【作者】 杨进；

【导师】 胡绍燕；

【作者基本信息】 苏州大学 ， 儿科学（血液病）， 2017， 硕士

【摘要】 第一部分儿童T系急性淋巴细胞白血病的临床特征及早期治疗反应与预后的相关性分析目的:回顾分析了从2008年到2016年在我院就诊的97例T系急性淋巴细胞白血病(T-ALL)患儿的临床生物学特征及早期治疗反应与预后的相关性。方法:入组从2008年7月至2016年12月于苏州大学附属儿童医院连续就诊的小于15岁的97例初治T-ALL患儿。所有病人均接受CCLG-ALL-2008方案为骨架的化疗方案,分析临床特征和早期治疗反应与预后的相关性。结果:97名T-ALL患儿的3年总生存率(3-year OS)和3年无事件生存率(3-year EFS)分别为68.1%±5.3%和59.3%±5.5%。高危组病人3-year OS和3-year EFS明显低于中危组(3-year OS IR:HR 94.7%±5.1%vs 61.3%±6.2%,P=0.012)(3-year EFS IR:HR 88.1%±7.9%vs 51.6%±6.3%,P=0.005)。初治白细胞≥100×10~9/L的病人3-year OS(58.2%±7.5%)较细胞数<100×10~9/L的病人(81%±6.7%)低(P=0.045),但是3-year EFS两组没有显著统计学意义(P=0.124)。强的松耐药病人的3-year OS及3-year EFS明显低于强的松敏感病人(44.5%±8.4%vs 89.5%±4.4%,P<0.001;41.6%±7.9%vs75.3%±6.8%,P=0.003)。治疗第15天(D15)骨髓白血病细胞≥25%(M3)的病人3-year EFS低于骨髓白血病细胞<5%(M1)和介于5%到25%(M2)的病人(48.5%±8.4%vs66%±7%,P=0.021),3-year OS未达到统计学意义(P=0.326)。多因素分析提示3-year OS中,只有强的松治疗反应是独立预后相关因素(P=0.028,HR=3.619,95%CI1.152-11.368)。3-year EFS中,各种因素均未达到统计学意义。结论:初诊外周血白细胞≥100×10~9/L、泼尼松试验不敏感以及D15骨髓M3状态影响T-ALL病人对CCLG-2008方案的治疗效果,需要更有效的治疗方案提高疗效。第二部分儿童T系急性淋巴细胞白血病的全基因组分析目的:通过全基因组测序(WGS)及转录组(WTS)测序,在完善T-ALL基因谱学的同时,更好的认识儿童T系急淋侵袭性表型的遗传学基础,发现新的预后相关的生物学标记。方法:本研究中我们随机选取了从2012年到2015年在苏州大学附属儿童医院治疗的31位初诊儿童。31位患儿的初诊样品及对应缓解期的样品进行了全基因组测序,30位患儿的初诊样品还进行了转录组测序。结果:编码区体细胞突变方面平均每个病人有6.4个编码区突变。NOTCH1是体细胞基因组改变中突变频率最高的,主要是激活突变。其次是FBXW7,USP7,PTEN,WT1,以及BCL11B。拷贝数改变方面平均每个病人有9.9个拷贝数缺失,3.2个拷贝数获得。最常见的是9p缺失导致的CDKN2A(p16)和CDKN2B(p15)失活。融合基因方面最常见的是1p32内80kb的缺失形成的STIL/TAL1融合基因(n=11)。其他发现的融合基因包括TCF7/SPI1(n=1),TCRA/TLX1(n=1),SET/NUP214(n=1),NUP214/ABL1(n=1),TCRB/LMO2(n=1),KMT2A/NUTM2D(n=1),KMT2A/MLLT1(n=1),TLX3/CDK6(n=1)。另外4个病人TAL1非编码区存在杂合性插入性突变。结论:我们的基因组学研究得到的结果与以往报道类似,也发现了一些新的融合基因及突变,为进一步完善儿童T-ALL精准诊疗和预后参数奠定基础。第三部分TAL1基因非编码区突变与儿童T系急性淋巴细胞白血病临床预后的相关性研究目的:本研究旨在探讨TAL1癌基因非编码区突变在儿童T系急性淋巴细胞白血病(T-ALL)中的发生率,分析伴有TAL1基因非编码区突变的T-ALL的临床特征。方法:运用聚合酶链式反应(PCR)扩增89例T-ALL患者初诊肿瘤细胞DNA中的TAL1基因,Sanger基因测序法检测TAL1基因突变。结果:8例T-ALL患者中检测到TAL1非编码区存在杂合性插入性突变,存在三种插入类型,发生率为9%(8/89)。8例TAL1基因非编码区突变患儿均表现为初诊就有较高的外周血白细胞(P=0.003)、强的松耐药(P=0.012)。诱导缓解D15,TAL1突变组75%(6/8)骨髓状态为M3(P=0.049)。巩固治疗前12W TAL1突变患者M2/M3的病人占37.5%(3/8)(P=0.007)。生存分析结果TAL1突变组2-year OS和2-year EFS均明显低于TAL1突变阴性患儿(14.6%±13.5%vs 77.3%±5.1%,P<0.005;12.5%±11.7%vs 63.2%±5.6%,P=0.001)。多因素分析提示强的松治疗反应(P=0.027,HR=4.711,95%CI 1.19-18.66)和TAL1突变(P=0.031,HR=3.153,95%CI 1.109-8.967)均是影响2-year OS的独立预后相关因素。结论:TAL1非编码区的插入性突变是T-ALL预后不良的分子标志之一,临床上应早期筛查,尽早干预。第四部分几种重现性染色体异位与儿童T系急性淋巴细胞白血病临床预后的相关性分析目的:研究STIL/TAL1,TCF7/SPI1以及TCRαδ和TCRβ与多种癌基因形成的融合基因在儿童T-ALL中的发生率及其与临床预后的关系。方法:STIL/TAL1,TCR融合基因主要根据临床检测染色体核型,实时荧光定量PCR以及二代测序结果确定。TCF7/SPI1的检测是通过聚合酶链式反应(PCR)扩增76例T-ALL患者初诊白血病细胞c DNA标本,Sanger基因测序法确定阳性条带病人的融合基因断裂点位置。结果:96例T-ALL患者中,22例有STIL-TAL1融合基因,阳性率22.9%。与STIL/TAL1阴性组比,STIL/TAL1融合基因组D15 81.5%(4/22)的病人骨髓状态为M1/M2(P=0.045),D33全部达到完全缓解(P=0.106)。STIL/TAL1组3-year OS低于阴性患儿,但是差异没有统计学意义(60.1%±11.1%vs70.6%±6%,P=0.465)。84例T-ALL患者中,9例形成了TCR融合基因,阳性率10.5%,其中LMO2/TCRαδ4例(4.8%),LMO1/TCRαδ1例(1.2%),MLLT3/TCRαδ1例(1.2%),TLX1/TCRαδ1例(1.2%),MYC/TCRαδ1例(1.2%),LMO2/TCRβ1例(1.2%)。9例病人中8例融合位点为14q11(TCRαδ),该融合位点的病人的长期生存率与无此融合位点的病人并无明显差别(3-year OS:66.7%±19.2%vs69.4%±5.6%,P=0.903)(3-year EFS45±18.8%vs 60.4%±5.9%,P=0.392)。76例T系病人中4例病人存在TCF7/SPI1融合基因,阳性率5.3%,3名男性,1名女性。四例病人均对激素敏感,D15评估骨髓状态均为M1/M2,D33及12W骨髓状态均为M1,四例现全部存活,两例生存期超过六年。均无不良事件发生。结论:这三种染色体异位的临床亚型的预后意义目前仍不明确,还需要进一步扩大标本验证。更多还原

【Abstract】 Part 1.Prediction of outcomes by clinical characteristics and early treatment esponses in childhood T-cell acute lymphoblastic leukemia.Objectives:To explore the predictive values of clinical characteristics and early treatment responses in childhood T-cell acute lymphoblastic leukemia in single center.Methods:From 2008 to 2016,97 consecutive patients aged≤15 years with newly diagnosed T-ALL were treated with CCLG2008 in Soochow children’s hospital.Predictive value of clinical characteristics and early treatment responses which include prednisone response,bone marrow morphology at day 15 and day 33 during induction chemotherapy and week 12 before consolidation therapy were analyzed.Results:The 3-year overall survival rate(OS)and event free survival rate(EFS)for these patients were 68.1%±5.3%and 59.3%±5.5%,respectively.Patients with initial white blood cell count more than100×10~9/L have a lower 3-year OS than those with less WBC count.Prednisone poor responder was strongly associated with decreased survival rates.And patients who got M3 bone marrow morphology at D15 tend to have a lower 3-year EFS than those who achieved M1/M2.Conclusion:Our study demonstrated that prednisone response was still one of the most powerful predictor of treatment outcome in childhood T-ALL patients and conventional morphological assessments of treatment response still played important roles in predicting treatment outcomes and tailoring treatment intensity especially in countries with inadequate skills or financial resources for MRD monitoring.Part 2.Whole genome analysis of pediatric T-cell acute lymphoblastic leukemia.Objectives:To explore the genomic landscape and identify prognostic biomarkers in childhood T-ALL through the next-generation sequencing technology.Methods: Whole genome sequencing(WGS)was performed in matched germline and tumor samples from 31 Chinese children with T-ALL,30 of whom were also profiled by transcriptome sequencing(RNA-seq).Results: On average,we observed 6.4 coding mutations per patient with 9.9 and 3.2 copy number losses and gains,respectively.Consistent with previous reports,NOTCH1 was most frequently targeted by somatic genomic alterations(primarily as activating sequencing mutations),followed by FBXW7,USP7,PTEN,WT1,and BCL11 B.Intrachromosomal rearrangement proximal to the TAL1 gene was also frequent(11 of 31 patients),leading to robust expression of the STIL-TAL1 fusion,as described previously.The other fusion genes identified were TCF7/SPI1,TCRA/TLX1,SET/NUP214,NUP214/ABL1,TCRB/LMO2,KMT2A/NUTM2 D,KMT2A/MLLT1,TLX3/CDK6.And four patients with insertion in the noncoding elements of TAL1 were also recognized.Conclusion: We need to validate our findings in more clinical patients’ sample.Part 3.Noncoding mutation in TAL1 predicts a poor clinical outcome in pediatric T-cell acute lymphoblastic leukemiaObjective : The present study was designed to investigate the incidence and clinical significance of TAL1 noncoding mutation in childhood T-cell acute lymphoblastic leukemia(T-ALL).Methods: The incidence of TAL1 noncoding mutation was analyzed by polymerase chain reaction(PCR)and Sanger sequencing in 89 children with T-ALL.Results : We have eight patients(9%)presented with heterozygous somatic mutation,three types of insertion,in noncoding elements of TAL1.All eight patients showed resistance to prednisone treatment.These cases with TAL1 mutation had a higher white blood cell(WBC)count than the cases without the type of mutation.Survival analysis showed patients with the mutation had lower overall survival rate(OS)and event free survival rate(EFS)than those without the abnormality.Cox analysis for 2-year OS identified TAL1 mutation and prednisone response as independent prognostic factors.Conclusions: It is concluded that TAL1 noncoding mutation is not rare in pediatric T-ALL patients.And patients with such type of mutation tend to have a poor clinical outcome.Part 4.A Clinical features and outcomes of pediatric T-cell acute lymphoblastic leukemia with a few recurrent chromosome rearrangements.Objective: To investigate predictive value of some chromosome alterations including STIL/TAL1,translocations involving T cell receptor(TCR)genes and TCF/SPI1.Methods: STIL/TAL1,translocations involving T cell receptor(TCR)genes were identified through karyotype,q RT-PCR and WGS/WTS.The incidence of TCF/SPI1 was analyzed by polymerase chain reaction(PCR)and Sanger sequencing in 76 children with T-ALL.Results: 22 out of 96 patients have STIL/TAL1 deletion.Patients with STIL/TAL1 tend to have a good early treatment response.The 3-year OS was lower in this fusion gene group,but it didn’t reach significant importance.We have 9 patients out of 89 presented with genetic alterations involving TCR genes,which are LMO2/TCRαδ(n=4),LMO1/TCRαδ(n=1),MLLT3/TCRαδ(n=1),TLX1/TCRαδ(n=1),MYC/TCRαδ(n=1),LMO2/TCRβ.4 out of 76 patients have TCF7/SPI1 fusion.All these four patients respond well to the chemotherapy and are alive.No adverse events happen to them.Conclusion: The prognostic significance of these alterations remain unclear.We still need to collect more samples for validation.But all these alterations make perfect candidates for minimal residual disease monitoring.更多还原