Epidermal Growth Factor Receptor Mutation and Anaplastic Lymphoma Kinase Gene Fusion: Detection in Malignant Pleural Effusion by RNA or PNA Analysis

• Published in: PloS one 2016-06, Vol.11 (6), p.e0158125-e0158125
• Main Authors: Chen, Yi-Lin, Lee, Chung-Ta, Lu, Cheng-Chan, Yang, Shu-Ching, Chen, Wan-Li, Lee, Yang-Cheng, Yang, Chung-Hsien, Peng, Shu-Ling, Su, Wu-Chou, Chow, Nan-Haw, Ho, Chung-Liang
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Adult; Aged; Aged, 80 and over; ALK protein; Analysis; Biology and Life Sciences; Biomarkers, Tumor - genetics; Biomarkers, Tumor - standards; Cancer therapies; Case-Control Studies; Cell Line, Tumor; Chemotherapy; Clinical medicine; Complementary DNA; Deoxyribonucleic acid; DNA; DNA sequencing; Enrichment; Epidermal growth factor; Epidermal growth factor receptors; Epidermal growth factors; Female; Gene Fusion; Gene sequencing; Genetic aspects; Hospitals; Humans; Lung cancer; Lung diseases; Lymphoma; Lymphomas; Male; Medical laboratories; Medical prognosis; Medicine; Medicine and Health Sciences; Middle Aged; Molecular Diagnostic Techniques - standards; Mutation; Oncology; Pathology; Peptide nucleic acids; Pleural effusion; Pleural Effusion, Malignant - genetics; Pleural Effusion, Malignant - metabolism; Pleural Effusion, Malignant - pathology; Pneumoviridae; Polymerase chain reaction; Protein-tyrosine kinase; Proteins; Receptor Protein-Tyrosine Kinases - genetics; Receptor, Epidermal Growth Factor - genetics; Research and analysis methods; Reverse transcription; Ribonucleic acid; Risk factors; RNA; Sensitivity analysis; Sensitivity and Specificity; Sequence Analysis, RNA - standards; Ship accidents & safety; Storms; Transcription (Genetics); Tumor cells; Workflow
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0158125

Analyzing EGFR mutations and detecting ALK gene fusion are indispensable when planning to treat pulmonary adenocarcinoma. Malignant pleural effusion (MPE) is a devastating complication of lung cancer and sometimes the only source for mutation analysis. The percentage of tumor cells in the pleural effusion may be low; therefore, mutant enrichment is required for a successful analysis. The EGFR mutation status in MPE was determined using three methods: (1) PCR sequencing of genomic DNA (direct sequencing), (2) mutant-enriched PCR sequencing of genomic DNA using peptide nucleic acid (PNA-sequencing), and (3) PCR sequencing of cDNA after reverse transcription for cellular RNA (RNA-sequencing). RT-PCR was also used to test cases for ALK gene fusion. PNA-sequencing and RNA-sequencing had similar analytical sensitivities (< 1%), which indicates similar enrichment capabilities. The clinical sensitivity in 133 cases when detecting the common EGFR exon 19 and exon 21 mutations was 56.4% (75/133) for direct sequencing, 63.2% (84/133) for PNA-sequencing, and 65.4% (87/133) for RNA-sequencing. RT-PCR and sequencing showed 5 cases (3.8%) with ALK gene fusion. All had wild-type EGFR. For EGFR analysis of MPE, RNA-sequencing is at least as sensitive as PNA-sequencing but not limited to specific mutations. Detecting ALK fusion can be incorporated in the same RNA workflow. Therefore, RNA is a better source for comprehensive molecular diagnoses in MPE.