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

<div><p>Analyzing <i>EGFR</i> mutations and detecting <i>ALK</i> 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 <i>EGFR</i> 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 <i>ALK</i> gene fusion.</p><p>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 <i>EGFR</i> 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 <i>ALK</i> gene fusion. All had wild-type <i>EGFR</i>. For <i>EGFR</i> analysis of MPE, RNA-sequencing is at least as sensitive as PNA-sequencing but not limited to specific mutations. Detecting <i>ALK</i> fusion can be incorporated in the same RNA workflow. Therefore, RNA is a better source for comprehensive molecular diagnoses in MPE.</p>