儿童或随后的第二原发性肿瘤中散发性第一原发性肿瘤的病因和大多数危险因素仍然未知。与治疗相关的第二原发性肿瘤的一个确定的因果因素是在放射治疗期间暴露于电离辐射作为癌症治疗的支柱。在德国首次诊断后的30年内,所有癌症幸存者中有8%发生第二原发性肿瘤,但是内在敏感性的潜在因素尚未明确。因此,这项巢式病例对照研究的目的是调查和比较仅患有第一原发性肿瘤或至少有一个随后的第二原发性肿瘤的儿童癌症幸存者的原发性成纤维细胞中的基因表达和受影响的途径。和暴露于低剂量和高剂量电离辐射后没有肿瘤的对照。
原代成纤维细胞是从52名成人捐献者的皮肤活检中获得的,这些捐献者在儿童时期有首次原发性肿瘤(N1),52与至少一个额外的原发性肿瘤(N2+),以及来自KiKme研究的52例无癌(N0)。将培养的成纤维细胞暴露于高[2Gray(Gy)]和低剂量(0.05Gy)的X射线。暴露后4小时提取信使核糖核酸并进行Illumina测序。差异表达基因(DEGs)使用limma计算R,在0.05的错误发现率水平下选择,并使用IngenuityPathwayAnalysis进一步分析途径富集(右尾Fisher精确检验)和(in-)激活(z≥|2|)。
0.05Gy后,在N0中发现最小DEG(n=236),与N1(n=653)和N2(n=694)相比。在所有供体组的成纤维细胞中,关于调整后的p值的顶部DEGs上调(SESN1,MDM2,CDKN1A,TIGAR,BTG2,BLOC1S2,PPM1D,PHLDB3,FBXO22,AEN,TRIAP1和POLH)。这里,我们在N0中观察到p53信号的激活,在N1中的程度较小,但在N2中没有。仅在N0中,DNA(切除-)修复(涉及的基因:CDKN1A,PPM1D,和DDB2)被预测为下游函数,N2+中的分子网络与癌症有关,以及伤害和异常(除其他外,下调MSH6、CCNE2和CHUK)。2Gy后,所有供体组的成纤维细胞中DEGs的数量相似,并且具有最高绝对log2倍数变化的基因在整个过程中上调(CDKN1A,TIGAR,HSPA4L,MDM2,BLOC1SD2,PPM1D,SESN1,BTG2,FBXO22,PCNA,和TRIAP1)。这里,p53信号通路在所有供体组成纤维细胞中均被激活.在N1和N2中,Polo样激酶途径的有丝分裂作用被灭活。癌症的分子机制在所有供体组成纤维细胞中均受影响。预测P53是所有供体组的成纤维细胞和N1和N2+中的E2F1的上游调节因子。下游分析结果为N0和N2+衰老,转化细胞在N0,在N1无显著影响。7个基因在对2Gy的反应中差异表达,取决于供体组(LINC00601,COBLL1,SESN2,BIN3,TNFRSF10A,EEF1AKNMT,和BTG2)。
我们的结果显示N1/N2+和N0之间辐射响应的剂量依赖性差异。虽然在所有组中高剂量后,针对遗传毒性应激的机制被激活到相同的程度,在N1/N2+低剂量后辐射反应受损,提示不良反应的风险增加,包括致癌作用,特别是在N2+。
The etiology and most risk factors for a sporadic first primary neoplasm in childhood or subsequent second primary neoplasms are still unknown. One established causal factor for therapy-associated second primary neoplasms is the exposure to ionizing radiation during radiation therapy as a mainstay of cancer treatment. Second primary neoplasms occur in 8% of all cancer survivors within 30 years after the first diagnosis in Germany, but the underlying factors for intrinsic susceptibilities have not yet been clarified. Thus, the purpose of this nested case-control
study was the investigation and comparison of gene expression and affected pathways in primary fibroblasts of childhood cancer survivors with a first primary neoplasm only or with at least one subsequent second primary neoplasm, and controls without neoplasms after exposure to a low and a high dose of ionizing radiation.
Primary fibroblasts were obtained from skin biopsies from 52 adult donors with a first primary neoplasm in childhood (N1), 52 with at least one additional primary neoplasm (N2+), as well as 52 without cancer (N0) from the KiKme
study. Cultured fibroblasts were exposed to a high [2 Gray (Gy)] and a low dose (0.05 Gy) of X-rays. Messenger ribonucleic acid was extracted 4 h after exposure and Illumina-sequenced. Differentially expressed genes (DEGs) were computed using limma for R, selected at a false discovery rate level of 0.05, and further analyzed for pathway enrichment (right-tailed Fisher\'s Exact Test) and (in-) activation (z ≥|2|) using Ingenuity Pathway Analysis.
After 0.05 Gy, least DEGs were found in N0 (n = 236), compared to N1 (n = 653) and N2+ (n = 694). The top DEGs with regard to the adjusted p-value were upregulated in fibroblasts across all donor groups (SESN1, MDM2, CDKN1A, TIGAR, BTG2, BLOC1S2, PPM1D, PHLDB3, FBXO22, AEN, TRIAP1, and POLH). Here, we observed activation of p53 Signaling in N0 and to a lesser extent in N1, but not in N2+. Only in N0, DNA (excision-) repair (involved genes: CDKN1A, PPM1D, and DDB2) was predicted to be a downstream function, while molecular networks in N2+ were associated with cancer, as well as injury and abnormalities (among others, downregulation of MSH6, CCNE2, and CHUK). After 2 Gy, the number of DEGs was similar in fibroblasts of all donor groups and genes with the highest absolute log2 fold-change were upregulated throughout (CDKN1A, TIGAR, HSPA4L, MDM2, BLOC1SD2, PPM1D, SESN1, BTG2, FBXO22, PCNA, and TRIAP1). Here, the p53 Signaling-Pathway was activated in fibroblasts of all donor groups. The Mitotic Roles of Polo Like Kinase-Pathway was inactivated in N1 and N2+. Molecular Mechanisms of Cancer were affected in fibroblasts of all donor groups. P53 was predicted to be an upstream regulator in fibroblasts of all donor groups and E2F1 in N1 and N2+. Results of the downstream analysis were senescence in N0 and N2+, transformation of cells in N0, and no significant effects in N1. Seven genes were differentially expressed in reaction to 2 Gy dependent on the donor group (LINC00601, COBLL1, SESN2, BIN3, TNFRSF10A, EEF1AKNMT, and BTG2).
Our results show dose-dependent differences in the radiation response between N1/N2+ and N0. While mechanisms against genotoxic stress were activated to the same extent after a high dose in all groups, the radiation response was impaired after a low dose in N1/N2+, suggesting an increased risk for adverse effects including carcinogenesis, particularly in N2+.