Studies of Benign and Malignant Thyroid Disease

背景：甲状腺微小癌（TMCs）是一种直径1cm的肿瘤，其特征是惰性。然而，高达10%的TMC可能存在临床显著的淋巴结（LNM）或远处转移（DM）。对易发生端粒酶的TMC的分子特征进行表征可能有助于制定管理方案。 方法：采用KAPA RNA HyperPrep Kit对95例组织标本的RNA进行全基因组测序。11个样品未通过质量检查。使用LCDB工作流程进行差异表达分析，调整后的p值0.1具有显著性。 结果：研究队列包括19例侧颈LNM和/或DM患者（N1 b组）和17例无转移患者（N 0/Nx组）。研究组之间的组织学分类（p=0.5）、中位原发性肿瘤大小（0.6 cm vs 0.8 cm，p=0.27）、年龄（39岁vs 46岁，p=0.54）、性别（p=0.32）无差异。pN 1b患者与pN 0/pNx患者的原发性肿瘤中有93个差异表达基因（DEG）（8个上调和85个下调; padj0.1）。N1 b组原发性肿瘤的特征是抗原结合免疫组分的显著下调（CD 19，CD 79 A，CR2; log 2FC>-2.6; padj<0.05），免疫球蛋白受体结合/结构（IGHV 1，3，4-家族，FCRLA; log 2FC>-2.6; padj<0.05）或在B细胞的发育和分化中（PAX 5，MS 4A 1; log 2FC>-2.7; padj<0.05），以及膜的组成成分（NUP 210 L，TMEM 145; log 2FC>2和padj<0.05）和胞外蛋白酶（ADAMTS 4; log 2FC>2; padj<0.05）的显著上调。 LNM与原发性肿瘤的配对分析显示518个DEG（322个上升和196个下降; padj 0.1）。LNM的特征是紧密连接蛋白编码基因的显著下调（CLDN 6，CLDN 4; log 2FC>-0.9; padj<0.001），电压门控钾通道的组分（KCNJ 12，KCNA 1; log 2FC>-0.8; padj<0.05），和G蛋白偶联受体信号传导（ARHGEF 35，GPRC 5A; log 2FC>-0.5; padj<0.05），而上调的基因包括非受体酪氨酸激酶家族成员（BLK，TXK; log 2FC>1.7; padj<0.01）、免疫组分（MS 4A 1，CR2，FCRL 2，FCMR; log 2FC>1.9; padj<0.01）和整合膜组分（TMEM 272，PCDH 15，SHISAL 2A; log 2FC>1.7; padj<0.01）。 结论：与典型的惰性TMC相比，与LNM和/或DM相关的TMC的特征在于失调的免疫景观，这表明免疫应答降低是转移的易感因素。转移性病变的特征在于作为进展标志之一的参与信号转导的紧密连接减少。

Background: Thyroid microcarcinomas (TMCs) are tumors measuring 1cm characterized by an indolent behavior. However, up to 10% of TMCs may present with clinically significant lymph node (LNM) or distant metastases (DM). The characterization of molecular signature of metastases-prone TMCs may help strategizing the management options. Methods: We performed whole transcriptome sequencing on the RNA extracted from 95 tissue samples, using KAPA RNA HyperPrep Kit. Eleven samples failed quality check. Differential expression analysis was performed using the LCDB workflow, with adjusted p-value0.1 rendered significant. Results: The study cohort consisted of 19 patients with lateral neck LNM and/or DM (N1b group) and 17 patients without metastases (N0/Nx). There was no difference in the histology breakdown (p=0.5), median primary tumor size (0.6cm vs 0.8cm, p=0.27), age (39y vs 46y, p=0.54), gender (p=0.32) between the study groups. There were 93 differentially expressed genes (DEGs) in the primary tumors of pN1b patients versus pN0/pNx (8-up and 85-down-regulated; padj0.1). Primary tumors from N1b group were characterized by a significant downregulation in antigen binding immune components (CD19, CD79A, CR2; log2FC>-2.6; padj<0.05), immunoglobulin receptor binding/structure (IGHV1,3,4-family, FCRLA; log2FC>-2.6; padj<0.05) or in the development and differentiation of B-cells (PAX5, MS4A1; log2FC>-2.7; padj<0.05), and a significant upregulation of integral components of the membrane (NUP210L, TMEM145; log2FC>2 and padj<0.05) and extracellular protease (ADAMTS4; log2FC>2; padj<0.05). The paired analysis of LNM vs primary tumor revealed 518 DEGs (322-up and 196-down; padj 0.1). LNM were characterized by a significant downregulation of the genes coding for tight junction proteins (CLDN6, CLDN4; log2FC>-0.9; padj<0.001), components of voltage-gated potassium channels (KCNJ12, KCNA1; log2FC>-0.8; padj<0.05), and G-protein coupled receptor signaling (ARHGEF35, GPRC5A; log2FC>-0.5; padj<0.05), while the up-regulated genes included members of non-receptor tyrosine kinase families (BLK, TXK; log2FC>1.7; padj<0.01), immune components (MS4A1, CR2, FCRL2, FCMR; log2FC>1.9; padj<0.01) and integral membrane components (TMEM272, PCDH15, SHISAL2A; log2FC>1.7; padj<0.01). Conclusions: TMCs associated with LNM and/or DM are characterized by dysregulated immune landscape compared with classic indolent TMCs, suggesting a decreased immune response as a factor predisposing to metastases. Metastatic lesions are characterized by decreased tight junctions that are involved in signal transduction as one of the hallmarks of progression.