Utility of Fine Needle Aspiration Supernatant for Real-Time PCR-Based Mutation Analysis in Non-Small Cell Lung Cancer

The emergence of personalized therapy and molecular sequencing has significantly transformed the management of advanced non-small cell lung carcinoma (NSCLC). Supernatants obtained from fine needle aspiration (FNA) have been shown to be a reliable source of high-quality nucleic acid for molecular testing. One pre-analytic factor that may influence the success of molecular analysis is the collection medium used for the FNA rinse.

This prospective study evaluated 31 FNA specimens collected in three different media-CytoRich Red, 10% neutral buffered formalin, or 10% bovine serum albumin (BSA)-to assess their impact on DNA yield and real-time polymerase chain reaction (RT-PCR) outcomes for KRAS (Kirsten rat sarcoma viral oncogene homolog) and/or EGFR (epidermal growth factor receptor) mutations. DNA extraction was performed using the QIAamp DNA FFPE (formalin-fixed paraffin-embedded) Tissue Kit (Qiagen), and RT-PCR was conducted using the QIAGEN therascreen EGFR or KRAS RGQ PCR kits. Concurrent direct smears or formalin-fixed, paraffin-embedded tissue from a cell block or core biopsy served as standard controls.

In the CytoRich Red group, 11 specimens (9 adenocarcinomas and 2 NSCLC) were analyzed. All 11 specimens generated results concordant with standard controls. One specimen required repeat DNA extraction due to initial pre-test quality control failure. The formalin group included 12 specimens (7 squamous cell carcinomas [SCC], 3 adenocarcinomas, 1 NSCLC, and 1 large cell neuroendocrine carcinoma). Nine specimens produced concordant results with controls. Two specimens were negative for EGFR mutations, although the corresponding control tissues were not tested. Six required repeat DNA extraction because of initial pre-test failure, and one specimen remained invalid after re-extraction. Eight specimens were collected in BSA (5 adenocarcinomas, 2 NSCLC, and 1 SCC). Five produced concordant results with standard controls, while three specimens failed PCR pre-tests despite repeat DNA extraction.

In this small cohort, CytoRich Red demonstrated reliable performance for RT-PCR-based mutation testing. FNA supernatant represents a practical alternative specimen for lung cancer molecular analysis and may optimize specimen utilization while preserving cell block material.