MYCN和PHOX2B基因联合血浆游离DNA检测在高危神经母细胞瘤危险度分层及预后评估中的作用

The role of MYCN and PHOX2B gene combined with plasma cell-free DNA detection in risk stratification and predicting the prognosis of high-risk neuroblastoma

目的探讨MYCN基因、PHOX2B基因及血浆游离DNA(cfDNA)水平用于高危神经母细胞瘤(NB)危险度分层及预后评估的作用和意义。方法对2017年8月至2018年12月首都医科大学附属北京儿童医院收治的94例高危NB患儿进行前瞻性研究,分别于初诊时、化疗4个疗程和6个疗程后检测MYCN基因、PHOX2B基因及cfDNA水平,观察治疗过程中3项指标的变化,采用χ^(2)检验和Kaplan-Meier生存分析法评价其与疗效的关系。结果94例患儿中MYCN基因扩增14例(14.9%),PHOX2B基因阳性76例(80.8%),cfDNA水平>100μg/L者56例(59.6%)。MYCN基因扩增患儿初诊高乳酸脱氢酶(LDH,≥1500 U/L)比例(6/14例)显著高于基因正常组患儿(9/80例)(P=0.009);PHOX2B基因阳性患儿多部位转移病例(54/76例)及高神经元特异性烯醇化酶(NSE,≥370μg/L)比例(37/76例)显著高于基因阴性组患儿(5/14例,2/14例)(P=0.015、0.020);cfDNA高水平患儿初诊高LDH及高NSE比例(13/37例,28/37例)显著高于cfDNA低水平组患儿(2/48例,10/48例)(均P<0.001)。治疗过程中,随着肿瘤负荷减小,PHOX2B基因拷贝数和cfDNA水平较初诊时显著降低[0(0~719.6)拷贝比1723.5(0~186000.0)拷贝;19.0(1.1~225.5)μg/L比200.6(8.0~5247.4)μg/L](均P<0.001)。初诊时,MYCN基因扩增组患儿2年无事件生存(EFS)率显著低于MYCN基因正常组患儿[(33.3±13.1)%比(58.5±7.1)%,P=0.020];PHOX2B基因阳性组患儿2年EFS率显著低于阴性组患儿[(47.9±7.1)%比(79.1±11.1)%,P=0.043];cfDNA高水平组(≥229.6μg/L)2年EFS率显著低于cfDNA低水平组[(38.6±9.8)%比(71.7±8.2)%,P=0.001]。6个疗程后PHOX2B基因阳性组患儿2年EFS率显著低于基因阴性组患儿[(16.7±14.4)%比(60.6±6.6)%,P=0.014];维持治疗前间碘苄胍(MIBG)核素扫描阳性组患儿2年EFS率显著低于阴性组患儿[(35.2±11.7)%比(65.8±7.1)%,P=0.037]。治疗过程中,MYCN基因和cfDNA水平与患儿预后无显著相关性。将6个疗程后的PHOX2B基因及维持治疗前MIBG结果联合分组进行生存分析(PHOX2B+/MIBG+,PHOX2B+或MIBG+,PHOX2B-/MIBG-),组间比较差异有统计学意义[0比(53.6±1.2)%比(65.5±7.4)%,P=0.003]。结论MYCN和PHOX2B基因及cfDNA水平在高危NB危险度分层及预后评估中具有重要作用;与MYCN和cfDNA水平相比,PHOX2B基因更适于治疗过程中的疗效监测,PHOX2B与维持治疗前的MIBG结果联合分析,用于判断患儿治疗效果,评估体内残余病灶更精准。

Objective To explore the clinical significance of the MYCN gene,PHOX2B gene and plasma cell-free DNA(cfDNA)in risk stratification and predicting the prognosis of high-risk neuroblastoma(NB).Methods This was a prospective study involving 94 high-risk NB children admitted to Beijing Children′s Hospital,Capital Medical University from August 2017 to December 2018.Relative levels of MYCN and PHOX2B and cfDNA at diagnosis,and 4 and 6 cycles of chemotherapy were detected,and their differences were compared by the Chi-square test.Kaplan-Meier survival analysis was performed to explore their prognostic potential in high-risk NB.Results Among the 94 high-risk NB children,14 cases(14.9%)had MYCN amplification,76 cases(80.8%)had positive expression of PHOX2B and 56 cases(59.6%)had cfDNA level higher than 100μg/L.The proportion of high lactate dehydrogenase(LDH,≥1500 U/L)level in the MYCN gene amplification group(6/14 cases)was higher than that in the normal group(9/80 cases)(P=0.009).The proportion of multi-site metastasis(54/76 cases)and high neuron specific enolase(NSE)level(NSE≥370μg/L,37/76 cases)in PHOX2B positive group were significantly higher than those in the negative group(5/14 cases,2/14 cases)(P=0.015,0.020).The proportion of high LDH and high NSE in high cfDNA concentration(≥229.6μg/L)group(13/37 cases,28/37 cases)were significantly higher than those in low cfDNA concentration group(2/48 cases,10/48 cases)(all P<0.001).With the decreased tumor burden during the treatment,the copy number of PHOX2B gene and cfDNA level were significantly lower than those at the initial diagnosis[0(0-719.6)copies vs.1723.5(0-186000.0)copies;19.0(1.1-225.5)μg/L vs.200.6(8.0-5247.4)μg/L,all P<0.001].The 2-year event-free survival(EFS)rate of the MYCN gene amplification group was significantly lower than that of the normal group[(33.3±13.1)%vs.(58.5±7.1)%,P=0.020].The 2-year EFS rate of PHOX2B positive group was significantly lower than that of the negative group[(47.9±7.1)%vs.(79.1±11.1)%,P=0.043].EFS rate in high cfDNA concentration group was significantly lower than that in cfDNA low concentration group[(38.6±9.8)%vs.(71.7±8.2)%,P=0.001].After 6 cycles of chemotherapy,EFS rate in the PHOX2B positive group was significantly lower than that in the negative group[(16.7±14.4)%vs.(60.6±6.6)%,P=0.014];which was significantly lower in the Metaiodobenzylguanidine(MIBG)positive group than that of the negative group[(35.2±11.7)%vs.(65.8±7.1)%,P=0.037].The MYCN gene and cfDNA concentration were not correlated with the prognosis of high-risk NB.Survival analysis of the combination of PHOX2B and MYCN gene(PHOX2B+/MIBG+,PHOX2B+or MIBG+,PHOX2B-/MIBG-)showed a significant difference in the survival among three groups[0 vs.(53.6±1.2)%vs.(65.5±7.4)%,P=0.003].Conclusions The MYCN and PHOX2B gene and cfDNA concentration are of significance in risk stratification and predicting the prognosis of high-risk NB.Compared with the MYCN gene and cfDNA concentration,the PHOX2B gene is more suitable for monitoring the curative effect of chemotherapy on high-risk NB.A combined analysis of PHOX2B gene and MIBG before treatment can be more accurate in evaluating the treatment effect and residual lesions.