Studies of Benign and Malignant Thyroid Disease

背景：甲状腺微小癌(TMCs)是一种直径为1 cm的肿瘤，以惰性行为为特征。然而，多达10%的TMC可能出现临床显著的淋巴转移(LNM)或远处转移(DM)。易于转移的TMC分子特征的表征可能有助于制定治疗方案。 方法：采用KAPA RNAHyperPrep试剂盒对95例组织标本中提取的RNA进行全序列测定。有11件样品质量检验不合格。使用LCDB工作流程进行差异表达分析，调整后的p值0.1显著。 结果：研究队列包括19例颈外侧淋巴结转移和/或糖尿病患者(N1b组)和17例无转移的患者(N0/NX组)。两组在组织学分类(p=0.5)、原发肿瘤中位大小(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)方面没有差异。在pN1b患者的原发肿瘤中有93个差异表达基因(Deg)，而pN0/pnx(8个上调和85个下调；padj0.1)。N1b组原发肿瘤的特征是抗原结合免疫成分(CD19，CD79A，CR2；log2FC&gt；-2.6；PADJ&lt；0.05)、免疫球蛋白受体结合/结构(IGH71，3，4-Family，FCRLA；log2FC&gt；-2.6；padj&lt；0.05)或B细胞的发育和分化(PAX5，MS4A1；log2FC&gt；-2.7；padj&lt；0.05)显著上调，膜整体成分(NUP210L，TMEM145；log2FC&gt；0.05)显著上调。和胞外蛋白酶(ADAMTS4；log2FC&gt；2；padj&lt；0.05)。 对LNM和原发肿瘤的配对分析显示518°(向上322°和向下196°；PADJ0.1)。LNM的特征是编码紧密连接蛋白(CLDN6，CLDN4；log2FC&gt；-0.9；padj&lt；0.001)、电压门控钾通道组件(KCNJ12，KCNA1；log2FC&gt；-0.8；padj&lt；0.05)和G蛋白偶联受体信号(ARHGEF35，GPRC5A；log2FC&gt；-0.5；padj&lt；0.05)的基因显著下调，而上调的基因包括非受体酪氨酸激酶家族成员(BLK，TXK；log2FC&gt；1.7；padj&lt；免疫成分(MS4A1、CR2、FCRL2、FCMR；log2FC&gt；1.9；PADJ&lt；0.01)和整膜成分(TMEM272、PCDH15、SHISAL2A；log2FC&gt；1.7；PADJ&lt；0.01)。 结论：与典型的惰性TMCs相比，与LNM和/或DM相关的TMCs具有免疫功能紊乱的特点，提示免疫反应降低是易发生转移的一个因素。转移性病变的特征是参与信号转导的紧密连接减少，这是进展的标志之一。

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.