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

Abstract

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.

Molecular testing of residual cytology samples: Rethink, reclaim, repurpose

“…One hypothesis that has been proposed is that, due to the high turnover of tumor cells, the supernatant fluid is enriched in tumor‐derived cell‐free DNA . However, a qualitative analysis of the DNA extracted from supernatant fluid samples in our previous study demonstrated that the majority of the DNA retrieved from these samples comprised high molecular weight DNA, suggestive of cellular DNA, with only a small component likely representing cell‐free DNA . Therefore, our hypothesis is that the supernatant fluid DNA is likely derived from tumor cells that are lysed during the FNA procedure or during processing of the needle rinse at the time of centrifugation.…”

“…15 However, a qualitative analysis of the DNA extracted from supernatant fluid samples in our previous study demonstrated that the majority of the DNA retrieved from these samples comprised high molecular weight DNA, suggestive of cellular DNA, with only a small component likely representing cell-free DNA. 18 Therefore, our hypothesis is that the supernatant fluid DNA is likely derived from tumor cells that are lysed during the FNA procedure or during processing of the needle rinse at the time of centrifugation. The DNA yield and variant allelic frequency observed on NGS in our study 18 correlated with the tumor percentage noted in the cellular component of the FNA.…”

“…18 Therefore, our hypothesis is that the supernatant fluid DNA is likely derived from tumor cells that are lysed during the FNA procedure or during processing of the needle rinse at the time of centrifugation. The DNA yield and variant allelic frequency observed on NGS in our study 18 correlated with the tumor percentage noted in the cellular component of the FNA. Hence, the cellularity of the FNA smears may be used as a surrogate for correlation when interpreting a negative mutational analysis result; however, further studies using larger numbers of cases are needed for confirmation.…”

Ancillary studies in cytology: Secondary or necessary?

“…For example, one recent study rigorously analyzed NGS of cytologic direct smears or liquid‐based preparations and found them to be comparable to FFPE core biopsies or cell blocks . Another intriguing potential sample source for NGS is the supernatant from fine needle aspiration needle rinses …”

“…25 Another intriguing potential sample source for NGS is the supernatant from fine needle aspiration needle rinses. 26…”