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Updated: Jun 8, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
EML4-ALK fusion gene assessment using metastatic lymph node samples obtained by endobronchial ultrasound-guided
Yuichi Sakairi1, Takahiro Nakajima, Kazuhiro Yasufuku
1Division of Thoracic Diseases, Chiba Cancer Center, Chiba, Japan.
Purpose:
Anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes for non-small cell lung cancers (NSCLC). Several ALK inhibitors have been developed, and are now being evaluated in ALK-positive NSCLC. The feasibility of detecting ALK fusion genes in samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was determined. The clinicopathologic characteristics of ALK-positive lung cancer were also analyzed.
Experimental Design:
From April 2008 to July 2009, NSCLC cases with hilar/mediastinal lymph node metastases detected by EBUS-TBNA were enrolled. Positive expression of ALK fusion protein was determined using immunohistochemistry, and ALK gene rearrangements were further examined to verify the translocation between ALK and partner genes using fluorescent in situ hybridization and reverse transcription-PCR. Direct sequencing of PCR products was performed to identify ALK fusion variants.
Results:
One hundred and nine cases were eligible for the analysis using re-sliced samples. Screening of these specimens with immunohistochemistry revealed ALK positivity in seven cases (6.4%), all of which possessed echinoderm microtubule-associated protein-like 4-ALK fusion genes as detected by fluorescent in situ hybridization and reverse transcription-PCR. All ALK-positive cases had an adenocarcinoma histology and possessed no EGFR mutations. Compared with ALK-negative cases, ALK-positive cases were more likely to have smaller primary tumors (P < 0.05), to occur at a younger age (<60 years; P < 0.05), and to occur in never/light smokers (smoking index < 400; P < 0.01). Mucin production was frequently observed in ALK-positive adenocarcinomas (29.4%; P < 0.01).
Conclusions:
EBUS-TBNA is a practical and feasible method for obtaining tissue from mediastinal and hilar lymph nodes that can be subjected to multimodal analysis of ALK fusion genes in NSCLC.
Insights
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a feasible method for detecting anaplastic lymphoma kinase (ALK) fusion genes in non-small cell lung cancer (NSCLC). This technique aids in identifying ALK-positive NSCLC, crucial for targeted therapy.
Area of Science:
- Oncology
- Pulmonology
- Molecular Diagnostics
Background:
- Anaplastic lymphoma kinase (ALK) fusion genes are key oncogenic drivers in a subset of non-small cell lung cancer (NSCLC).
- Targeted therapies inhibiting ALK are effective treatments for ALK-positive NSCLC.
- Accurate detection of ALK fusions is critical for patient selection.
Purpose of the Study:
- To assess the feasibility of detecting ALK fusion genes using samples from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA).
- To analyze the clinicopathologic characteristics of ALK-positive NSCLC identified through EBUS-TBNA.
Main Methods:
- One hundred and nine NSCLC cases with lymph node metastases identified via EBUS-TBNA were analyzed.
- Immunohistochemistry (IHC) was used for initial ALK fusion protein screening.
- Fluorescent in situ hybridization (FISH) and reverse transcription-PCR (RT-PCR) confirmed ALK gene rearrangements and identified fusion variants.
Main Results:
- ALK positivity was detected in 6.4% (7/109) of cases via IHC, confirmed by FISH and RT-PCR.
- All ALK-positive cases exhibited echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) fusions.
- ALK-positive NSCLC cases were associated with adenocarcinoma histology, absence of EGFR mutations, smaller tumors, younger age, and never/light smoking history.
Conclusions:
- EBUS-TBNA is a practical and feasible method for obtaining mediastinal and hilar lymph node tissue for multimodal ALK fusion gene analysis in NSCLC.
- This approach facilitates the identification of patients who may benefit from ALK-targeted therapies.
