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Updated: Aug 29, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Cell block-based RNA next generation sequencing for detection of gene fusions in lung adenocarcinoma: An
Shuanzeng Wei1, Jacqueline N Talarchek1, Min Huang1
1Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.
Objective:
Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test.
Methods:
Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS.
Results:
A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material.
Conclusions:
Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.
Insights
RNA next-generation sequencing (NGS) on cytology cell blocks reliably detects gene fusions in lung adenocarcinoma. This method offers a convenient and effective approach for molecular testing in clinical practice.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Targeted therapy for lung adenocarcinoma requires testing for specific gene fusions like EGFR, ALK, ROS1, RET, and NTRK.
- Traditional methods for detecting these gene fusions include fluorescence in situ hybridisation (FISH) and immunohistochemistry.
- The efficacy of RNA next-generation sequencing (RNA-NGS) and the utility of cytology cell blocks for this testing remain areas of investigation.
Purpose of the Study:
- To evaluate the accuracy of RNA-NGS in detecting gene fusions in pulmonary adenocarcinoma.
- To determine if cytology cell blocks are effective specimens for RNA-NGS-based gene fusion detection.
Main Methods:
- Retrospective retrieval of 129 lung adenocarcinoma cytology specimens from 2019-2022.
- Hybrid capture-based targeted RNA-NGS covering 507 fusion genes, including ALK, ROS1, RET, and NTRKs.
- Confirmation of RNA-NGS results using DNA NGS, FISH, and chromosomal microarray analysis.
Main Results:
- Overall success rate for RNA sequencing was 93% (120/129) from cytology cell blocks.
- Eight percent (10/120) of cases were positive for gene fusions (7 ALK, 2 ROS1, 1 RET).
- FISH testing on 22 cell blocks showed a 50% failure rate due to inadequate material, highlighting challenges with traditional methods.
Conclusions:
- Cytology cell blocks are a viable primary source for molecular testing in lung cancer.
- RNA-based NGS on cytology cell blocks provides a convenient and reliable method for detecting gene fusions in routine practice.
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