【作者】 张晓；

【导师】 胡绍燕；

【作者基本信息】 苏州大学 ， 儿科学（专业学位）， 2023， 硕士

【摘要】 目的:本文回顾性分析了同源盒(homeobox gene,HOX)基因家族在儿童急性T淋巴细胞白血病(T-cell acute lymphoblastic leukemia,T-ALL)中的表达模式,及其与患儿的临床特征及预后的关系,以期为儿童T-ALL的精准化、个体化诊疗提供依据。方法:收集2012年7月1日至2022年9月30日期间就诊并治疗于苏州大学附属儿童医院的115例初诊T-ALL儿童临床标本,采用全转录组测序(transcriptome sequencing,RNA-seq)检测儿童T-ALL中HOX基因家族表达量,并回顾性分析临床资料,比较39个HOX家族基因高表达组与低表达组患儿的临床特征及治疗反应。HOX基因家族共39个成员,分别为HOXA1、HOXA2、HOXA3、HOXA4、HOXA5、HOXA6、HOXA7、HOXA9、HOXA10、HOXA11、HOXA13、HOXB1、HOXB2、HOXB3、HOXB4、HOXB5、HOXB6、HOXB7、HOXB8、HOXB9、HOXB13、HOXC4、HOXC5、HOXC6、HOXC8、HOXC9、HOXC10、HOXC11、HOXC12、HOXC13、HOXD1、HOXD3、HOXD4、HOXD8、HOXD9、HOXD1O、HOXD11、HOXD12、HOXD13,分析每个病人39个HOX基因表达量。通过GraphPad Prism 8.0.2软件进行统计描述,将截断值设置在所有患者HOX基因表达量的第75百分位数[1]。利用SPSS 24.0软件进行统计学分析,P<0.05具有统计学差异。结果:T-ALL患者中,39个HOX基因只有19个基因有高表达,分别为HOXA1-11、HOXB2-7、HOXC4-6。115例初诊T-ALL患儿,其中男孩93例,女孩22例,中位年龄7.8岁(年龄范围为1.2～16.8岁),初诊白细胞中位数109.5(1.08～693.8)x10^9/L,初诊血红蛋白中位数102(37～159)g/L,初诊血小板中位数72(10～412)x10^9/L。其中74例成功完成了染色体核型分析,42例检出异常染色体核型;通过荧光原位杂交技术(fluorescence in situ hybridization,FISH)、聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)、RNA-seq 检测手段,检出SIL::TAL1融合基因阳性者31例,检出MLL重排阳性5例,其中3例MLL::ENL(KMT2A::MLLT1),1例 MLL::AF9,2 例PICALM::MLLT10,1 例 SET::NUP214 融合基因,2 例HOXA13::CDK6融合基因。入组115例T-ALL患儿,随访中位生存时间49.2(1.8～119.7)个月。失访15例,死亡22例,复发22例,移植52例,10年总生存率(Overall survival,OS)为 77.3±4.5%,1 0 年无复发生存率(Relapse-free survival,RFS)为 73.6±5.0%。HOXA簇基因高表达组更易检出MLL重排(MLL::ENL、MLL::AF9)、HOX重排(HOXA13::CDK6),PICALM::MLLT10 和SET::NUP214重排(P值均<0.05);且HOXA7-11基因高表达组中,上述重排阳性患儿对激素反应性明显较差(P值均<0.05)。HOXA3-7和HOXA11基因低表达组更易检出SIL::TAL1融合基因(P值均<0.05);在HOXA1、HOXA9基因低表达组,SIL::TAL1融合基因阳性患儿D15 MRD<1×10^-2和D15骨髓涂片幼稚细胞<25%的数量多于SIL::TAL1融合基因阴性组;HOXA2、HOXA11基因低表达组中,SIL::TAL1融合基因阳性患儿的10年RFS高于SIL::TAL1融合基因阴性患儿(P值均<0.05)。HOXB2、HOXB4、HOXB7基因低表达组D15骨髓涂片幼稚细胞≥25%的患儿明显多于高表达组(P值均<0.05)。HOXC4基因高表达组患儿对激素敏感性比低表达组差(P=0.011),高表达组D33骨髓MRD≥1×10^-4的患儿数量多于HOXC4基因低表达组(P=0.048)。对患儿预后因素分析显示:患儿的10年OS在D15骨髓MRD≥1×10^-2和MRD<1×10^-2 两组有明显统计学差异(67.3±12.6%vs 100.0±0.0%;P=0.012);10 年 RFS在D33骨髓涂片幼稚细胞占≥5%和骨髓幼稚细胞<5%两组有显著的统计学差异(25.0±21.0%vs 76.7±5.5%;P=0.006);19 个HOX基因表达程度均不影响 T-ALL患儿的预后。多因素分析仅发现D33骨髓涂片幼稚细胞≥5%是复发的独立危险因素,风险系数(HR)及 95%置信区间为 3.595[95%CI(1.171-11.035),(P=0.023)]。结论:(1)人类39个HOX基因,19个在T-ALL患者中有高表达,但HOXD簇基因在T-ALL中几乎不表达。(2)HOXA簇基因更多与基因重排相关,HOXA7-A11高表达患儿同时具有MLL和HOXA重排、SET::NUP214、PICALM::MLLT10融合基因表现为激素耐药;SIL::TAL1融合基因阳性的患儿在HOXA1、HOXA9基因低表达组早期治疗反应较好,在HOXA2、HOXA11基因低表达组中,具有较长的无复发生存。(3)HOXB和HOXC簇基因与治疗反应有关,其中HOXB2、HOXB4、HOXB7基因低表达组D15天骨髓缓解差,而HOXC4基因高表达组患儿激素敏感性较差,且D33骨髓MRD≥1× 10^-4的患儿明显多于HOXC4基因低表达组,早期治疗反应较差。(4)D15骨髓MRD≥1×10^-2患儿预后差,总生存期短;D33骨髓涂片幼稚细胞≥5%是复发的独立危险因素;HOX家族对T-ALL预后没有影响。更多还原

【Abstract】 Objective:This study retrospectively analyzed the expression pattern of HOX genes family in pediatric T-cell acute lymphoblastic leukemia,and the association with the clinical features and patients’ prognosis.This study aims to provide evidence for the precise and personalised therapy of pediatric T-ALL.Methods:A total of 115 pediatric T-ALL treated at the children’s hospital of Soochow university between July 1,2012 and September 30,2022 were recruited in this retrospective study.The expression of HOX gene was detected by transcriptome sequencing(RNA-seq),and the clinical data were retrospectively analyzed.The patients were categorized into high-HOX and low-HOX expression groups according to their expression of HOX family genes,and their clinical characteristics and treatment response were compared.HOX family genes include 39 members,namely HOXA1、HOXA2、HOXA3、HOXA4、HOXA5、HOXA6、HOXA7、HOXA9、HOXA10、HOXA11、HOXA13、HOXB1、HOXB2、HOXB3、HOXB4、HOXB5、HOXB6、HOXB7、HOXB8、HOXB9、HOXA13、HOXC4、HOXC5、HOXC6、HOXC8、HOXC9、HOXC10、HOXC11、HOXC12、HOXC13、HOXDI、HOXD3、HOXD4、HOXD8、HOXD9、HOXD10、HOXD11、HOXD12、HOXD13.The expression level of 39 HOX genes from each patients were collected and the cut-off of each gene was set as the 75th percentile of the overall HOX gene RNA expression in these patients.GraphPad Prism 8.0.2 software was used for statistical description.Statistical analysis was performed using SPSS 24.0 software.Results:In pediatric T-ALL,only 19 of the 39 HOX genes were highly expressed,including HOXA1-11,HOXB2-7 and HOXC4-6.115 patients diagnosed with T-ALL were enrolled,which including 93 boys and 22 girls.The median age was 7.8 years(1.2-16.8years);The median leukocyte of initial diagnosis was 109.5(1.08～693.8)x10^9/L,the median hemoglobin was 102(37～159)g/L,and the median platelet was 72(10～412)x10^9/L.74 cases got chromosomal karyotype analysis,of which 42 cases harboring abnormal karyotype.By combining the testing resulst of fluorescence in situ hybridization(FISH),reverse transcription polymerase chain reaction(RT-PCR)and RNA-seq,31 cases of SIL::TAL1 fusion,2 cases of PICALM::MLLT10,1 case of SET::NUP214,and 2 cases of HOXA13::CDK6,5 case MLL rearrangement,including 3 cases of MLL::ENL(KMT2A::MLLT1)and 1 case of MLL::AF9 fusion were detected.The median follow-up time was 49.2 months(1.8～119 months).There were 15 cases loss of follow-up,22 cases died,22 cases relapsed and 52 cases received hematopoietic stem cell transplantation.The 10-year overall survival(OS)was 77.3±4.5%,10-year relapse-free survival(RFS)was 73.6±5.0%.Patients with MLL(MLL::ENL,MLL::AF9),HOX(HOXA13::CDK6),PICALM::MLLT10 and SET::NUP214 rearrangements were more likely to have high-HOXA cluster gene expression(P<0.05).Moreover,in the high HOXA 7-11 expression group,the patients with the above rearrangements had a significantly worse response to glucocorticoid(P<0.05).Patients with SIL::TAL1 fusion gene was more likely to have low expression of HOXA3-7 and HOXA11 genes(P<0.05).In the HOXA1 and HOXA9 low expression group,the case number of D15 MRD<1×10^-2 and D15 BM blasts<25%in SIL::TAL1 positive patients were more than those in SIL::TAL1 negative patients.In the HOXA2 and HOXA11 gene low expression group,the 10-years RFS of children with SIL::TAL1 fusion gene was higher than those without SIL::TAL1 fusion gene(P<0.05).In the low HOXB2,HOXB4 and HOXB7 gene expression groups,the proportion of children with D15 bone marrow(BM)blasts≥25%were significantly higher than those in the high expression groups(P<0.05).The children with high expression of HOXC4 gene had a worse response to glucocorticoid than those with low expression of HOXC4 gene(P=0.011).The number of children with D33 BM MRD≥1x 10^-4 in HOXC4 gene high expression group was more than those in low expression group(P=0.048).Analysis of prognostics factors showed that the 10-year OS in MRD≥1×10^-2 and MRD<1×10^-2 were 67.3±12.6%and 100.0±0.0%,and the difference was statistically significant(P=0.012);the 10-year RFS showed statistical significance between BM blast>5%and<5%groups(25.0±21.0%vs 76.7±5.5%;P=0.006);the expression level of the 19 HOX genes was not significantly associated with the prognosis of T-ALL patients.Multivariate analysis showed that BM blast≥5%on day 33[HR=3.595,95%CI(1.171-11.035)]was an independent risk factor for RFS.Conclusion:(1)Of the 39 HOX genes,19 genes were highly expressed in T-ALL patients,and the HOXD cluster genes were merely expressed.(2)HOXA cluster genes expression were associated with chromosome rearrangement.Children with high HOXA7-A11 expression,together with MLL-,HOXA rearrangement,SET::NUP214,and PICALM::MLLT10 fusion genes showed glucocorticoid resistance.In the low HOXA1 and/HOXA9 expression group,patients with SIL::TAL1 fusion gene had a better early treatment response.In the low HOXA2 and HOXA11 expression group,patients with SIL::TAL1 fusion gene had a longer recurrence free survival.(3)HOXB and HOXC cluster genes were associated with treatment response,in which the low HOXB2,HOXB4,HOXB 7 gene expression group had poor bone marrow remission at day 15.Patients with high HOXC4 gene expression had poorer sensitivity to hormone,and number of cases with MRD≥1×10^-2 on day 33 were significantly higher than the low HOXC4 expression group,which had poor early treatment response.(4)Patients with MRD≥1×10^-2 on day 15 had poorer prognosis and shorter overall survival.BM blasts≥5%on day33 is an independent risk factor for relapse.The expreesion level of HOX gene family have no significant effect on the prognosis of T-ALL.更多还原