Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)-from morphology to molecular testing

Abstract

In recent years, endobronchial ultrasound-guided TBNA (EBUS-TBNA) has emerged as an innovative technique for diagnosis and staging of lung cancer and has been successfully introduced into daily clinical practice with several advantages including minimally invasive approach, safe, cost-effective, real time image guidance, broad sampling capability, and rapid on-site evaluation (ROSE). Both cytological and histological approach could be useful to have material for diagnosis, immunohistochemical and molecular analyses which may be very important for targeted therapy with successful rate ranging from 89% to 98%. The utility of ROSE during EBUS-TBNA has been matter of debate. Indeed, although some evidence concluded that ROSE does not increase the diagnostic efficacy of EBUS-TBNA, other demonstrated that it improves the diagnostic yield of the procedure up to 30%, allows to avoid repetition of additional diagnostic procedures and reduces risk of complications. Furthermore the sample preparation by cytopathologist is optimized with the aid of direct macroscopic inspection, optimal smearing techniques, and triage of the sample permitting to obtain adequate tissue for diagnosis, ancillary techniques and molecular testing, when needed. Some pathological issues on EBUS-TBNA are reviewed and discussed with particular focus on ROSE and molecular testing.

Keywords: ALK; EGFR; Endobronchial ultrasound-guided (EBUS); immunohistochemistry (IHC); lung cancer; transbronchial needle aspiration (TBNA).

Figure 1

A 67-year-old female with a history of previous lung adenocarcinoma presented with a PET positive nodule in a para-tracheal lymph node. (A) The ROSE smear showed abundant cellular material with mostly isolated cells. The cells showed enlarged nuclei, prominent nucleoli and cytoplasmic vacuoles. The material was sufficient for diagnosis (working diagnosis: non-small cell metastatic lung carcinoma) (toluidine blue staining, 200×); (B) the same smear stained with Papanicolaou for definitive evaluation confirmed the ROSE diagnosis (Papanicolaou staining, 200×); (C) immunocytochemical expression for CK7 on the same de-stained slide was intense. The final cytological diagnosis was: lymph-node metastatic lung adenocarcinoma (CK7 immunostaining, 200×). In case of need, the same slide could be used for molecular analysis, either scraping the slides or using laser capture microdissection.

Figure 2

Histological biopsy of a lymph node with a metastasis from a pulmonary adenocarcinoma. Tumor cells are grouped in cluster (A, ematoxilin & eosin, 100×) and are positive for cytokeratin 7 (B, immunostaining, 100×) and TTF1 (C, immunostaining, 100×), whilst are negative for p40 (D, immunostaining, 100×).

Figure 3

Histological biopsy of a lymph node with a metastasis from a pulmonary squamous cell carcinoma. Tumor cells show the typical features of a squamous cell carcinoma (A, ematoxilin & eosin, 100×). The diagnosis is confirmed by the immunoreactivity for p40 (B, immunostaining, 100×) and the lack of TTF1 expression (C, immunostaining, 100×).

Figure 4

Example of a metastasis form a poorly differentiated neuroendocrine lung carcinoma. Neoplastic cells are atypical with hyperchromic nuclei (A, ematoxilin & eosin, 100×) and show chromogranin A immunoreactivity (B, immunostaining, 100×).

Figure 5

Trans-bronchial biopsy of a mediastinal enlarged lymph node. Neoplastic cells are monomorphic showing a lymphoid appearance (A, ematoxilin & eosin, 40×). They are positive for CD20 (B, immunostaining, 40×) and cyclin D1 (C, immunostaining, 40×), while are negative for CD3, which shows some residual non neoplastic T-lymphocytes (D, immunostaining, 40×). The final diagnosis was of mantle cell lymphoma.

Figure 6

Trans-bronchial biopsy of an enlarged lymph node. Normal tissue is replaced by epithelioid granulomas suggesting the diagnosis of sarcoidosis (ematoxilin & eosin, 40×).

Figure 7

EGFR pyrosequencing diagrams showing a case with an activating EGFR mutations in exon 21 (L858R) and a resistance mutation in exon 20 (T790M). Arrows show the point of the curve where the point mutation occurred.

Figure 8

Lung adenocarcinoma case with ALK determination. Smear specimen showing neoplastic epithelial cells arranged in pseudo-papillary structures (A, Giemsa stain, 200×). Cell block picture showing large amount of adenocarcinoma neoplastic tissue (B, ematoxilin & eosin, 100×). ALK immunohistochemistry (IHC) (clone 5A4) showing the majority of cells with moderate (2+) intensity and some cells with high (3+) intensity score (C, immunostaining 200×). ALK rearrangement was confirmed by FISH analysis (D, fluorochrome, 1,000×).