Targeted high throughput RNA sequencing for detection of gene fusions and gene expression markers in thyroid cancer

Background. Detection of fusion genes is important in the preoperative differential diagnostics of thyroid cancer, as well as for tyrosine kinase inhibitors prescription. It is assumed that the assessment of the 5’/3’ expression imbalance, which indirectly reflects the presence of gene rearrangements, and gene expression markers, might widen the range of markers and would thereby improve the sensitivity of the malignant thyroid tumors diagnostics. High throughput RNA sequencing, which allows determining the full range of rearrangements in combination with expression markers, is supposed to be promising application and is under active implementation into clinical practice.

Aim. To assess the detection of rearrangements and expression markers by targeted high throughput RNA sequencing in thyroid cancer.

Methods. 64 samples of thyroid cancer and 16 samples of benign thyroid lesions with a cytological diagnosis of Bethesda III-V were examined. Presence of RET, ALK, NTRK1, NTRK3, PPARG, THADA, LTK, MET, BRAF, C15orf55, ERBB4, OFD1, ROS1 gene rearrangement transcripts, 5’/3’ gene expression imbalance and expression marker levels were evaluated by high throughput sequencing using AmpliSeq technology on the custom primer panel.

Results. Fusion genes were found in 12% of cancer samples. Their list includes CCDC6-RET, ETV6-NTRK3, TPM3-NTRK1, STRN-ALK, and PAX8-PPARG. The proportion of samples with identified rearrangements meets the values expected according to the literature. No rearrangements were found in the benign samples. 5’/3’ imbalance in cancer samples was detected for the RET, NTRK1, NTRK3, MET, and THADA genes, while it was absent in benign lesions. Among the studied expression markers, the KRT7, KRT20, CHGA, CITED1 genes had significant overexpression in cancer samples, with no aberrant expression in benign neoplasms.

Conclusions. Targeted high throughput RNA sequencing based on AmpliSeq technology using the developed primer panel allows to determine gene fusions, 5’/3’ expression imbalance and expression markers with high specificity regarding the malignancy of the neoplasm. Inclusion of 5’/3’ expression imbalance and gene expression markers can improve the accuracy of differential diagnostics of malignant neoplasms.

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