The study was divided into 2 parts. The first was a descriptive study for which 176 patients with mediastinal/hilar LN undergoing EBUS in the Hospital Universitario Son Espases between 2009 and 2012 for lung cancer staging or investigation of suspected malignant LN infiltration were screened. Of these patients, 141 were included in the study, and 2 raters who were unaware of the final diagnosis independently analyzed the 208 LN images. The second part was a prospective study for which 39 consecutive patients were recruited and two raters predicted the probability of malignant LN involvement in real time on the basis of the previously validated criteria. Consecutive patients scheduled to undergo EBUS to study mediastinal/hilar LN observed on a computed tomography (CT) of the chest or hypermetabolic LN identified in a positron emission tomography with fluorodeoxyglucose (PET-FDG) were included.

The indication for performing invasive mediastinum staging by EBUS, endoscopic ultrasound (EUS), or a combination of both techniques is established by a local multidisciplinary lung cancer committee, according to the following standardized criteria12: (a) enlarged discrete mediastinal LN with PET uptake; (b) PET activity in a mediastinal LN and nodes with normal appearance on CT, and (c) suspected N2,3 involvement with a radiographically normal mediastinum (determined by CT and PET) and a central or N1 tumor. Patients with any contraindication for bronchoscopy according to the recommendations of the American Thoracic Society13 or a high anesthetic risk (American Society of Anesthesiologists physical status >3)14 were excluded. A full clinical history was obtained and chest CT and PET-FDG images were analyzed by a multidisciplinary lung cancer committee that established the indication for performing EBUS with transbronchial needle aspiration (TBNA) or transtracheal aspiration (TTA), following international guidelines.15 Patients were then evaluated by the anesthesiology team before the intervention. Informed consent was obtained from all patients.

EBUS was performed using an EB-1970UK (Pentax, 10.0-5.0MHz, Tokyo, Japan) endoscope and the Hitachi Digital Ultrasound Scanner EUB-7000HV. The procedure was performed via the oral route under deep sedation with propofol, midazolam and fentanyl with anesthesiologist-controlled spontaneous ventilation in all study patients. The intervention was performed as described by Yasufuku et al.2 Enlarged LNs (greater than 5mm) were identified by the measurements made in the digitally captured ultrasound images. Fixed images were captured for subsequent analysis. Blood vessels were confirmed with the Doppler modality. For TBNA and/or TTA, a 22-caliber needle was used. The needle was introduced via the working channel of the EBUS through the bronchial wall, and the LN was aspirated under ultrasound guidance to obtain a tissue biopsy. A smear of the aspirated material was prepared on a glass slide, air-dried and immediately stained with standard hematoxylin and eosin for rapid on-site evaluation (ROSE) by a cytopathologist. Three consecutive biopsies of a reactive LN were considered negative for malignant disease. The aspirated materials were also collected in liquid formol, and the cell block underwent a final histological evaluation by the pathologist. The final pathological diagnosis was the standard reference for positive malignant LN infiltration. Patients with negative pathology results on EBUS in whom malignant LNs were confirmed after surgery (mediastinoscopy or lung resection with systematic lymphadenectomy) were also considered positive in the sonographic analysis of the LNs.

All ultrasound images were evaluated to determine the foregoing sonographic features11 with the aim of validating a score predictive of malignancy. The following sonographic features associated with malignity were evaluated11:

• 1.

  Round shape, defined as a ratio of <1.5 between 2 perpendicular axes.

• 2.

  Distinct and well defined, distinguished by a marked white line delimiting the LN.

• 3.

  Appearance of heterogeneous echogenicity as a dichotomous variable (as opposed to homogeneous).

• 4.

  Absence of visible CHS in the form of a central linear structure with high echogenicity, with or without a blood vessel.

• 5.

  Small axis ≥10mm.

• 6.

  Hyperechogenic density in the interior of the LN (Figs. 1 and 2).

  
  

  Fig. 1.

  Series of EBUS images of the mediastinal lymph node showing the different criteria: (A) heterogeneous echogenicity with a distinct margin in a reactive lymph node. (B) Homogeneous echogenicity with an indistinct margin in a reactive lymph node. (C) Heterogeneous echogenicity with a distinct margin in adenocarcinoma. (D) Hyperechogenic density in the interior of a lymph node (arrow) in small cell carcinoma. The lymph node images (A–D) show the absence of central hilar structure.

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  Fig. 2.

  Series of EBUS images of the mediastinal lymph node showing the different criteria (continued). (A) Presence of central hilar structure (arrow) in reactive lymph node. (B and C) Presence of central hilar structure with blood vessel (arrow) in another reactive lymph node; demonstration in Doppler mode in (C). (D) Indistinct margin with heterogeneous echogenicity in extrathoracic metastasis with the absence of central hilar structure.

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The interrater agreement (IRA) was calculated for each sonographic criteria. These criteria were correlated with the final histopathological results, and the sensitivity, specificity and positive and negative predictive values (PPV and NPV, respectively) were calculated from the receiver operating characteristics (ROC) curve and the area under the curve (AUC).

According to the reproducibility of the above-mentioned criteria, a simplified 5-item score was generated and calculated for prediction of LN malignancy. Each item was scored as follows: heterogeneous (1.5 points) versus homogeneous (0 points) echogenicity; absence of CHS (1.5 points) versus presence (0 points); round shape (1 versus 0 points); small LN axis ≥10mm (1 point); and well-defined LN margin (1 point) versus indistinct margin (0 points). A total score was generated from the sum of each separate criterion and plotted relative to the pathological diagnosis on ROC curves.

The previously modified score was applied prospectively to the ultrasound images of the suspect LN during the EBUS procedure by the two bronchoscopists performing the examination. The sonographic analysis was performed by a pathologist before the ROSE. The score was calculated and correlated with the histopathological results. A Doppler ultrasound was performed to evaluate blood flow within the LN. The utility of the proposed score was evaluated for the prediction of LN malignancy in real-time clinical application.

The data are presented as mean±standard deviation (SD) or number (percentage), as appropriate. Malignant and non-malignant sonographic features were compared using the Mann–Whitney test. Statistical tests were performed with two-tailed p values, with p<0.05 being considered statistically significant. ROC curves were used to calculate sensitivity and specificity, followed by the calculation of PPV and NPV for the score and for each criterion in relation to the final pathological diagnosis. AUC, C-index values and 95% confidence intervals (CI) are presented. The IRA was calculated using the interval-to-interval method, which is calculated by adding the total number of agreements in the intervals (for both the presence and the absence of malignancy) and dividing by the sum of the number of agreements and discrepancies in the intervals16; an IRA of 80% was considered a positive agreement. MedCalc® (version 9.2.1.0, Acacialaan 22, B-8400 Ostend, Belgium) statistical software was used.

Patient characteristics and principal results are summarized in Table 1. After correlation with the pathology data was established, the images obtained from 141 patients, including 208 LNs from various node stations, were analyzed. The subcarinal LN (station 7) was the most frequently aspirated node (45.7%), followed by stations 4R and 4L (24% and 9.6%, respectively) (Table 1).

A definitive diagnosis of malignant disease in the LNs analyzed was obtained in 50.5% of the subjects compared to 49.5% with reactive lymphadenitis. The final pathology of the biopsies evaluated is presented in Table 1, showing that adenocarcinoma was the most common diagnosis in the study cohort (22.7%), followed by squamous cell carcinoma (12.8%).

The analysis of the LN sonographic features, with their corresponding sensitivity, specificity, PPV and NPV for malignant disease, is shown in Table 2. The evaluation of the different sonographic criteria revealed that both heterogeneous echogenicity and the absence of CHS with or without central blood vessel were the most sensitive factors for predicting malignant disease (99% in both cases), while hyperechogenic density in the interior of the LN had the highest specificity for predicting malignant LNs (78%). Moreover, heterogeneous echogenicity showed an NPV of 94.4%, followed by the absence of CHS (90%). None of the proposed sonographic criteria had a clinically acceptable PPV. A positive IRA for the proposed criteria was recorded, with the maximum agreement being observed for heterogeneous echogenicity (92.2%) (Table 2).

The difference between the malignant and non-malignant infiltration of the LN for each criterion was statistically significant (p<0.01), with the exception of the presence of hyperechogenic density in the interior of the LN (p>0.05). Consequently, scoring was generated using only 5 criteria, as described above in the methodology section. With the ROC analysis, the combined score of >5 has a sensitivity of 73.3% and a specificity of 68% (AUC=0.738; 95% CI: 0.673–0.796; p=0.0001) for the prediction of malignant LN infiltration (Fig. 3). The PPV and NPV for the generated score were 70% and 71.4%, respectively.

Fig. 3.

ROC curve of the score for the prediction of malignant lymph node infiltration. (A) ROC curve for the retrospective study showing different sensitivities and specificities of the combined positive criteria (AUC=0.738; 95% CI: 0.673–0.796; p=0.0001). (B) ROC curve for the prospective study (AUC=0.852; 95% CI: 0.743–0.928; p=0.0001).

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A real-time evaluation of the LNs during EBUS was performed in 39 patients, and 65 LNs were examined during the procedure. The prevalence of malignant disease in the LNs examined was 35.4% and the most common pathological diagnosis was adenocarcinoma (15.4%). The subcarinal LN was the most frequently aspirated station (46.2%), followed by station 4R (24.6%) and then station 10R or 10L (18.5%). Stations 4L, 2R and 2L represented 7.7%, 1.5% and 1.5% of the aspirations, respectively. When the previously validated sonographic criteria were applied in real time, the absence of CHS, a round shape and a short axis ≥10mm yielded significantly statistical differences between non-malignant and malignant LNs (p=0.0012; p=0.012 and p=0.023, respectively). Neither heterogeneous echogenicity nor a distinct margin showed a statistically significant difference between benign and malignant LNs (p>0.05), as indicated in the retrospective analysis of the ultrasound images.

With the combination of positive criteria during the procedure, a score of >5 was found to have a sensitivity of 78% and specificity of 86% in the detection of a malignant LN (AUC=0.852; 95% CI: 0.743–0.928; p=0.0001), with a greater specificity than that obtained from the analysis of images. PPV and NPV were 75% and 88%, respectively.