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.

BACKGROUND: Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid-based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from thyroid FNA samples (n = 156) were evaluated. A 50-gene next-generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. RESULTS: All 156 samples yielded adequate DNA (median, 135 ng; range, 11-3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow-up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n = 8) showed 100% concordance. CONCLUSIONS: This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management.

Introduction: Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations.Methods: Supernatants from lung carcinoma FNA samples (n ¼ 150) were evaluated. Samples were further analyzed using nextgeneration sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n ¼ 67) and available plasma liquid biopsy assay (n ¼ 45).Results: All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance.Conclusions: FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

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