysplasia and carcinoma in-situ (CIS) is increased to 88% after using AFB, up from the median 40% detection rate that conventional white-light bronchoscopy provides [161,162]. However, as already stated, both premalignant lesions and various inflammatory conditions have similar appearances in AFB. Therefore, the specificity is rather low, with a high rate of false positive investigations [163,164].
Since diffuse reflectance spectroscopy measures directly the changes in the path of white light through tissue scattering and absorption levels, some have theorized that combining autofluorescence with diffuse reflectance probes may enhance the specificity of this method. Preliminary findings have shown that a combination between the two can significantly improve the positive predictive value of AFB without altering the already attained high sensitivity for premalignant and early malignant lesions [165] ( Bard MPL et al, 2005). Other attempts to improve specificity by local spectroscopical measurements have shown promising results, making way for one-stop techniques which would combine two types of wavelength measurement and fluorophore weighting [153,165].
One study investigated the use of AFB in primary lung cancer, after treatment of head and neck cancer, detecting 29% (12/44 patients) of all second primary lung cancers, while detecting early lesions in two patients which would otherwise be missed by conventional imaging methods [166]. Early reports of Lam and his team in asbestos and diesel-exposed individuals showed an 86% sensitivity for moderate to severe dysplasia and CIS in the case of AFB, compared to just 52% for white light bronchoscopy [167]. A multicenter study performed by Lam in 1998 with the LIFE system, on 173 patients (142 biopsy-proven cases of severe dysplasia, CIS or invasive cancer) showed that AFB has a relative sensitivity of 2.71 compared to conventional bronchoscopy, successfully detecting 91 cases in comparison to only 35 with bronchoscopy alone. When considering intraepithelial lesions alone, the relative sensitivity increases to 6.1 for AFB compared to standard bronchoscopy [164].
A correlation between the loss of fluorescence and the grade of dysplasia; therefore, a trained endoscopist can differentiate these gradations to some extent, being even able to identify inflammatory or granulomatous lesions [155]. This would In turn greatly improve the specificity of AFB. A different approach consists in the usage of computer assisted analysis of signals of different wavelengths, represented in a spectrogram which in turn can classify benign from malignant lesions.

Narrow-Band Imaging (NBI)
NBI is a novel imaging technique capable of improving the visualization of superficial structures of the respiratory mucosa [168,169]. NBI is used for an accurate classification of lesions, even though it does not have an established role during routine bronchoscopy [170][171][172]. High magnification bronchoscopy in combination with NBI can visualize the altered micro-vascularization which is formed in dysplastic or neoplastic lesions [173]. As the tumor progresses, it requires an adequately enlarged blood supply, therefore in most cancers, including those of the lungs and airways, neo angiogenesis is a constant phenomenon through which newly formed vessels are constantly produced in order to supplement local needs. These vessels form irregular aberrant patterns, having unequal diameters, tortuous configurations and uneven segment lengths.
The principle of NBI is simple: three optical filters segment the RGB (red-green-blue) light spectrum in sequence, thus narrowing the bandwidth of the spectral transmittance [174]. This allows for visual marking of capillary structures of the sub-mucosa in deep red color, allowing an easier identification and characterization, as different wavelengths penetrate the tissue at different depths. The filtering system is placed in the optical illumination system and basically contains two narrow wavelength components: NBI-B corresponding to the 400-430 nm range (blue and green visualization) and NBI-G for the 530-550 nm range (red visualization), while a third filter can be installed for an accurate segmentation of the bluegreen features, operating in the 430-460 nm range; the optical light-source usually operates in the 400-700 nm wavelength range. All current models include a 2-filter system, which emphasizes capillaries in the superficial mucosal layers by the 400-430 nm light, coloring them in shades of brown, while deeper mucosal or submucosal vessels are displayed in cyan by using the 530-550 nm filter. Modern systems allow for easy switch between the normal WLI and the enhanced NBI operations [168].
Limitations of the technology arise from the fact that not all lung malignancies develop submucosal neo vascularization visible from the airways, therefore potential false-negative NBI findings can be frequent when used in combination with WLB. Its sensitivity may be however increased when used in conjunction with other specific techniques such as AFB; however, its role in the screening process for lung malignancies is yet to be determined. A step further in this direction would be the standardization of imaging findings, based on clinical descriptions and classification of abnormal airway vascularity. High magnification bronchoscopy can improve the specificity of the technique, as it may allow a more clear description of the vascular patterns [168]. Good prospects come from new techniques such as probebased confocal laser endomicroscopy which can provide an in situ diagnosis of malignancy if used in conjunction with any endoscopic technique.

Probe-based Confocal Laser Endomicroscopy (pCLE)
Gastrointestinal applications of pCLE are numerous, the system being successfully tested in real-life conditions [175]. It can be used for identifying premalignant lesions otherwise invisible to classic endoscopic techniques, such as Barrett's esophagus [176], classification of polipous lesions at colonic level [177], gastric metaplasia or early stage lesions at all levels of the digestive tracts [178,179]. The technique requires injection of fluorescein or another fluorophore, followed by endoscopic imaging of the dye to identify mucosal details such as mucosal and vessel architecture to distinguish among normal, dysplastic, and neoplastic tissue. The use of fluorescein as an in-vivo contrast agent for detecting vascular structures has been deemed safe by several studies [180]. It thus allows for better visualization of vascular structures, serving as an overall tissue contrasting agent as it may leak beyond capillary confinement.
The imaging microprobe is connected to 30,000 fiber-optic threads that enable point-topoint real-time detection at 12 frames/sec. The microprobe's flexibility and size (1.5 mm in diameter) allow for great user maneuverability to scan tissues in situ at angles that would not be possible with any available confocal microscope objective. The mini-probe is inserted through a working channel of any standard bronchoscope and can reach the alveolar duct. A laser generator emits 488 nm blue argon laser light which is transmitted through the optical fibers and excites the elastin scaffold of the acinus. In turn, a real time image of the elastic fibers which sustain the alveoli, as well as the microvessel architecture is revealed. The probe can be translated over a larger tissue sample, and the images later reconstructed in order to extend the field of view covered [181]. A monitor attached to an imaging unit is available for real-time visualization of cellular images, the device being capable of recording full length movies which can be recorded and later on analyzed with dedicated software [182].
This technology is still in its early stages; however, it was deemed safe by regulatory organisms in the United States of America for applications in patient settings. In conjunction with safe contrasting agents such as fluorescein [183] or methilen blue [184], it can be used for the in vivo histologic assessment of the bronchial epithelium at all levels of the airway system [185][186][187]. Several studies reported good initial results in control groups of healthy smokers or patients with chronic obstructive pulmonary disease for diagnosing various forms of parenchymal lung disease [188,189]. A pCLE investigation seems to be a quick and safe procedure for patients with various pulmonary conditions, with minimal side-effects related to both the technique and the contrasting fluorescent agents used for imaging. General consensus is that further studies are needed in order to extent the diagnostic capabilities and enhance its sensibility in comparison with standard cytological and histological techniques.

Author details
Mihai