Application of molecular diagnostics in thyroid cancer

背景：卵巢甲状腺肿是一种罕见的卵巢畸胎瘤，其特征是50%的肿瘤内有甲状腺组织存在。大多数SO是良性的；然而，高达5%的病例会发生恶变。良性和恶性SO的分子基础尚不清楚。因此，这项研究的目标是对良性和恶性SO进行第一次全面的基因组和转录分析。 材料和方法：我们对福尔马林固定石蜡包埋的SO肿瘤组织提取的DNA和RNA进行了全外显子测序(WES)和靶向RNA测序(SEQ)。WES文库使用Agilents SureSelect XT HS2试剂盒制备，有4个样品没有通过质量评估(QA)。从GATK处理的WES数据中调用变体，并使用VEP(与ClinVar和COSMIC数据库)进行注释。目标RNA-Seq文库是使用TruSight RNA泛癌小组试剂盒制备的，涵盖1385个癌症基因，所有样本都通过了QA。研究队列的临床特征通过分类变量的百分比和连续变量的中位数(四分位数范围为25%-75%)来总结。 结果：共收集31例甲状腺组织标本，其中良性病变21例，恶性病变10例，其中乳头状癌6例，滤泡型甲状腺癌3例，滤泡性甲状腺癌1例。良性SO的发病年龄中位数为39岁~33~54岁，肿瘤大小为3.1 cm 2.5~5.8；恶性SO的发病年龄为45~28~54岁，肿瘤大小为6 cm~0.85~14，转移率为30%(3/10)，其中腹膜转移2例，盆腔淋巴结转移1例。E1a结合蛋白P300、EP300(6/27)和异柠檬酸脱氢酶IDH2(5/27)是SO患者中突变最多的基因。恶性SO以致病变异KRAS(pQ61L和pG12V)、NRAS(PQ61R)、TP53(剪接位点)突变和核受体结合集结构域蛋白1(NSD1)融合为最常见的分子驱动因素。在良性SO样本中，最常见的驱动因素是甲状腺球蛋白(TG)与鸟核苷酸结合蛋白(GNAS)或RAC家族小GTPase 1(RAC1)的融合。 差异表达分析显示，抑癌基因家族成员肿瘤蛋白63(TP63)下调幅度最大(Log2FC=-3)，而与生殖细胞肿瘤发生有关的双性和单抗-3相关转录因子1(DMRT1)在恶性SO中表达最高(Log2FC=2.1；PADJ&lt；0.05)。 结论：与以BRAFV600E为最常见突变特征的甲状腺癌不同，恶性So属于RAS样瘤。抑癌基因的下调和DMRT1的上调可能参与了SO的恶变过程。

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.