Single-cell RNA sequencing analysis of thyroid nodule fine needle biopsy aspiration

Objective

• Molecular testing with bulk RNA and DNA sequencing is used for thyroid nodule fine needle aspiration (FNA) biopsies with indeterminate cytology. While the sensitivity of these tests is excellent, specificity and prognostic information could be improved.
• Single cell RNA sequencing (scRNAseq) is a novel approach used to identify cell subpopulations in tumor microenvironments and has the potential to identify diagnostic and prognostic molecular data in cell clusters not identified in bulk sequencing.
• There is scarce data on the diagnostic potential for scRNAseq for thyroid cancer, especially from preoperative samples.
• We sought to determine the feasibility of using scRNAseq on thyroid nodule FNA samples.

Methods

1. Adults referred for thyroid nodule FNA biopsy or undergoing thyroidectomy were enrolled in the study.
2. FNA biopsies were performed under US guidance for a total of 3-4 FNA passes per nodule.
3. Material was collected for both bulk RNA seq (Afirma GSC) and scRNAseq in separate tubes.
4. Digestion and RBC lysis were optimized for scRNAseq with >70% cell viability and capture of follicular-derived cells.
5. Bioinformatics analysis was done using standard scRNAseq data analysis pipeline in Seurat package in R.

Results

Enrollment Data

• Total of 18 subjects were enrolled; 16 completed scRNAseq; 10 had high quality data (presence of follicular cells).
• Average age was 54.8 years.
• 6 women and 4 men.
• 4 biopsies were performed in clinic; 6 in OR (prior to neck incision).

ScRNAseq Data

Conclusions

• We developed a protocol that demonstrates the feasibility of scRNAseq on thyroid nodule FNA samples.
• Our scRNAseq analysis showed that most cells from FNA are immune cells, likely from peripheral blood. Importantly, we were able to differentiate benign and malignant samples by follicular cell clustering.
• Furthermore, our scRNAseq analysis showed that more aggressive cancers seem to demonstrate unique clusters with less differentiated cells, which could enhance FNA molecular prognosis.
• Future studies will evaluate the concordance of scRNAseq from different cell populations with the Afirma GSC bulk RNAseq results.