Molecular profiling of breast cancer pleural effusions using cytology specimens: Impact of sample source and cytological features on next‐generation sequencing performance

BACKGROUND: Malignant effusions in breast cancer are a minimally invasive source for molecular profiling. Effusion-derived cytology specimens, cell blocks (CB) and post-ThinPrep PreservCyt (PTPC) supernatant, are reliable sources for molecular testing but differ in DNA concentration and turnaround time (TAT). This study evaluates mutational profiles, cytology characteristics, and clinical correlates to assess the utility of effusion-based molecular testing in breast cancer. METHODS: The authors screened 2400 malignant effusion cases, identifying 22 cases of breast adenocarcinoma effusions that underwent next-generation sequencing (NGS). Both CB and PTPC supernatant were evaluated for tumor cellularity, fluid volume, DNA concentration, TAT, and mutation detection. Associations with clinicopathological features and outcomes were explored. RESULTS: NGS was successful in all 22 cases (mean volume, 426.5 mL; mean tumor cellularity, 50%). Successful sequencing was achieved in low volume samples with high tumor cellularity. Higher tumor cellularity (>50%) was associated with bloody effusions (p = .016). PTPC supernatant yielded 5-fold higher DNA (mean 177.4 ng/µL vs. 33.8 ng/µL) and shorter TAT by 9 days (15 vs. 24 days). TP53 mutations were more common in estrogen receptor (ER)-negative and triple-negative breast effusions (p = .025). Mutations in PIK3CA, TP53, CCND1, and AKT were reliably detected. Time from diagnosis to effusion development was significantly shorter in ER negative disease (p = .001) CONCLUSIONS: Effusion cytology using PTPC supernatant, support successful NGS even in low-volume samples. ER-negative and triple-negative effusions demonstrate more aggressive molecular features including enrichment of TP53 mutations and earlier effusion development. Effusion-based molecular testing yields clinically relevant genomic information in advanced breast cancer.