Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity. Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. Here we characterize in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and after induction chemotherapy using paired samples from 6 patients with primary refractory AML. Overall design: Primary human AML were from the Manchester Cancer Research Centre Tissue Biobank (approved by the South Manchester Research Ethics Committee). Their use was authorized by the Tissue Biobank’s scientific sub-committee, with the informed consent of donors. Blasts were flow sorted according to either a CD45low/intCD34+ (n=5) or CD45low/intCD117+ (n=1) immunophenotype. Total RNA was extracted from 10^3-10^5 sorted blasts using QIAshredder spin columns and an RNeasy® Plus Micro kit (Qiagen). Prior to sequencing RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA yield from the sorted populations ranged from 3.5-475ng. To ensure consistency the sample with the lowest yield was used to define the input (3.5ng) for amplification of all samples. Amplification was performed using a SMARTer Stranded Total RNA-Seq Pico input kit (Clontech, Mountain View, CA). Sequencing was performed using a NextSeq desktop sequencing system (Illumina). A single run (400M reads) of 151bp paired-end sequencing produced a mean of 31.4M reads per sample (range 29.3-33.5M).

急性髓系白血病（acute myeloid leukemia, AML）治疗后疾病复发仍较为常见，其根源在于具备疾病重建潜能的白血病细胞产生了化疗耐药性。迄今为止，针对体内耐药白血病母细胞（blast）特征的研究多聚焦于初诊与复发时白血病细胞的对比分析。然而复发后往往仍可观察到后续治疗应答，提示大部分白血病母细胞仍保持化疗敏感性。本研究通过分析6例原发难治性AML患者的配对样本，从诱导化疗前后的白血病母细胞转录组与遗传特征层面，完成了体内化疗耐药母细胞的特征解析。总体实验设计：本研究中的原代人AML样本均取自曼彻斯特癌症研究中心组织生物样本库（已获得南曼彻斯特研究伦理委员会批准），样本使用已通过生物样本库科学小组委员会授权，并遵循供者的知情同意原则。研究人员按照CD45low/intCD34+（n=5）或CD45low/intCD117+（n=1）的免疫表型，对白血病母细胞进行流式分选。使用QIAshredder离心柱与RNeasy® Plus Micro试剂盒（Qiagen），从10^3至10^5个分选得到的母细胞中提取总RNA。测序前，采用2100生物分析仪（Agilent Technologies，美国加利福尼亚州圣克拉拉市）检测RNA完整性。分选所得细胞群的总RNA产出量介于3.5ng至475ng之间。为保证实验一致性，以产出量最低的样本（3.5ng）作为所有样本扩增的起始投入量。采用SMARTer Stranded Total RNA-Seq Pico input试剂盒（Clontech，美国加利福尼亚州芒廷维尤市）完成扩增。使用NextSeq桌面测序系统（Illumina）进行测序：单次运行（4亿条reads）采用151bp双端测序策略，单样本平均测序reads数为31.4M（区间为29.3M至33.5M）。