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Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Targeted Gene Next-Generation Sequencing Panel in Patients with Advanced Lung Adenocarcinoma: Paving the Way for
Maria Gabriela O Fernandes1,2,3, Maria Jacob4, Natália Martins4,5,6
1Pulmonology Department, Centro Hospitalar Universitário de São João, Alameda Prof. Hernani Monteiro, 4200-319 Porto, Portugal. gfernandes@med.up.pt.
Abstract:
Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.
Insights
Next-Generation Sequencing (NGS) is superior to traditional Sanger sequencing and FISH for detecting actionable genomic mutations in advanced lung adenocarcinoma. NGS significantly reduces the unclassifiable rate, improving treatment selection for patients.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Accurate molecular profiling is critical for targeted therapy selection in advanced lung adenocarcinoma.
- Traditional methods like Sanger sequencing and FISH have limitations in comprehensive genomic mutation detection.
Purpose of the Study:
- To compare the efficacy of Next-Generation Sequencing (NGS) against Sanger sequencing and Fluorescence In Situ Hybridization (FISH) for identifying actionable genomic alterations.
- To evaluate the clinical utility of NGS in both experimental and real-world clinical settings for advanced lung adenocarcinoma.
Main Methods:
- A sequential approach using Sanger sequencing and FISH was compared with a comprehensive NGS-based approach in an experimental cohort (EC) of 117 patients.
- NGS was subsequently implemented in a clinical practice cohort (IC) of 123 patients using a combined DNA and RNA panel.
- Assessment included identification of gene variants, translocations, and mutations, alongside concordance analysis and impact on clinical outcomes.
Main Results:
- NGS identified at least one gene variant in 47.9% of the EC, significantly reducing the unclassifiable rate by 24.8% compared to Sanger/FISH.
- In the EC, NGS detected various mutations including EGFR, KRAS, BRAF, ERBB2, STK11, TP53, and ALK, with 12% presenting synchronous alterations.
- In the IC, NGS detected actionable genomic variants in 64.2% of patients, including EML4-ALK, KIF5B-RET, CD74-ROS1, TPM3-NTRK translocations, and MET mutations, with 36% being treatable alterations.
Conclusions:
- Next-Generation Sequencing (NGS) demonstrates superior performance in detecting actionable genomic alterations in advanced lung adenocarcinoma.
- NGS significantly improves the molecular classification of patients, thereby facilitating more precise and effective targeted therapy selection.
- The study strongly supports the adoption of NGS as a first-line diagnostic tool for comprehensive genomic profiling in advanced lung adenocarcinoma.
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