数字PCR检测循环肿瘤DNA中HER2基因扩增对胃癌的预后评估

Prognostic assessment of gastric cancer by detection of HER2 gene amplification in circulating tumor DNA by digital PCR

目的探讨基于数字PCR(d PCR)检测循环肿瘤DNA(ctDNA)中人类表皮生长因子受体2(HER2)基因扩增在胃癌进展监测及预后评估的临床应用价值。方法选取2018年11月至2020年12月华中科技大学协和深圳医院收治的60例胃癌患者为病例组,以同期20名健康体检者为对照组。建立dPCR技术方法检测胃癌患者ctDNA中HER2基因扩增及阳性阈值,评价其与免疫组织化学(IHC)联合荧光原位杂交技术(FISH)病理诊断HER2表达结果一致性,分析胃癌患者术前ctDNA中HER2基因扩增与临床病理特征的关系。同时选择24例Ⅲ、Ⅳ期的进展期胃癌患者纳入随访研究,分析术后监测ctDNA中HER2基因扩增与无进展生存时间的关系。结果HER2基因扩增比值的阳性阈值设为1.40,dPCR技术定量检测ctDNA中HER2基因扩增的敏感度、特异性分别达到0.90、0.94;胃癌患者dPCR检测HER2基因扩增、IHC与FISH联合检测HER2表达阳性率分别为20%(12/60)、16.7%(10/60),两种方法检测结果符合率一致性较好(k=0.778,P<0.01)。dPCR检测胃癌患者术前ctDNA的HER2基因扩增阳性患者的肿瘤组织高-中分化、远处转移比例明显高于HER2阴性者,差异有统计学意义(P<0.05)。进展转移组患者ctDNA中HER2基因扩增比值为(1.88±0.76),明显高于无进展转移组的(1.14±0.42),差异有统计学意义(P<0.05)。HER2基因扩增阳性患者的中位无进展生存时间(PFS)为8.5月,明显低于HER2阴性患者的18.9月,风险比率(hazard ratio,HR)为0.321(95%CI:0.12,0.90),差异有统计学意义(P<0.05)。结论利用dPCR技术不仅可以定量检测ctDNA中HER2基因扩增比值水平,还适用于晚期或进展转移胃癌患者术后监测HER2基因扩增变化,对于胃癌进展及预后评估具有一定临床应用意义。

Objective To investigate the clinical application of human epidermal growth factor receptor 2(HER2)gene amplification in circulating tumor DNA(ctDNA)based on digital PCR(dPCR)in gastric cancer progression monitoring and prognosis evaluation.Methods The 60 gastric cancer patients admitted to Huazhong University of Science and Technology Union Shenzhen Hospital from November 2018 to December 2020 were selected as the case group,and 20 healthy subjects in the same period were selected as the control group.Digital PCR method was established to detect HER2 gene amplification and positive threshold in ctDNA of gastric cancer patients,and its consistency with immunohistochemistry(IHC)combined with fluorescence in situ hybridization(FISH)pathological diagnosis of HER2 expression was evaluated.The relationship between HER2 gene amplification and clinicopathological features in preoperative ctDNA of patients with gastric cancer was analyzed.Meanwhile,24 patients with stageⅢandⅣadvanced gastric cancer were included in the follow⁃up study to analyze the relationship between HER2 gene amplification in postoperative ctDNA and progression⁃free survival time.Results The positive threshold of the HER2 gene amplification ratio was set at 1.40.The sensitivity and specificity of HER2 gene amplification in ctDNA were 0.90 and 0.94,respectively.The positive rate of HER2 gene amplification detected by dPCR and HER2 expression by IHC combined with FISH was 20%(12/60)and 16.7%(10/60),respectively.The coincidence rate of the two methods was good(k=0.778,P<0.01).The proportion of tumor tissues with high⁃moderate differentiation and distant metastasis in gastric cancer patients with preoperative ctDNA positive amplification of HER2 gene detected by dPCR was significantly higher than that in HER2⁃negative patients,and the difference was statistically significant(P<0.05).The ratio of HER2 gene amplification in the ctDNA in the patients with progressive metastasis was(1.88±0.76),which was significantly higher than that in the non⁃progressive metastasis group(1.14±0.42),and the difference was statistically significant(P<0.05).The median progression⁃free survival(PFS)of HER2 gene amplification⁃positive patients was 8.5 months,which was significantly lower than that of HER2⁃negative patients of 18.9 months,with a hazard ratio(HR)of 0.321(95%CI:0.12,0.90),The difference was statistically significant(P<0.05).Conclusion Using dPCR technology can not only quantitatively detect the HER2 gene amplification ratio level in ctDNA,but also is suitable for postoperative monitoring of HER2 gene amplification changes in patients with advanced or advanced metastatic gastric cancer,which has certain clinical significance for the evaluation of gastric cancer progression and prognosis.