Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling.

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling. In this study, we evaluated the post-centrifuged supernatant from 25 malignant and 10 benign fine needle aspiration needle rinses. The mean and median DNA yields from the supernatants were 445 ng and 176.4 ng (range, 15.1-2958 ng), respectively. Next generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 detected somatic mutations in all 25 malignant samples. No mutations were detected in any of the benign samples tested. When available, mutations detected in the supernatant fluid were compared to the next generation sequencing analysis performed on a prior or concurrent surgical specimen from the same patient and showed 100% concordance. In a subset of cases (n = 19) mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS were successfully confirmed by droplet digital PCR, providing an orthogonal platform for mutation analysis. In summary, in this study we show that post centrifuged supernatants from fine needle aspiration needle rinses can provide a robust substrate for expanded mutation profiling by next generation sequencing, as well as hotspot mutation testing by droplet digital PCR. The ability to detect somatic mutations from otherwise discarded supernatant fluids offers the ability to triage and effectively utilize limited volume fine needle aspiration samples when multiple molecular tests are requested, without the need to re-biopsy for additional tissue samples.