Application of molecular diagnostics in thyroid cancer

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Background: Struma Ovarii (SO) is a rare ovarian teratoma characterized by the presence of thyroid tissue in >50% of the tumor. Majority of SO are benign; however, malignant transformation occurs in up to 5% of the cases. The molecular foundations of benign and malignant SO are grossly unknown. Therefore, the goal of this study was to perform the first comprehensive genomic and transcriptomic analysis of the benign and malignant SO. Material and method: We performed whole-exome sequencing (WES) and targeted RNA-sequencing (seq) on the DNA and RNA extracted from formalin-fixed paraffin-embedded SO tumor tissue samples. WES library was prepared using Agilents SureSelect XT HS2 kit, with 4 samples failing the quality assessment (QA). Variants were called from GATK processed WES data and annotated using VEP (with ClinVar and COSMIC databases). The targeted RNA-Seq library was prepared using the TruSight RNA Pan-Cancer Panel kit covering 1385 cancer genes, with all samples passing QA. The clinical characteristics of the study cohort were summarized by percentages for categorical variables and medians with 25-75% interquartile ranges for continuous variables. Results: The study included 31 tissue samples 21 benign and 10 malignant, including 6 cases of papillary thyroid cancer (PTC), 3 of follicular variant of PTC and 1 of follicular thyroid cancer. Patients with benign SO were characterized by the median age at diagnosis of 39 years 33-54, tumor size of 3.1 cm 2.5-5.8, while the patients with malignant SO presented at age of 45 28-54, tumor size of 6 cm 0.85-14 and metastatic disease in 30% (3/10) 2 patients with peritoneal metastases and 1 patient with pelvic lymph node metastases. The E1A Binding Protein P300, EP300 (6/27) and Isocitrate dehydrogenase, IDH2 (5/27) were the topmost mutated genes in the SO samples. Malignant SO samples were characterized with the presence of pathogenic variants of KRAS (pQ61L and pG12V), NRAS (pQ61R), TP53 (splice site) mutations and Nuclear Receptor Binding SET Domain Protein 1 (NSD1) fusion as the most common molecular drivers. Among benign SO samples, the most common driver was Thyroglobulin (TG) fusion with either Guanine Nucleotide binding protein (GNAS) or Rac Family Small GTPase 1 (RAC1). Differential expression analysis showed that the member of tumor suppressor family - tumor protein 63 (TP63) was the most downregulated (Log2FC = -3), while Double-sex and Mab-3 Related Transcription Factor 1 (DMRT1), implicated in the development of germ cell tumors, was the most upregulated gene in malignant SO samples over benign (Log2FC = 2.1; padj<0.05). Conclusions: In contrast to cancer arising from the thyroid gland, characterized by BRAFV600E as the most common mutation, malignant SO belongs to RAS-like tumors. The downregulation of tumor suppressors and upregulation of DMRT1 might be implicated in the malignant transformation of SO.