【作者】 李倩；

【导师】 牛挺；

【作者基本信息】 四川大学 ， 内科学（血液病）（专业学位）， 2021， 博士

【摘要】 背景和目的:慢性髓性白血病(Chronic Myeloid Leukemia,CML)是一种起源于骨髓多能造血干细胞的骨髓增殖性肿瘤,以9号和22号染色体易位形成的含BCR-ABL1融合基因的费城染色体为特征。CML自然病程分为三期,慢性期(Chronic Phase,CML-CP)、加速期(Accelerated Phase,CML-AP)和急变期(Blast Crisis,CML-BC)。CML-BC患者整体预后差,未行异基因造血干细胞移植(Allogeneic Hematopoietic Stem Cell Transplantation,Allo-HSCT)治疗的患者预后尤其差。目前关于CML-BC的治疗,国内外指南都推荐治疗缓解后尽快行Allo-HSCT治疗,但是各指南对移植前的治疗推荐却各有不同,同时各指南对具体酪氨酸激酶抑制剂(Tyrosine Kinase Inhibitor,TKI)类别、具体化疗方案、具体TKI和化疗的组合的推荐都相对模糊,目前国际上没有统一共识。因为Allo-HSCT治疗受供者、患者病情、移植相关风险、医疗条件及患者经济等多种因素制约,目前中国大部分CML-BC患者没有条件及时行Allo-HSCT治疗,所以待移植患者移植前的治疗选择以及不移植患者的治疗选择都尤为重要且待探究。但是,近年来比较各类治疗、探究疗效及生存相关影响因素的相关研究较少且病例数有限,且多为国外研究。然而,国内外CML-BC患者在急变发病率、急变年龄、初诊即为急变期占比、急变后不同治疗的可及性、治疗依从性、经济能力等方面都有较大差异,亟待更多的真实世界数据反映中国CML-BC患者现况、治疗实践及对应的疗效及生存结局。因此,我们总结了12年内于四川大学华西医院住院部治疗的所有CML-BC患者的临床特征、治疗、疗效评价及生存现况,探究了不同治疗方案及其他因素对CML-BC患者疗效和生存结局的影响,以期为中国CML-BC患者临床诊治和相关指南的制定提供帮助。虽然CML-BC预后差,但是CML-CP/CML-AP患者治疗缓解率高,预后佳。探究CML-CP/AP进展至CML-BC的机制,对于控制CML进展和探索新的治疗靶点都有重要意义。因此,我们从美国基因表达综合数据库(Gene Expression Omnibus Database,GEO)中获取CML-CP/AP/BC三期患者的基因表达数据,分别筛选CML-BC和CML-CP/CML-AP间的差异表达基因,对差异表达基因进行富集分析,挖掘出进展至CML-BC过程中的表达改变的中枢基因,为探究在临床上有意义的生物标志物提供备选基因,也为探究CML进展至急变期机制研究提供思路并为后续CML-BC的治疗提供了潜在靶点。方法:我们首先对2009年1月1日至2020年11月30日就诊于四川大学华西医院住院部的所有CML-BC患者进行临床分析。疗效评价共涉及主要血液学缓解(Major Hematologic Response,MHR)、完全血液学缓解(Complete Hematologic Response,CHR)、完全细胞遗传学缓解(Complete cytogenetic response,CCy R)、分子学缓解1(Molecular response 1,MR~1)、分子学缓解2(Molecular response 2,MR~2)和主要分子学缓解(Major molecular response,MMR)。总生存时间(Overall Survival,OS)定义为初次确诊CML-BC至患者死亡或随访结束。一线治疗无失败生存时间(Failure Free Survival,FFS)定义为CML-BC一线治疗开始至缓解后首次复发、启用二线治疗、死亡或随访结束。治疗分组包括总体治疗模式分组和一线治疗分组,各四组。比较各组缓解率差异时采用卡方检验或Fisher精确检验。采用二项Logistic回归探究各变量对MHR的影响。比较生存资料时采用Kaplan-Meier法并进行Log-Rank检验。运用Cox比例风险模型探究各变量对OS及FFS的影响。随后,为了分析CML-BC和CML-AP/CML-CP两期的差异表达基因,我们在GEO数据库中下载系列号为GSE4170的表达芯片数据。借助R语言的limma包,分别筛选出CML-BC vs CML-AP,以及CML-BC vs CML-CP间的差异表达基因,并进行GO分析、KEGG分析及HALLMARK基因集富集分析。画维恩图筛选出两组比较的共同差异表达基因,借助STRING在线数据库进行蛋白互作分析,最后用Cytoscape的插件cytoh Hubba分析蛋白互作网络,从蛋白互作网络中挑选出与周围基因具有高度连通性的前15个基因,作为中枢基因。结果:本研究临床分析共纳入191例CML-BC患者。2013年后年新发CML-BC患者较前减少。CML-BC诊断中位年龄为40.0岁。男性占比70.2%。CML初诊断为CP/AP/BC的占比分别是75.9%/5.2%/18.8%。急髓变/急淋变/混合变/分型不明占比分别为63.9%/31.9%/3.1%/1.0%。仅83例患者有染色体核型分析结果,其中伴附加染色体异常的患者28例,检出频率较高的有双Ph+染色体、+8、i17q、-7等。仅78例患者有ABL激酶区域突变结果,其中ABL突变阳性者34例,检出频率最高的突变是T315I突变。CML-BC患者治疗后血液学缓解可评价者共174例(91.1%);其中获得MHR的共77例(44.3%),获得CHR的共52例(29.9%)。急变治疗后细胞遗传学缓解可评价者仅有17例(8.9%);其中获得CCy R的共9例(52.9%)。急变治疗后分子学缓解率可评价者共81例(42.4%);其中获得MR~1的共39例(48.1%),获得MR~2的共30例(37.0%),获得MMR的共25例(30.9%)。总体治疗模式分组四组间MHR、CHR、MR~1、MR~2和MMR五个水平的缓解率最高的都是移植治疗组,其次是TKI+联合化疗组,然后是单用TKI治疗组,最后是非积极治疗组,五次比较差异均有统计学意义(P<0.050)。急变后一线治疗分组四组间MHR和CHR两个水平缓解率最高的都是一线TKI+联合化疗组,其次是一线单用TKI组,然后是一线联合化疗组,最后是一线非积极治疗组,两次比较差异均有统计学意义(P<0.050)。二项Logistic回归分析发现以单用TKI治疗为参照,TKI+联合化疗为MHR的独立促进因素(OR=5.770,P=0.010);以2007-2012年段诊断为参照,2013-2020年段诊断为MHR的独立促进因素(OR=5.914,P=0.013);以急变前有TKI暴露史为参照,无暴露史为MHR的独立促进因素(OR=8.743,P=0.003);以急髓变为参照,急淋变为MHR的独立促进因素(OR=4.192,P=0.034)。191例CML-BC患者中位OS为8.7月(95%CI 5.7-11.7)。总体治疗模式分组的移植治疗组未及中位OS,TKI+联合化疗组、单用TKI治疗组和非积极治疗组三组中位OS依次12.5月(95%CI 7.9-17.1)、7.8月(95%CI 5.4-10.3)和0.3月(95%CI 0.1-0.5),四组间两两比较时,仅TKI+联合化疗组和单用TKI治疗组间OS差异无统计学意义(P=0.140),其余任意两组间差异均有统计学意义(P<0.008)。一线TKI+联合化疗组、一线单用TKI治疗组、一线联合化疗组和一线非积极治疗组四组中位OS依次为12.3月(95%CI 9.4-15.2)、9.7月(95%CI3.7-15.6)、未及中位OS和0.3月(95%CI 0.1-0.5),一线非积极治疗组OS差于其他三组且差异均有统计学(P<0.001),其他三组间OS差异均无统计学意义(P≥0.008)。急变治疗后获得MHR、CHR、CCy R、MR~1、MR~2以及MMR组OS均优于对应未获得缓解组,且差异均有统计学意义(P<0.050);急变治疗后可评价细胞遗传学反应或分子学反应组OS均优于对应未能评价组,且差异均有统计学意义(P<0.050)。多因素COX回归分析发现以移植治疗为参照,其他三种治疗均为患者死亡的独立促进因素(HR>1.000且P<0.050);以2007-2012年段为参照,2013-2020年段为患者死亡的独立阻碍因素(HR=0.473,P=0.002);以Hb<60g/L为参照,Hb≥60g/L为患者死亡的独立阻碍因素(HR=0.526,P=0.042);以LDH<715 IU/L为参照,LDH≥715 IU/L为患者死亡的独立促进因素(HR=2.074,P=0.005);以初诊断分期为CML-CP/AP为参照,初诊即为CML-BC为患者死亡的独立阻碍因素(HR=0.552,P=0.042)。共188例CML-BC患者可评价一线治疗FFS,中位FFS为3.0月(95%CI2.2-3.8)。一线TKI+联合化疗组、一线单用TKI治疗组、一线联合化疗组和一线非积极治疗组四组中位FFS依次为4.7月(95%CI 2.9-6.5)、3.5月(95%CI2.7-4.3)、1.0月(95%CI 0.4-1.6)和0.3月(95%CI 0.1-0.5),两两比较时,一线TKI+联合化疗组和一线单用TKI治疗组FFS均优于其他两组且差异均有统计学意义(P<0.008)。多因素COX回归分析发现以一线TKI+联合化疗为参照,其他三种一线治疗均为患者FFS终点的独立促进因素(HR>1.000),但是仅一线非积极治疗组有统计学意义(P<0.001);以2007-2012年段为参照,2013-2020年段为患者FFS终点的独立阻碍因素(HR=0.399,P=0.001);以骨髓原始细胞百分比<39%组为参照,骨髓原始细胞百分比≥39%组为患者FFS终点的独立促进因素(HR=2.661,P<0.001);以急变前无TKI暴露史组为参照,有TKI暴露史组为患者FFS终点的独立促进因素(HR=2.015,P=0.017)。本研究中转录组学分析时,我们在GSE4170数据集中筛选了CML-BC和CML-AP间的差异表达基因总共478个,包括315个下调基因和163个上调基因。其中上调前五的基因分别是IQCF2、C6orf165、C10orf68、IRF4、NES;下调前五的基因分别是CEACAM7、IL8RB、GGTA1、FLJ31978、HP。GO分析发现CML-BC相对CML-AP的上调的基因主要富集于突触膜等部位,参与突触组织等生物学过程;下调的基因主要富集于分泌颗粒等部位,参与中性粒细胞脱颗粒、中性粒细胞激活等相关生物学过程。KEGG分析发现发现CML-BC相对CML-AP的上调基因主要富集于核糖体、核糖体生物合成、RNA转运及氨酰-t RNA生物合成等相关通路中;下调基因主要富集于Toll样受体信号通路等免疫相关通路中。HALLMARK基因集富集分析发现CML-BC相对CML-AP的上调基因主要富集于Myc＿Targets＿V2,Myc＿Targets＿V1以及氧化磷酸化的通路中。我们也在GSE4170数据集中筛选了CML-BC和CML-CP间的差异表达基因总共205个,包括181下调基因和24个上调基因。其中上调前五的基因分别是EMP1、ZBTB8、HOP、IFI27、HLA-DPB1;下调前五的基因分别是S100A12、LCN2、HP、OLFM4、MNDA。我们通过GO分析发现CML-BC相对CML-CP的上调基因主要富集在粘着斑和细胞-基质连接等部位,参与细胞生长及调节等生物学过程;下调的基因主要富集在分泌颗粒等部位,参与中性粒细胞脱颗粒、中性粒细胞激活等相关生物学过程。KEGG分析发现发现CML-BC相对CML-CP的上调基因主要富集于核糖体、氨酰-t RNA生物合成等相关通路中;下调基因主要富集于Fcγ受体介导的吞噬作用等免疫相关通路中。HALLMARK基因集富集分析发现CML-BC相对CML-AP的上调基因主要富集于Myc＿Targets＿V2和Myc＿Targets＿V1通路中。我们通过画维恩图取交集筛选出CML-BC vs CML-AP和CML-BC vs CML-CP两组比较的共同差异表达基因,初步获得了5个共同差异表达的上调基因和137个共同差异表达的下调基因。137个共同下调的基因主要富集在中性粒细胞脱颗粒、髓系白细胞激活等免疫相关通路中。构建好共同差异表达基因的蛋白互作网络图,筛选出蛋白互作网络中排名前15位的中枢基因,分别是ITGAM、S100A12、TYROBP、MNDA、ITGB2、TLR8、PTGS2、CYBB、FPR1、PRTN3、RNASE2、LCN2、CEACAM8、LILRA1、RNASE3。结论:1、CML-BC总体治疗优先推荐Allo-HSCT治疗。移植前推荐TKI+联合化疗或单用TKI治疗,尽快获得血液学缓解后再行Allo-HSCT治疗。2、无条件行Allo-HSCT治疗时,总体治疗优先推荐TKI+联合化疗治疗,然后是单用TKI治疗。总体治疗含TKI治疗时,建议发现ABL突变或治疗不耐受时及时更换TKI治疗;病情控制不佳时更换TKI治疗或加用联合化疗治疗。总体治疗含联合化疗时,建议尽可能完成两周期及以上的联合化疗;CML-LBC患者推荐VD/VP加用柔红霉素/去甲氧柔红霉素联合化疗;CML-MBC患者推荐DA/IDA化疗或HA化疗。3、TKI+联合化疗、2013-2020年段诊断、急变诊断前无TKI暴露史、急淋变均为急变后获得MHR的独立促进因素。移植治疗、2013-2020年段诊断、Hb≥60g/L、LDH<715 IU/L、初诊即为CML-BC均为患者死亡的独立阻碍因素。4、一线治疗优先推荐一线TKI+联合化疗,然后是一线单用TKI治疗。一线TKI+联合化疗、2013-2020年段诊断、骨髓原始细胞百分比<39%、急变诊断前无TKI暴露史均为急变后一线治疗FFS终点的独立阻碍因素。5、治疗反应的全面监测,尤其细胞遗传学反应、分子学反应、ABL激酶突变等,真实世界数据存在不足,提示今后临床实践规范性有待加强。6、相较于CML-AP,CML-BC中IQCF2、CFAP206、CCDC7、IRF4、NES等基因表达上调;CEACAM7、CXCR2、GGTA1、GLT1D1、HP等基因表达下调。7、相较于CML-CP,CML-BC中EMP1、ZBTB8、HOPX、IFI27、HLA-DPB1等基因表达上调;S100A12、LCN2、HP、OLFM4、MNDA等基因表达下调。8、CML-BC相对CML-CP或者CML-AP,都是MYC-TARGET通路上调,核糖体及蛋白质翻译相关通路上调,肿瘤细胞生长与增殖增加;下调的主要是各类免疫相关通路,各类感染风险增加。9、ITGAM、S100A12、TYROBP、MNDA、ITGB2、TLR8、PTGS2、CYBB、FPR1、PRTN3、RNASE2、LCN2、CEACAM8、LILRA1、RNASE3是CML-CP和CML-AP进展至CML-BC的中枢基因,可能是导致CML-BC发生的关键基因。更多还原

【Abstract】 Objective:Chronic myeloid leukemia(CML)is a kind of clonal myeloproliferative neoplasm originating from a multipotent hematopoietic stem cell and is characterized by the Philadelphia chromosome(Ph+)containing BCR-ABL1 fusion gene formed by translocation of chromosomes 9 and 22.The natural course of CML is divided into three phases,chronic phase(CML-CP),accelerated phase(CML-AP)and blast crisis(CML-BC).The prognosis of CML-BC patients is poor,especially for patients who are not treated with Allogeneic Hematopoietic Stem Cell Transplantation(Allo-HSCT).For the treatment of CML-BC,domestic and overseas guidelines all recommend Allo-HSCT once remission.However,these guidelines have different recommendations for treatment before Allo-HSCT and there is currently no international consensus on recommendations for specific TKI,chemotherapy regimens and TKI and chemotherapy combinations.As Allo-HSCT is restricted by multiple factors such as the donor,patient’s condition,transplant-related risks,medical conditions,and patient economics and so on,most CML-BC patients in China can’t receive Allo-HSCT treatment in time.Hence,treatment before transplantation and the treatment options for non-transplant patients are particularly important and need to be explored.However,in recent years,there are a few related studies comparing various treatments,exploring efficacy and survival-related factors,and the number of cases is limited,and most of them are foreign studies.However,CML-BC patients at home and abroad differ in incidence,age,the proportion of blast crisis at first diagnosis of CML,the availability of different treatments,treatment compliance,and economic ability.Thus,more real-world studies,reflecting current status,treatment and corresponding efficacy and survival outcomes of CML-BC patients in China,are needed.Therefore,we summarized the clinical characteristics,treatment,efficacy and survival status of all CML-BC patients treated in the inpatient department of West China Hospital of Sichuan University within 12 years,and then explored prognostic factors of efficacy and survival outcome of CML-BC patients with a view to providing help for the clinical diagnosis and treatment of CML-BC in China and the formulation of relevant guidelines.Although CML-BC has a poor prognosis,CML-CP/CML-AP own high treatment remission rate and good prognosis.Exploring the mechanism of the progression of CML-CP/AP to CML-BC is of great significance for controlling the progression of CML and exploring new therapeutic targets.Therefore,we obtained CML-CP/AP/BC three-stage gene expression data from the Gene Expression Omnibus Database(GEO),screened the differentially expressed genes between CML-BC and CML-CP/CML-AP respectively,did enrichment analysis and then found out the key differentially expressed genes,and thus provided candidate genes for exploring clinically meaningful biomarkers,ideas for exploring the mechanism of CML progression to the BC and potential targets for subsequent treatment of CML-BC.Methods:We performed a clinical analysis of all CML-BC patients who were admitted to the inpatient department of West China Hospital of Sichuan University from January1,2009 to November 30,2020.Efficacy evaluation contains major hematologic response(MHR),complete hematologic response(CHR),complete cytogenetic response(CCy R),molecular response 1(MR1),molecular response 2(MR2)and major molecular response(MMR).Overall survival(OS)is defined as the initial diagnosis of CML-BC to the death of the patient or the end of follow-up.Failure Free Survival(FFS)was assessed from the beginning of first-line treatment for CML-BC to the date of first relapse,date of switch to second line therapy,date of death,or date of last follow up.The treatment grouping included the overall treatment mode grouping and the first-line treatment grouping,each with four groups.Chi-square test or Fisher’s exact test was used to compare the differences in remission rates of each group.Binomial Logistic regression was used to identify specific characteristics of CML-BC affecting MHR.The Kaplan-Meier method and Log-Rank test were used when comparing survival data.Use the Cox proportional hazard model to explore the impact of various variables on OS and FFS.Subsequently,we download GSE4170 profile datasets from GEO database to analyze the differentially expressed genes between CML-BC and CML-AP/CML-CP.With the help of the limma package of R language,we screened the differentially expressed genes between CML-BC and CML-AP/CML-CP and did GO analysis,KEGG analysis and HALLMARK gene set enrichment analysis.By drawing a Venn diagram.We screened out the common differentially expressed genes of CML-BC vs CML-AP and CML-BC vs CML-CP and we performed protein interaction analysis on the differentially expressed genes with STRING online database and selected the first 15 genes with high connectivity with surrounding genes from the protein interaction network as the central genes with Cytoscape plug-in cytoh Hubba.Results:A total of 191 CML-BC patients were included in the clinical analysis of this study.The number of annual new CML-BC patients decreased after 2013.The median age at diagnosis of CML-BC was 40.0 years.Men accounted for 70.2%.The proportion of CML initially diagnosed as CP/AP/BC was 75.9%,5.2%,18.8%,respectively.The proportions of acute myeloid transformation,acute lymphoid transformation,mixed transformation and unknown type were 63.9%,31.9%,3.1%and 1.0%,respectively.83 patients had chromosomal karyotype analysis results and28 patients had additional Chromosomal Alterations/Philadephia Chromosome(ACA/Ph+).ACA/Ph+ with higher frequency were double Ph+ chromosome,+8,i17 q,-7 and so on.78 patients had analysis results for mutations in the ABL kinase region and 34 were positive for ABL mutations.The most frequent mutations were T315 I mutation.A total of 174 cases(91.1%)of CML-BC patients were able to evaluate the hematological remission rate.Among them,77 cases(44.3%)achieved MHR,and52 cases(29.9%)achieved CHR.Only 17 cases(8.9%)were evaluable for cytogenetic remission.Among them,9 cases(52.9%)obtained CCy R.A total of 81patients(42.4%)were able to evaluate the hematological remission rate.39 patients(48.1%)obtained MR1,30 patients(37.0%)obtained MR2,and 25 patients(30.9%)obtained MMR.The highest remission rates for MHR,CHR,CCy R,MR1,MR2 and MMR,were all in the transplantation group,followed by the TKI+ combined chemotherapy group,followed by the TKI alone group,and finally the non-active treatment group and the differences of the five comparisons were statistically significant(P<0.050).For first-line treatment grouping,the highest remission rates for MHR and CHR were in the first-line TKI+ combined chemotherapy group,followed by the first-line TKI alone group,then the first-line combined chemotherapy group,and finally the first-line non-active treatment group(P<0.050).Binary logistic regression analysis found that TKI+combined chemotherapy was the independent promoting factor of MHR(OR=5.770,P=0.010)with reference to TKI alone group;diagnosed in 2013-2020 was the independent promoting factor of MHR(OR=5.914),P=0.013)with reference to the group diagnosed in 2007-2012;no history of exposure was the independent promoting factor of MHR(OR=8.743,P=0.003)with reference to history of TKI exposure before and lymphoid BC became a independent promoting factor of MHR(OR=4.192,P=0.034)with reference to myeloid BC.The median OS of 191 CML-BC patients was 8.7 months(95%CI 5.7-11.7).Transplantation group didn’t reach its median OS and the median OS of TKI+combined chemotherapy group,TKI alone group,and non-active treatment group were 12.5 months(95%CI 7.9-17.1),7.8 months(95%CI 5.4-10.3)and 0.3 months(95%CI 0.1-0.5).There was no significant difference in OS between the TKI+combined chemotherapy group and the TKI alone group(P=0.140),and the difference between any other two groups was statistically significant(P<0.008).The median OS of the four groups in the first-line TKI+ combined chemotherapy group,first-line TKI alone group,first-line combined chemotherapy group,and first-line non-active treatment group were 12.3 months(95%CI 9.4-15.2),9.7months(95%CI 3.7-15.6).),unreached median OS and 0.3 months(95%CI 0.1-0.5).The OS of the first-line non-active treatment group was worse than the other three groups and the difference was statistically significant(P<0.001).There was no statistically difference in OS among the other three groups(P≥0.008).OS of groups whose patients reached MHR,CHR,CCy R,MR1,MR2 and MMR were better than the corresponding non-remission group,and the difference was statistically significant(P<0.050).The OS of the group with cytogenetic response evaluation or molecular response evaluation after treatment was better than that of the group without evaluation and the differences was statistically significant(P<0.050).The results of multivariate COX regression analysis showed that the other three treatments are all independent contributing factors to patient death(HR>1.000 and P<0.050)with reference to transplantation treatment;the period 2013-2020 is the independent hindering factor of patient death(HR= 0.473,P=0.002)with reference to the period 2007-2012;Hb≥60g/L as the independent hindering factor of patient death(HR=0.526,P=0.042)with reference to Hb<60g/L;LDH≥715 IU/L is the independent contributing factor of patient death(HR=2.074,P=0.005)with reference to LDH<715 IU/L and the first diagnosis is that CML-BC is the independent hindering factor of patient death(HR=0.552,P =0.042)with reference to initial diagnosis stage CML-CP/AP.A total of 188 CML-BC patients can be evaluated for first-line FFS,with a median FFS of 3.0 months(95% CI 2.2-3.8).The median FFS of first-line TKI+combined chemotherapy group,first-line TKI alone group,first-line combined chemotherapy group,and first-line non-active treatment group were 4.7 months(95%CI 2.9-6.5),3.5 months(95%CI 2.7-4.3),1.0 month(95%CI 0.4-1.6)and 0.3month(95%CI 0.1-0.5).The FFS of the first-line TKI+ combined chemotherapy group and the first-line TKI alone group are better than the other two groups and all the differences were statistically significant(P<0.008).The results of multivariate COX regression analysis showed that the other three treatments are all independent contributing factors to the FFS endpoint(HR>1.000)with reference to first-line TKI+combined chemotherapy,but only the first-line non-active treatment group has statistical significance(P<0.001);the 2013-2020 period is the independent obstructive factor of the FFS endpoint(HR=0.399,P=0.001)with reference to the period 2007-2012;the bone marrow blast cell% ≥39% is the independent promoting factor of FFS endpoint(HR=2.661,P<0.001)with reference to the bone marrow blast %<39%;history of TKI exposure was the independent promoting factor of the FFS endpoint(HR=2.015,P=0.017)with reference to no history.During the transcriptomics analysis in this study,we screened a total of 478 differentially expressed genes between CML-BC and CML-AP in the GSE4170 data set,including 315 down-regulated genes and 163 up-regulated genes.The top five up-regulated genes were IQCF2,C6orf165,C10orf68,IRF4,and NES;the top five down-regulated genes were CEACAM7,IL8 RB,GGTA1,FLJ31978,and HP.GO analysis found that the up-regulated genes of CML-BC relative to CML-AP are mainly enriched in synaptic membranes and other parts of the synapse,and participate in biological processes such as synaptic organization and that down-regulated genes are mainly enriched in secretory granules and other parts and participate in neutrophils related biological processes such as degranulation and neutrophil activation.KEGG analysis found that the up-regulated genes of CML-BC relative to CML-AP are mainly enriched in ribosomes,ribosome biosynthesis,RNA transport and aminoacyl-t RNA biosynthesis and other related pathways;down-regulated genes are mainly enriched in Toll-like receptors Signaling pathways and other immune-related pathways.HALLMARK gene set enrichment analysis found that the up-regulated genes of CML-BC relative to CML-AP are mainly enriched in Myc＿Targets＿V2,Myc＿Targets＿V1 and oxidative phosphorylation pathways.We also screened a total of 205 differentially expressed genes between CML-BC and CML-CP in the GSE4170 dataset,including 181 down-regulated genes and 24 up-regulated genes.The top five genes up-regulated were EMP1,ZBTB8,HOP,IFI27,and HLA-DPB1;the top five genes down-regulated were S100A12,LCN2,HP,OLFM4,MNDA.Through GO analysis,we found that the up-regulated genes of CML-BC relative to CML-CP are mainly enriched in focal adhesion and cell-substrate junction,and participate in biological processes such as cell growth and regulation and that down-regulated genes are mainly enriched in secretory granules and other parts,participate in related biological processes such as neutrophil degranulation and neutrophil activation.KEGG analysis found that the up-regulated genes of CML-BC relative to CML-CP are mainly enriched in related pathways such as ribosomes and aminoacyl-t RNA biosynthesis;down-regulated genes are mainly enriched in immune-related pathways such as phagocytosis mediated by Fcγ receptors.HALLMARK gene set enrichment analysis found that the up-regulated genes of CML-BC relative to CML-AP were mainly enriched in Myc＿Targets＿V2 and Myc＿Targets＿V1 pathways.By drawing the Venn diagram and taking the intersection,we screened out the common differentially expressed genes of CML-BC vs CML-AP and CML-BC vs CML-CP,and initially obtained 5 common differentially expressed up-regulated genes and 137 common differentially expressed down-regulated genes.The 137common-down-regulated genes are mainly enriched in immune-related pathways such as neutrophil degranulation and myeloid leukocyte activation.The protein interaction network diagram of 142 common differentially expressed genes was constructed,and the top 10 central genes in the protein interaction network were screened out,namely ITGAM、S100A12、TYROBP、MNDA、ITGB2、TLR8、PTGS2、CYBB、FPR1、PRTN3、RNASE2、LCN2、CEACAM8、LILRA1、RNASE3.Conclusions:1.The overall treatment of CML-BC is preferred to recommend Allo-HSCT treatment.Before transplantation,TKI+combined chemotherapy or TKI alone is recommended to get hematological remission as soon as possible.2.For CML-BC patients who are not eligible to undergo Allo-HSCT,TKI+combined chemotherapy is recommended followed by TKI alone treatment.When the overall treatment includes TKI,it is recommended to change TKI treatment in time when ABL mutation or treatment intolerance is found and to change TKI treatment or add combined chemotherapy treatment when the disease is not well controlled.When the overall treatment includes combined chemotherapy,it is recommended to complete two or more cycles of combined chemotherapy and we recommend VD/VP adding daunorubicin/ idarubicin treatment for patients with CML-LBC and DA/IDA or HA chemotherapy for patients with CML-MBC.3.TKI+ combined chemotherapy,diagnosis in the 2013-2020 period,no history of TKI exposure before the diagnosis of BC,and lymphoid BC are all independent contributing factors for obtaining MHR.Transplantation treatment,diagnosis in 2013-2020 period,Hb≥60g/L,LDH<715 IU/L and first diagnosis is that CML-BC are all independent hindering factors for the death of patients.4.For first-line treatment,first-line TKI+combined chemotherapy is first recommended,followed by first-line TKI alone treatment.First-line TKI+ combined chemotherapy,diagnosis in the 2013-2020 period,percentage of bone marrow blasts<39%,and no history of TKI exposure before the diagnosis of BC are all independent hindering factors for the FFS endpoint.5.The comprehensive monitoring of treatment response,especially cytogenetic response,molecular response,ABL kinase mutation,are insufficient in real-world.Recommend to strengthen comprehensive monitoring in the future clinical practice.6.Compared with CML-AP,the expression of IQCF2,CFAP206,CCDC7,IRF4,NES and other genes are up-regulated in CML-BC;the expression of CEACAM7,CXCR2,GGTA1,GLT1D1,and HP is down-regulated in CML-BC.7.Compared with CML-CP,EMP1,ZBTB8,HOPX,IFI27,HLA-DPB1 and other genes are up-regulated in CML-BC;S100A12,LCN2,HP,OLFM4,MNDA and other genes are down-regulated in CML-BC.8.Compared with CML-CP or CML-AP,CML-BC is up-regulated in the MYC-TARGET pathway,ribosome and protein translation related pathways are up-regulated,thus promote tumor cell growth and proliferation;is down-regulated mainly in various immune-related pathways,thus increase the risk of infection.9.ITGAM、S100A12、TYROBP、MNDA、ITGB2、TLR8、PTGS2、CYBB、FPR1、PRTN3、RNASE2、LCN2、CEACAM8、LILRA1、RNASE3 are the central genes for the progression to CML-BC,which may be the key gene leading to the occurrence of CML-BC.更多还原