微小残留白血病监测的研究
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摘要
研究背景
随着对白血病认识的不断深入,白血病的诊断、治疗和预后等水平也不断提高。儿童及成人急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)的5年无病生存(disease-free survival,DFS)率分别达75%及35%甚至更高,急性髓细胞白血病(acute myeloid leukemia,AML)的完全缓解(complete remission,CR)率和5年DFS率分别为60%~70%和30%,其中急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)的CR率达85%。但是,目前仍有部分经治疗后获得完全缓解的患者在数年之内复发。
白血病初诊时,患者体内的白血病细胞数量约为1012,经化疗取得临床及血液学完全缓解后,体内仍存在106~108个残留白血病细胞,这些存在于体内的形态上不能检测到的白血病细胞称为微小残留白血病(Minimal Residual Disease,MRD),是引起白血病复发的主要原因之一。因此,在白血病治疗过程中准确测定MRD并分析其意义,对临床追踪病情、判断预后、制定治疗方案等,具有重要意义。
在白血病治疗过程中准确测定MRD并分析其意义,具有如下意义:①根据体内白血病细胞的负荷决定是继续治疗还是停止治疗;②更早地发现化疗药物的耐药性;③更早地预测白血病复发;④评价骨髓移植的预处理效果;⑤为体内残留白血病细胞分布和增殖动力学的研究提供可能性。
白血病MRD检测,必须符合以下条件:①敏感度至少为10-3(即可在103个正常细胞中检出一个白血病细胞);②所采用的检测标志比较稳定,不会随病程改变而导致假阴性或假阳性结果;③不同实验室之间的检查结果具有可重复性及可比性;④检测方法易于在临床推广应用。
目前用于检测MRD的方法主要有核型分析、荧光原位杂交(fluorescence in situ hybridization,FISH)、流式细胞术(flow cytometry,FCM)和聚合酶链反应(polymerasechain reaction,PCR)等,不同方法各有利弊。核型分析、FISH和FCM等的敏感性分别为10-1~10-2、10-2~10-3和10-3~10-4,难以满足对MRD诊断的需要,且都存在或操作费时,或费用较高,或对样本要求较高等不足之处。FQ-PCR (fluorescent quantitative PCR,FQ-PCR)是在PCR基础上发展的核酸分子定量技术,该技术以Ig/TCR重排、染色体易位断裂融合区为标志,结合探针和引物的合理搭配,利用荧光信号的变化,实时检测PCR扩增反应中每一个循环扩增产物量的变化,通过循环阈值(cycle threshold, Ct)和标准曲线的分析对起始模板进行定量分析,具有敏感度高、准确定量、简便快速,污染较少等优点,是目前最为理想的MRD定量检测方法。
检测白血病MRD的靶标志之一融合基因是由于染色体结构改变导致的分子水平的异常,目前已发现超过50种与白血病有关的融合基因异常,这些异常已成为不同类型白血病的分子生物学特异性标志,可作为临床上对白血病进行分型、预后判断和残留病追踪的依据。由欧洲10个国家26个实验室共同参与研究制定的“欧洲防癌计划”(Europe Against Cancer program,EAC),通过以FQ-PCR方法检测9种主要的白血病特异融合基因,建立了一套标准化的MRD定量检测方法,并初步评价该方法的敏感度、特异性和重复性,为大规模的临床实验研究建立了方法学基础。该标准化方法在儿童及成人ALL及AML中的检出率达30%～40%,在CML中超过95%。
尽管以FQ-PCR检测白血病融合基因已应用于临床,但仍存在下列问题:①对一项检测方法的评价,应在严格的质量控制的前提下,对实验结果进行真实性(包括敏感性、特异性和约登指数)和可靠性(主要以变异系数表示)等方面的评价,而目前的文献中,仅见对FQ-PCR检测白血病融合基因的检测方法进行敏感性、特异性和可靠性的评价,而未见质量控制等方面的报道;②在引物、探针、内参基因的选择等技术问题上尚未达成一致;③目前国际上尚未统一各种白血病融合基因的表达水平与临床分级和预后的关系。
我们通过前期研究,已分别构建了含BCR-ABL M-bcr、BCR-ABL m-bcr、BCR-ABLμ-bcr、PML-RARα、AML1-ETO、TEL-AML1、CBFβ-MYH11、E2A-PBX1和MLL-AF4等9种白血病相关融合基因的重组质粒,以及含ABL内参基因的重组质粒,并建立了其FQ-PCR检测的标准曲线及标准方程。本研究的目的,是在上述前期研究的基础上,以K562细胞(含BCR-ABL融合基因)、REH细胞(含TEL-AML1融合基因)、NB4细胞(含PML-RARα融合基因)和KASUMI-1细胞(含AML1-ETO融合基因)等4种细胞为阳性细胞,以HL-60细胞系(不含上述白血病相关融合基因)作为阴性细胞,以含BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种白血病相关融合基因的重组质粒为标准品,以FQ-PCR方法检测阳性细胞中BCR-ABL M-bcr、TEL-AML1、PML-RARα和AML1-ETO等4种白血病融合基因的表达量,并通过对检测结果进行室内质量控制和误差分析,以及真实性和可靠性的判断,对FQ-PCR检测白血病融合基因进行方法学的评价。在此基础上,应用FQ-PCR检测初诊白血病患儿的相关融合基因,并在治疗过程中对融合基因的表达量进行初步追踪观察,为临床应用FQ-PCR监测白血病MRD奠定基础。
研究目的
1.通过细胞水平的研究,对FQ-PCR定量检测白血病融合基因进行全面的方法学评价。
2.以FQ-PCR定量检测初诊白血病患儿的相关融合基因,对阳性者,追踪观察其在白血病治疗过程中的变化,为进一步临床研究奠定基础。
研究内容
1.选取K562细胞(含BCR-ABL融合基因)、REH细胞(含TEL-AML1融合基因)、NB4细胞(含PML-RARα融合基因)和KASUMI-1细胞(含AML1-ETO融合基因)作为阳性细胞,以HL-60细胞(不含上述白血病相关融合基因)作为阴性细胞,将上述4种细胞分别梯度稀释为1:100～1:105 6个浓度,以FQ-PCR检测相应融合基因的相对表达量。
2.根据上述检测结果,通过计算SI值、灵敏度和变异系数(coefficient of variation,CV),对实验研究进行室内质量控制和误差控制的分析。
3.通过计算敏感性、特异性、约登指数和CV,判断检测方法的真实性和可靠性。
4.以FQ-PCR检测初诊白血病患儿的相关融合基因,阳性者,在治疗过程中继续追踪观察相关融合基因表达量的变化。
技术路线
1.细胞水平研究
2.临床观察
研究方法
1.细胞水平的研究
(1)培养K562细胞、REH细胞、NB4细胞和KASUMI-1细胞。
(2)用HL-60细胞作为阴性细胞梯度稀释上述4种细胞,其稀释浓度为1:100～1:105。
(3)提取上述细胞RNA,并逆转录为cDNA。
(4)以含BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种白血病相关融合基因的重组质粒为标准品,通过FQ-PCR建立标准曲线和标准方程。以FQ-PCR检测待检细胞的BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种融合基因,通过质粒标准品的标准曲线及标准方程得出融合基因的相对表达量(融合基因拷贝数与内参基因拷贝数之比),以NCN表示。
(5)上述实验每组设3个复管,连续3天重复实验,即每种细胞每一浓度可获得9个实验数据。从第三次实验开始进行“即刻法”质控,直至第九次实验结束。根据实验结果计算SI值、批内CV、批间CV和灵敏度,进行室内质量控制和误差控制的分析。
(6)计算敏感性、特异性、约登指数、批内CV和批间CV,评价检测方法的真实性和可靠性。
2.临床观察
(1)采集经骨髓细胞形态、免疫分型确诊的初诊白血病患儿骨髓并抽提RNA,制备cDNA。
(2)FQ-PCR定量检测7种白血病相关融合基因。
(3)白血病相关融合基因阳性者,于诱导缓解治疗后追踪融合基因表达量的变化。
研究结果
1.质粒标准品经FQ-PCR检测后,生成标准曲线、标准方程、相关系数r2及扩增效率(见表5)。
2.FQ-PCR检测白血病融合基因的结果显示:①当细胞浓度在1:100~1:105范围时,细胞浓度与待检融合基因的Ct值呈负相关;②细胞浓度每下降1个对数级,其待检融合基因的NCN值相应下降0.5~1个对数级,两者之间的相关系数r2在0.985~0.986之间, P<0.001 ,呈高度正相关;③内参基因的Ct值在21.86±0.05~26.48±0.73范围之间。
3.每种细胞每一浓度的第九次实验结束后,其NCN的SI上限值和SI下限值均小于Q2s。
4.FQ-PCR检测4种细胞系相关融合基因的灵敏度均为10-5。
5.FQ-PCR检测白血病融合基因,敏感度以TEL-AML1和AML1-ETO最高(100%),BCR-ABL M-bcr次之(97.92%),PML-RARα最低(89.58%);TEL-AML1、PML-RARα和AML1-ETO的特异度一致(93.75%),BCR-ABL M-bcr的特异度较低(81.25%);约登指数以TEL-AML1和AML1-ETO最接近理想(0.94),PML-RARα次之(0.83),BCR-ABL M-bcr最小(0.79)。
6.在被检的4种融合基因中,KASUMI-1细胞浓度为1:100～1:105时,其AML1-ETO融合基因NCN值的批内CV和批间CV均小于3%,可靠性最好;K562、REH和NB4细胞浓度为1:100～1:103时,其相关融合基因NCN值的批内CV和批间0CV均小于10%,可靠性较好,在细胞浓度为1:104～1:105时,其相关融合基因NCN值的批内CV小于6%,可靠性也较好,而批间CV则小于30%,可靠性逊于上述。
7.收集初诊白血病标本55例,FQ-PCR检测初诊白血病患者融合基因的检出率为28.57%,其中初诊ALL的检出率为15.38%,AML的检出率为14.29%,APL和CML的检出率均为100.00%。
8.对6例APL患儿予诱导缓解化疗,2例CML患儿予格列卫治疗,取得CR后继续以FQ-PCR追踪PML-RARα和BCR-ABL融合基因的NCN值,结果见表12和表13。
结论
1.每种白血病细胞稀释为1:100～1:105 6个浓度,每一浓度设3个复管,连续检测3天,所获得的全部数据均在可控范围内。
2.细胞浓度每下降1个对数级,其待检融合基因的NCN值相应下降0.5~1个对数级,两者之间的相关系数r2在0.985~0.986之间,P<0.001,呈高度正相关性。
3.FQ-PCR检测白血病融合基因的灵敏度为10-5,与国外文献报道一致。
4.FQ-PCR检测白血病融合基因,敏感度以TEL-AML1和AML1-ETO最高,为100%,BCR-ABL M-bcr次之(97.92%),PML-RARα最低(89.58%);TEL-AML1、PML-RARα和AML1-ETO的特异度一致(93.75%),BCR-ABL M-bcr的特异度较低(81.25%);约登指数以TEL-AML1和AML1-ETO最接近理想(0.94),PML-RARα次之(0.83),BCR-ABL M-bcr最小(0.79)。
5.当KASUMI-1细胞浓度在1:100~1:105范围时,FQ-PCR检测AML1-ETO融合基因的NCN值的批内CV和批间CV均小于3%,可靠性和精密度最好;K562、REH和NB4细胞浓度为1:100～1:103时,FQ-PCR检测BCR-ABL M-bcr、TEL-AML1和PML-RARα的NCN值的批内CV和批间CV均小于10%,可靠性和精密度较好,在细胞浓度为1:104～1:105时,上述相关融合基因NCN值的批内CV小于6%,可靠性和精密度也较好,而批间CV小于30%,但仍在可控范围之内。
6.收集55例初诊白血病患儿的骨髓标本,FQ-PCR检测初诊白血病患者融合基因的检出率为28.57%,其中初诊ALL的检出率为15.38%,AML的检出率为14.29%,APL和CML的检出率均为100.00%。初步追踪观察融合基因水平变化与治疗反应一致。
Background
As the recognition is more and more further,diagnosis,treatment and prognosis of leukemia is advancing. 5 years disease free survival (DFS) rate of childhood and adult ALL is more than 75% and 35%, complete remission (CR) rate and 5 years DFS rate in AML is 60% ~70% and 30% respectively, CR rate in APL is 85%. But some patients still suffer from relapse within a few years.
At the time of diagnosis, the number of malignant cells in vivo is about 1012 or more. After chemotherapy and achieved clinical and hematological CR, there are remain 106~108 malignant cells in vivo. The leukemia cells which can not be detected by morphological examination are defined minimal residual disease (MRD), and it is the primary reason that cause relapse.
Detecting MRD accurately during treatment of leukemia and analysis its meaning is significance to clinical disease condition monitoring, prognosis judgement and treatment protocols selection. The meaning of MRD detection lies in :①decide stop treatment or not according to the quantity of malignant cells in vivo;②reveal drug resistance;③predict relapse earlier;④evaluate the pretreatment effect of marrow transplantation;⑤provides research probability of distribution and proliferation of leukemia cells.
At present, the methods of MRD detection include karyotype analysis, fluorescence in situ hybridization (FISH), flow cytometry (FCM), polymerase chain reaction (PCR), etc. Each method has advantage and disadvantage. Sensitivity of karyotype analysis, FISH and FCM is 10-1~10-2, 10-2~10-3 and 10-3~10-4 respectively, can not satisfy the MRD diagnosis demand, and has disadvantage such as time consuming, expensive, high quality requirements of sample. Fluorescent quantitative PCR(FQ-PCR) is base on the PCR technology, it add fluorescent probe in the PCR system, detecting the change of fluorescence signal to estimate the increasing PCR product.FQ-PCR through cycle threshold(Ct) and standard curve to analysis the quantity of sample,and has advantage of sensitive, quantitive, convenient.It is the better method of MRD detection.
As one of the markers detected leukemia MRD, fusion gene is molecular abnomal resulted from the change of chromosome structure.Now it has found more than 50 leukemia-associated fusion genes,these abnomal is the mark of some types of leukemia,also it is useful for realize leukemia types, prognosis and MRD detection. The EAC program, instituted by 26 European university laboratories from 10 countries, had collaborated to establish a standardizaed protocol for TaqMan based FQ-PCR analysis of 9 main leukemia-associated fusion genes to quantify MRD, in order to evaluate leukemia curative effect and prognosis.Using the standardizaed protocol, 30%~40% of childhood and adult ALL and AML and more than 95% of CML patients can be detected.
We have constructed 9 recombinant plasmids containing leukemia relative fusion genes, and established the standard curve and standard equation of the recombinant plasmids. To evaluate the method of FQ-PCR detecting leukemia relative fusion genes, cellular experiment and clinical observation were performed in this study.
Objective
1.To evaluate the method of FQ-PCR detecting leukemia relative fusion genes by cellular experimental study.
2.To detect leukemia relative fusion genes of patients with leukemia by FQ-PCR at diagnosis, and observe the variation of quantity of leukemia relative fusion genes during treatment.
Contents
1.To dilute K562 cell(contain BCR-ABL M-bcr), REH cell(contain TEL-AML1), NB4 cell (contain PML-RARα) and KASUMI-1 cell(contain AML1-ETO) using HL-60 cell (without leukemia relative fusion genes) in 1:100~1:105, extract RNA, reverse transcript to cDNA, and detect the qualities of fusion genes by FQ-PCR, calculate NCN.
2.To calculate SI value and CV to evaluate quality control, and analyze the sensitivity, specificity, Youden’s index and CV to evaluate the validity and reliability of the experiment study of FQ-PCR.
3.To detect leukemia relative fusion genes of patients with leukemia by FQ-PCR at diagnosis, and observe the variation of quantity of leukemia relative fusion genes during treatment.
Technical Strategy
1.Cellular experimental study
1.Cellular experimental study
(1)To cultivate K562, REH, NB4 and KASUMI-1 cell lines.
(2)To dilute K562, REH, NB4 and KASUMI-1 cell lines using HL-60 cell line as negative cell in1:100 to 1:105.
(3)To extract RNA from the 6 diluted cells, then reverse transcription into cDNA.
(4)To establish standard curves and standard equations of recombinant plasmids which inserted TEL-AML1, PML-RARα, AML1-ETO and BCR-ABL M-bcr by FQ-PCR.
(5)To detect the fusion genes of diluted cells using FQ-PCR, calculate NCN values.
(6)To calculate the SI, intra-CV and extra-CV of NCN, in order to analyze quality control of the cellular experiment study using FQ-PCR.
(7)To estimate the sensitivity, specificity, Youden’s index, intra-CV and extra-CV, in order to evaluate the validity and reliability of the cellular experiment study using FQ-PCR.
2.Clinical observation
(1)Collect the bone marrow of patients at diagnosis.
(2)Extract RNAand reverse transcription into cDNA.
(3)Observe the variation of qualities of fusion genes after induction therapy.
Results
1.Standard curves and standard equations of the standard plasmids are established by FQ-PCR, the results are shown in the table 5 below.
2.The leukemia-associated fusion genes were detected by FQ-PCR, the results show that,①As the concentrations of cell lines within 1:100 to 1:105, the concentration has negative correlation with the Ct values of fusion genes detected from the cell lines.②the cell concentration decrease 1 log, the NCN of fusion genes decrease 0.5 to 1 log, the correlation coefficient (r2) of them are between 0.985 to 0.986.③the Ct values of control gene are between 21.86±0.05 to 26.48±0.73.
3.All of the SI values that calculated from experimental data are less than the value of Q2s.
4.To detect leukemia relative fusion genes using FQ-PCR, the detectable concentration of cell lines is at least 10-5.
5.To detect leukemia relative fusion genes using FQ-PCR, the sensitivity was 100.00% (both TEL-AML1 and AML1-ETO), 97.92% (BCR-ABL M-bcr) and 89.58% (PML-RARα).The specificity was 93.75% (TEL-AML1, PML-RARαand AML1-ETO) and 81.25% (BCR-ABL M-bcr). Youden’s index was0.94(both TEL-AML1 and AML1-ETO), 0.83 (PML-RARα), and 0.79 (BCR-ABL M-bcr).
6.As the concentrations of KASUMI-1cell line within 1:100～1:105, the intra-CV and extra-CV of the NCN of AML1-ETO fusion gene were less than 3%, and the repeatability and precision were the first. As the concentrations of K562, REH and NB4 cell lines within 1:100～1:103, the intra-CV and extra-CV of the NCN of BCR-ABL M-bcr、TEL-AML1 and PML-RARαwere less than 10%, and the repeatability and precision were second,as the concentrations within 1:104～1:105, the intra-CV was less than 6%, the extra-CV was less than 30%.
7.The leukemia relative fusion genes were detected using FQ-PCR in 55 childhood with leukemia at diagnosis, fusion genes were found in 28.57% of all the patients, 15.38% of ALL, 14.29% of AML, 100.00% of APL and CML. During every stage of treatment, the NCN of PML-RARαand BCR-ABL(shown in table 12 and 13) were consistent with the response to the chemotherapy.
Conclusions
1.All the NCN values are in the control range.
2.As the concentration of 4 cell lines decrease 1 log, the NCN values of the leukemia-relative fusion genes reduce 0.5 or 1 log.
3.To detect leukemia relative fusion genes using FQ-PCR, the detectable minimal concentration of cell lines is 10-5.
4.To detect leukemia relative fusion genes using FQ-PCR, the sensitivity was 100.00% (both TEL-AML1 and AML1-ETO), 97.92% (BCR-ABL M-bcr) and 89.58% (PML-RARα).The specificity was 93.75% (TEL-AML1, PML-RARαand AML1-ETO) and 81.25% (BCR-ABL M-bcr). Youden’s index was 0.94(both TEL-AML1 and AML1-ETO), 0.83 (PML-RARα), and 0.79 (BCR-ABL M-bcr).
5.As the concentrations of KASUMI-1cell line within 1:100～1:105, the intra-CV and extra-CV of the NCN of AML1-ETO fusion gene were less than 3%, and the repeatability and precision were the first. As the concentrations of K562, REH and NB4 cell lines within 1:100～1:103, the intra-CV and extra-CV of the NCN of BCR-ABL M-bcr、TEL-AML1 and PML-RARαwere less than 10%, and the repeatability and precision were second,as the concentrations within 1:104～1:105, the intra-CV was less than 6%, the extra-CV was less than 30%.
6.The leukemia relative fusion genes were detected using FQ-PCR in 55 childhood with leukemia at diagnosis, fusion genes were found in 28.57%,E2A-PBX1 TEL-AML1 and MLL-AF4 were found in 15.38% of ALL, CBFβ-MYH11 was found in 14.29% of AML, both PML-RARαand BCR-ABL M-bcr were found in 100.00% of APL and CML. During every stage of treatment, the NCN of PML-RARαand BCR-ABL were consistent with the response to the chemotherapy.
随着对白血病认识的不断深入,白血病的诊断、治疗和预后等水平也不断提高。儿童及成人急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)的5年无病生存(disease-free survival,DFS)率分别达75%及35%甚至更高,急性髓细胞白血病(acute myeloid leukemia,AML)的完全缓解(complete remission,CR)率和5年DFS率分别为60%~70%和30%,其中急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)的CR率达85%。但是,目前仍有部分经治疗后获得完全缓解的患者在数年之内复发。
白血病初诊时,患者体内的白血病细胞数量约为1012,经化疗取得临床及血液学完全缓解后,体内仍存在106~108个残留白血病细胞,这些存在于体内的形态上不能检测到的白血病细胞称为微小残留白血病(Minimal Residual Disease,MRD),是引起白血病复发的主要原因之一。因此,在白血病治疗过程中准确测定MRD并分析其意义,对临床追踪病情、判断预后、制定治疗方案等,具有重要意义。
在白血病治疗过程中准确测定MRD并分析其意义,具有如下意义:①根据体内白血病细胞的负荷决定是继续治疗还是停止治疗;②更早地发现化疗药物的耐药性;③更早地预测白血病复发;④评价骨髓移植的预处理效果;⑤为体内残留白血病细胞分布和增殖动力学的研究提供可能性。
白血病MRD检测,必须符合以下条件:①敏感度至少为10-3(即可在103个正常细胞中检出一个白血病细胞);②所采用的检测标志比较稳定,不会随病程改变而导致假阴性或假阳性结果;③不同实验室之间的检查结果具有可重复性及可比性;④检测方法易于在临床推广应用。
目前用于检测MRD的方法主要有核型分析、荧光原位杂交(fluorescence in situ hybridization,FISH)、流式细胞术(flow cytometry,FCM)和聚合酶链反应(polymerasechain reaction,PCR)等,不同方法各有利弊。核型分析、FISH和FCM等的敏感性分别为10-1~10-2、10-2~10-3和10-3~10-4,难以满足对MRD诊断的需要,且都存在或操作费时,或费用较高,或对样本要求较高等不足之处。FQ-PCR (fluorescent quantitative PCR,FQ-PCR)是在PCR基础上发展的核酸分子定量技术,该技术以Ig/TCR重排、染色体易位断裂融合区为标志,结合探针和引物的合理搭配,利用荧光信号的变化,实时检测PCR扩增反应中每一个循环扩增产物量的变化,通过循环阈值(cycle threshold, Ct)和标准曲线的分析对起始模板进行定量分析,具有敏感度高、准确定量、简便快速,污染较少等优点,是目前最为理想的MRD定量检测方法。
检测白血病MRD的靶标志之一融合基因是由于染色体结构改变导致的分子水平的异常,目前已发现超过50种与白血病有关的融合基因异常,这些异常已成为不同类型白血病的分子生物学特异性标志,可作为临床上对白血病进行分型、预后判断和残留病追踪的依据。由欧洲10个国家26个实验室共同参与研究制定的“欧洲防癌计划”(Europe Against Cancer program,EAC),通过以FQ-PCR方法检测9种主要的白血病特异融合基因,建立了一套标准化的MRD定量检测方法,并初步评价该方法的敏感度、特异性和重复性,为大规模的临床实验研究建立了方法学基础。该标准化方法在儿童及成人ALL及AML中的检出率达30%～40%,在CML中超过95%。
尽管以FQ-PCR检测白血病融合基因已应用于临床,但仍存在下列问题:①对一项检测方法的评价,应在严格的质量控制的前提下,对实验结果进行真实性(包括敏感性、特异性和约登指数)和可靠性(主要以变异系数表示)等方面的评价,而目前的文献中,仅见对FQ-PCR检测白血病融合基因的检测方法进行敏感性、特异性和可靠性的评价,而未见质量控制等方面的报道;②在引物、探针、内参基因的选择等技术问题上尚未达成一致;③目前国际上尚未统一各种白血病融合基因的表达水平与临床分级和预后的关系。
我们通过前期研究,已分别构建了含BCR-ABL M-bcr、BCR-ABL m-bcr、BCR-ABLμ-bcr、PML-RARα、AML1-ETO、TEL-AML1、CBFβ-MYH11、E2A-PBX1和MLL-AF4等9种白血病相关融合基因的重组质粒,以及含ABL内参基因的重组质粒,并建立了其FQ-PCR检测的标准曲线及标准方程。本研究的目的,是在上述前期研究的基础上,以K562细胞(含BCR-ABL融合基因)、REH细胞(含TEL-AML1融合基因)、NB4细胞(含PML-RARα融合基因)和KASUMI-1细胞(含AML1-ETO融合基因)等4种细胞为阳性细胞,以HL-60细胞系(不含上述白血病相关融合基因)作为阴性细胞,以含BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种白血病相关融合基因的重组质粒为标准品,以FQ-PCR方法检测阳性细胞中BCR-ABL M-bcr、TEL-AML1、PML-RARα和AML1-ETO等4种白血病融合基因的表达量,并通过对检测结果进行室内质量控制和误差分析,以及真实性和可靠性的判断,对FQ-PCR检测白血病融合基因进行方法学的评价。在此基础上,应用FQ-PCR检测初诊白血病患儿的相关融合基因,并在治疗过程中对融合基因的表达量进行初步追踪观察,为临床应用FQ-PCR监测白血病MRD奠定基础。
研究目的
1.通过细胞水平的研究,对FQ-PCR定量检测白血病融合基因进行全面的方法学评价。
2.以FQ-PCR定量检测初诊白血病患儿的相关融合基因,对阳性者,追踪观察其在白血病治疗过程中的变化,为进一步临床研究奠定基础。
研究内容
1.选取K562细胞(含BCR-ABL融合基因)、REH细胞(含TEL-AML1融合基因)、NB4细胞(含PML-RARα融合基因)和KASUMI-1细胞(含AML1-ETO融合基因)作为阳性细胞,以HL-60细胞(不含上述白血病相关融合基因)作为阴性细胞,将上述4种细胞分别梯度稀释为1:100～1:105 6个浓度,以FQ-PCR检测相应融合基因的相对表达量。
2.根据上述检测结果,通过计算SI值、灵敏度和变异系数(coefficient of variation,CV),对实验研究进行室内质量控制和误差控制的分析。
3.通过计算敏感性、特异性、约登指数和CV,判断检测方法的真实性和可靠性。
4.以FQ-PCR检测初诊白血病患儿的相关融合基因,阳性者,在治疗过程中继续追踪观察相关融合基因表达量的变化。
技术路线
1.细胞水平研究
2.临床观察
研究方法
1.细胞水平的研究
(1)培养K562细胞、REH细胞、NB4细胞和KASUMI-1细胞。
(2)用HL-60细胞作为阴性细胞梯度稀释上述4种细胞,其稀释浓度为1:100～1:105。
(3)提取上述细胞RNA,并逆转录为cDNA。
(4)以含BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种白血病相关融合基因的重组质粒为标准品,通过FQ-PCR建立标准曲线和标准方程。以FQ-PCR检测待检细胞的BCR-ABL M-bcr、PML-RARα、AML1-ETO和TEL-AML1等4种融合基因,通过质粒标准品的标准曲线及标准方程得出融合基因的相对表达量(融合基因拷贝数与内参基因拷贝数之比),以NCN表示。
(5)上述实验每组设3个复管,连续3天重复实验,即每种细胞每一浓度可获得9个实验数据。从第三次实验开始进行“即刻法”质控,直至第九次实验结束。根据实验结果计算SI值、批内CV、批间CV和灵敏度,进行室内质量控制和误差控制的分析。
(6)计算敏感性、特异性、约登指数、批内CV和批间CV,评价检测方法的真实性和可靠性。
2.临床观察
(1)采集经骨髓细胞形态、免疫分型确诊的初诊白血病患儿骨髓并抽提RNA,制备cDNA。
(2)FQ-PCR定量检测7种白血病相关融合基因。
(3)白血病相关融合基因阳性者,于诱导缓解治疗后追踪融合基因表达量的变化。
研究结果
1.质粒标准品经FQ-PCR检测后,生成标准曲线、标准方程、相关系数r2及扩增效率(见表5)。
2.FQ-PCR检测白血病融合基因的结果显示:①当细胞浓度在1:100~1:105范围时,细胞浓度与待检融合基因的Ct值呈负相关;②细胞浓度每下降1个对数级,其待检融合基因的NCN值相应下降0.5~1个对数级,两者之间的相关系数r2在0.985~0.986之间, P<0.001 ,呈高度正相关;③内参基因的Ct值在21.86±0.05~26.48±0.73范围之间。
3.每种细胞每一浓度的第九次实验结束后,其NCN的SI上限值和SI下限值均小于Q2s。
4.FQ-PCR检测4种细胞系相关融合基因的灵敏度均为10-5。
5.FQ-PCR检测白血病融合基因,敏感度以TEL-AML1和AML1-ETO最高(100%),BCR-ABL M-bcr次之(97.92%),PML-RARα最低(89.58%);TEL-AML1、PML-RARα和AML1-ETO的特异度一致(93.75%),BCR-ABL M-bcr的特异度较低(81.25%);约登指数以TEL-AML1和AML1-ETO最接近理想(0.94),PML-RARα次之(0.83),BCR-ABL M-bcr最小(0.79)。
6.在被检的4种融合基因中,KASUMI-1细胞浓度为1:100～1:105时,其AML1-ETO融合基因NCN值的批内CV和批间CV均小于3%,可靠性最好;K562、REH和NB4细胞浓度为1:100～1:103时,其相关融合基因NCN值的批内CV和批间0CV均小于10%,可靠性较好,在细胞浓度为1:104～1:105时,其相关融合基因NCN值的批内CV小于6%,可靠性也较好,而批间CV则小于30%,可靠性逊于上述。
7.收集初诊白血病标本55例,FQ-PCR检测初诊白血病患者融合基因的检出率为28.57%,其中初诊ALL的检出率为15.38%,AML的检出率为14.29%,APL和CML的检出率均为100.00%。
8.对6例APL患儿予诱导缓解化疗,2例CML患儿予格列卫治疗,取得CR后继续以FQ-PCR追踪PML-RARα和BCR-ABL融合基因的NCN值,结果见表12和表13。
结论
1.每种白血病细胞稀释为1:100～1:105 6个浓度,每一浓度设3个复管,连续检测3天,所获得的全部数据均在可控范围内。
2.细胞浓度每下降1个对数级,其待检融合基因的NCN值相应下降0.5~1个对数级,两者之间的相关系数r2在0.985~0.986之间,P<0.001,呈高度正相关性。
3.FQ-PCR检测白血病融合基因的灵敏度为10-5,与国外文献报道一致。
4.FQ-PCR检测白血病融合基因,敏感度以TEL-AML1和AML1-ETO最高,为100%,BCR-ABL M-bcr次之(97.92%),PML-RARα最低(89.58%);TEL-AML1、PML-RARα和AML1-ETO的特异度一致(93.75%),BCR-ABL M-bcr的特异度较低(81.25%);约登指数以TEL-AML1和AML1-ETO最接近理想(0.94),PML-RARα次之(0.83),BCR-ABL M-bcr最小(0.79)。
5.当KASUMI-1细胞浓度在1:100~1:105范围时,FQ-PCR检测AML1-ETO融合基因的NCN值的批内CV和批间CV均小于3%,可靠性和精密度最好;K562、REH和NB4细胞浓度为1:100～1:103时,FQ-PCR检测BCR-ABL M-bcr、TEL-AML1和PML-RARα的NCN值的批内CV和批间CV均小于10%,可靠性和精密度较好,在细胞浓度为1:104～1:105时,上述相关融合基因NCN值的批内CV小于6%,可靠性和精密度也较好,而批间CV小于30%,但仍在可控范围之内。
6.收集55例初诊白血病患儿的骨髓标本,FQ-PCR检测初诊白血病患者融合基因的检出率为28.57%,其中初诊ALL的检出率为15.38%,AML的检出率为14.29%,APL和CML的检出率均为100.00%。初步追踪观察融合基因水平变化与治疗反应一致。
Background
As the recognition is more and more further,diagnosis,treatment and prognosis of leukemia is advancing. 5 years disease free survival (DFS) rate of childhood and adult ALL is more than 75% and 35%, complete remission (CR) rate and 5 years DFS rate in AML is 60% ~70% and 30% respectively, CR rate in APL is 85%. But some patients still suffer from relapse within a few years.
At the time of diagnosis, the number of malignant cells in vivo is about 1012 or more. After chemotherapy and achieved clinical and hematological CR, there are remain 106~108 malignant cells in vivo. The leukemia cells which can not be detected by morphological examination are defined minimal residual disease (MRD), and it is the primary reason that cause relapse.
Detecting MRD accurately during treatment of leukemia and analysis its meaning is significance to clinical disease condition monitoring, prognosis judgement and treatment protocols selection. The meaning of MRD detection lies in :①decide stop treatment or not according to the quantity of malignant cells in vivo;②reveal drug resistance;③predict relapse earlier;④evaluate the pretreatment effect of marrow transplantation;⑤provides research probability of distribution and proliferation of leukemia cells.
At present, the methods of MRD detection include karyotype analysis, fluorescence in situ hybridization (FISH), flow cytometry (FCM), polymerase chain reaction (PCR), etc. Each method has advantage and disadvantage. Sensitivity of karyotype analysis, FISH and FCM is 10-1~10-2, 10-2~10-3 and 10-3~10-4 respectively, can not satisfy the MRD diagnosis demand, and has disadvantage such as time consuming, expensive, high quality requirements of sample. Fluorescent quantitative PCR(FQ-PCR) is base on the PCR technology, it add fluorescent probe in the PCR system, detecting the change of fluorescence signal to estimate the increasing PCR product.FQ-PCR through cycle threshold(Ct) and standard curve to analysis the quantity of sample,and has advantage of sensitive, quantitive, convenient.It is the better method of MRD detection.
As one of the markers detected leukemia MRD, fusion gene is molecular abnomal resulted from the change of chromosome structure.Now it has found more than 50 leukemia-associated fusion genes,these abnomal is the mark of some types of leukemia,also it is useful for realize leukemia types, prognosis and MRD detection. The EAC program, instituted by 26 European university laboratories from 10 countries, had collaborated to establish a standardizaed protocol for TaqMan based FQ-PCR analysis of 9 main leukemia-associated fusion genes to quantify MRD, in order to evaluate leukemia curative effect and prognosis.Using the standardizaed protocol, 30%~40% of childhood and adult ALL and AML and more than 95% of CML patients can be detected.
We have constructed 9 recombinant plasmids containing leukemia relative fusion genes, and established the standard curve and standard equation of the recombinant plasmids. To evaluate the method of FQ-PCR detecting leukemia relative fusion genes, cellular experiment and clinical observation were performed in this study.
Objective
1.To evaluate the method of FQ-PCR detecting leukemia relative fusion genes by cellular experimental study.
2.To detect leukemia relative fusion genes of patients with leukemia by FQ-PCR at diagnosis, and observe the variation of quantity of leukemia relative fusion genes during treatment.
Contents
1.To dilute K562 cell(contain BCR-ABL M-bcr), REH cell(contain TEL-AML1), NB4 cell (contain PML-RARα) and KASUMI-1 cell(contain AML1-ETO) using HL-60 cell (without leukemia relative fusion genes) in 1:100~1:105, extract RNA, reverse transcript to cDNA, and detect the qualities of fusion genes by FQ-PCR, calculate NCN.
2.To calculate SI value and CV to evaluate quality control, and analyze the sensitivity, specificity, Youden’s index and CV to evaluate the validity and reliability of the experiment study of FQ-PCR.
3.To detect leukemia relative fusion genes of patients with leukemia by FQ-PCR at diagnosis, and observe the variation of quantity of leukemia relative fusion genes during treatment.
Technical Strategy
1.Cellular experimental study
1.Cellular experimental study
(1)To cultivate K562, REH, NB4 and KASUMI-1 cell lines.
(2)To dilute K562, REH, NB4 and KASUMI-1 cell lines using HL-60 cell line as negative cell in1:100 to 1:105.
(3)To extract RNA from the 6 diluted cells, then reverse transcription into cDNA.
(4)To establish standard curves and standard equations of recombinant plasmids which inserted TEL-AML1, PML-RARα, AML1-ETO and BCR-ABL M-bcr by FQ-PCR.
(5)To detect the fusion genes of diluted cells using FQ-PCR, calculate NCN values.
(6)To calculate the SI, intra-CV and extra-CV of NCN, in order to analyze quality control of the cellular experiment study using FQ-PCR.
(7)To estimate the sensitivity, specificity, Youden’s index, intra-CV and extra-CV, in order to evaluate the validity and reliability of the cellular experiment study using FQ-PCR.
2.Clinical observation
(1)Collect the bone marrow of patients at diagnosis.
(2)Extract RNAand reverse transcription into cDNA.
(3)Observe the variation of qualities of fusion genes after induction therapy.
Results
1.Standard curves and standard equations of the standard plasmids are established by FQ-PCR, the results are shown in the table 5 below.
2.The leukemia-associated fusion genes were detected by FQ-PCR, the results show that,①As the concentrations of cell lines within 1:100 to 1:105, the concentration has negative correlation with the Ct values of fusion genes detected from the cell lines.②the cell concentration decrease 1 log, the NCN of fusion genes decrease 0.5 to 1 log, the correlation coefficient (r2) of them are between 0.985 to 0.986.③the Ct values of control gene are between 21.86±0.05 to 26.48±0.73.
3.All of the SI values that calculated from experimental data are less than the value of Q2s.
4.To detect leukemia relative fusion genes using FQ-PCR, the detectable concentration of cell lines is at least 10-5.
5.To detect leukemia relative fusion genes using FQ-PCR, the sensitivity was 100.00% (both TEL-AML1 and AML1-ETO), 97.92% (BCR-ABL M-bcr) and 89.58% (PML-RARα).The specificity was 93.75% (TEL-AML1, PML-RARαand AML1-ETO) and 81.25% (BCR-ABL M-bcr). Youden’s index was0.94(both TEL-AML1 and AML1-ETO), 0.83 (PML-RARα), and 0.79 (BCR-ABL M-bcr).
6.As the concentrations of KASUMI-1cell line within 1:100～1:105, the intra-CV and extra-CV of the NCN of AML1-ETO fusion gene were less than 3%, and the repeatability and precision were the first. As the concentrations of K562, REH and NB4 cell lines within 1:100～1:103, the intra-CV and extra-CV of the NCN of BCR-ABL M-bcr、TEL-AML1 and PML-RARαwere less than 10%, and the repeatability and precision were second,as the concentrations within 1:104～1:105, the intra-CV was less than 6%, the extra-CV was less than 30%.
7.The leukemia relative fusion genes were detected using FQ-PCR in 55 childhood with leukemia at diagnosis, fusion genes were found in 28.57% of all the patients, 15.38% of ALL, 14.29% of AML, 100.00% of APL and CML. During every stage of treatment, the NCN of PML-RARαand BCR-ABL(shown in table 12 and 13) were consistent with the response to the chemotherapy.
Conclusions
1.All the NCN values are in the control range.
2.As the concentration of 4 cell lines decrease 1 log, the NCN values of the leukemia-relative fusion genes reduce 0.5 or 1 log.
3.To detect leukemia relative fusion genes using FQ-PCR, the detectable minimal concentration of cell lines is 10-5.
4.To detect leukemia relative fusion genes using FQ-PCR, the sensitivity was 100.00% (both TEL-AML1 and AML1-ETO), 97.92% (BCR-ABL M-bcr) and 89.58% (PML-RARα).The specificity was 93.75% (TEL-AML1, PML-RARαand AML1-ETO) and 81.25% (BCR-ABL M-bcr). Youden’s index was 0.94(both TEL-AML1 and AML1-ETO), 0.83 (PML-RARα), and 0.79 (BCR-ABL M-bcr).
5.As the concentrations of KASUMI-1cell line within 1:100～1:105, the intra-CV and extra-CV of the NCN of AML1-ETO fusion gene were less than 3%, and the repeatability and precision were the first. As the concentrations of K562, REH and NB4 cell lines within 1:100～1:103, the intra-CV and extra-CV of the NCN of BCR-ABL M-bcr、TEL-AML1 and PML-RARαwere less than 10%, and the repeatability and precision were second,as the concentrations within 1:104～1:105, the intra-CV was less than 6%, the extra-CV was less than 30%.
6.The leukemia relative fusion genes were detected using FQ-PCR in 55 childhood with leukemia at diagnosis, fusion genes were found in 28.57%,E2A-PBX1 TEL-AML1 and MLL-AF4 were found in 15.38% of ALL, CBFβ-MYH11 was found in 14.29% of AML, both PML-RARαand BCR-ABL M-bcr were found in 100.00% of APL and CML. During every stage of treatment, the NCN of PML-RARαand BCR-ABL were consistent with the response to the chemotherapy.
引文
1.Gaynon PS.Childhood acute lymphoblastic leukaemia and relapse.Br J Haematol.2005, 131(5):579-87.
2.V.H.J.Vander Velden, N. Boeckx, E.R.Van Wering,et al. Detection of minimal residual disease in acute leukemia. J Biol Regul Homeost Agents. 2004, 18: 146-54.
3.J Moppett,G A A Bruke,C G Steward et al.The clinical relevance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia.J Clin Pathol .2003,56:249-253.
4.A Hochhaus. A further milestone towards comprehensive standardization of quantitative RT-PCR protocols for leukemic fusion gene transcripts has been reached. Leukemia. 2003, 17: 2383–2384.
5.VHJ van der Velden, A Hochhaus, G Cazzaniga.et al. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia. 2003, 17: 1013–1034.
6.何映谊,叶铁真.PCR检测儿童微小残留白血病的研究进展.中国实验血液学杂志.2007,15(3):652-656.
7.Gabert J, Beillard E, van der Velden VH,et al.Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.Leukemia.2003,17(12):2318-57.
8.钟南山.医学科研设计.广州:中山大学出版社,2000.
9.中华医学会儿科分会血液学组.儿童急性淋巴细胞白血病诊疗建议(第三次修订草案).中华儿科杂志. 2006,44(5):392-395.
10.H. C. Schouten. Diagnosis, staging and prognostic factors. Annals of Oncology. 2007, 18 (Supplement 1): i22–i28.
11. M Bruggemann, H White, P Gaulard,et al. Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936. Leukemia. 2007, 21:215–221.
12 . Van Dongen JJM, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations:Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia, 2003,17:2257-2317.
13.Beillard E, Pallisgaard N, van der Velden VH, et al.Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program.Leukemia.2003,17(12):2474-86.
14.黄鹏,陈建昌.目前临床检验室内质量控制方法的选择.中国现代实用医学杂志.2004,3(7):62-65.
15.李金明.临床分子诊断质量保证的重要性.中华检验医学杂志.2003,26(11):652-653.
16.临床实验室定量检测室内质量控制工作指南(讨论稿3).卫生部临床检验中心.2001.
17.王胜蓝,黄静.“即刻法”质控中出现“告警”时应该删去最大偏离值.中国输血杂志,2005,18(3):230-231.
18.Birgitte S. Preissa, Gitte B. Kerndrupa,*, Rikke K. Pedersen.et al. Contribution of multiparameter genetic analysis to the detection of genetic alterations in hematologic neoplasia. An evaluation of combining G-band analysis, spectral karyotyping, and multiplex reverse-transcription polymerase chain reaction (multiplex RT-PCR). Cancer Genetics and Cytogenetics. 2006, 165:1–8.
19.Wolfgang Kern, Claudia Haferlach, Torsten Haferlach,et al. Monitoring of Minimal Residual Disease in Acute Myeloid Leukemia. Cancer. 2008, 112(1): 4-16
20.Grimwade D. The significance of minimal residual disease in patients with t(15;17). Best practice & research. Clinical haematology. 2002, 15: 137-158.
21.Carlos Santamaría, Maria Carmen Chillón, Carina Fernández,et al. Using quantification of the PML-RARa transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia. Haematologica 2007; 92:315-322.
22.Yokota H, Tsuno NH, Tanaka Y,et al.Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using areal-time RT-PCR assay.Leukemia.2002,16(6):1167-75.
23.林丽丹,叶铁真.BCR-ABL阳性的儿童急性淋巴细胞性白血病的研究进展.国际输血及学业学杂志.2006,29(6):509-512.
24.V Asnafi, M-T Rubio, E Delabesse,et al. Prediction of relapse by day 100 BCR-ABL quantification after allogeneic stem cell transplantation for chronic myeloid leukemia. Leukemia.2006;20: 793–799.
25.Hughes TP, Deininger MW, Hochhaus A, Branford S, Radich JP, Kaeda J et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors– review and recommendations for‘harmonizing’current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37.
26.Miguel A. Sanz, David Grimwade, Martin S. Tallman, et al. Management of acute promyelocytic leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood, 2009;113:1875-1891.
1 Shaham J, Bomstein Y, Gurvich R, et al. DNA-protein crosslinks and p53 protein expression in relation to occupational exposure to formaldehyde. Occup Environ Med, 2003,60(6):403-409.
2 Bolufer P, Barragan E,Collado M,et al.Influence of genetic polymorphisms on the risk of developing leukemia and on disease progression. Leuk Res,2006,30(12):1471-1491
3 Krajinovic M,Labuda D,Richer C,et al.Susceptibility to childhood acute lymphoblastic leukemia: influence of CYP1A1,CYP2D6,GSTM1,and GSTT1 genetic polymorphisms. Blood, 1999,93(5):1496-1501
4 Canalle R,.Burim RV, Tone LG, et al. Genetic Polymorphisms and Susceptibility to Childhood Acute Lymphoblastic Leukemia. Environ Mol Mutagen,2004,43(2):100-109.
5 Aydin-Sayitoglu M, Hatirnaz O, ErensoyN,et al.Role of CYP2D6, CYP1A1, CYP2E1, GSTT1, and GSTM1 Genes in the Susceptibility to Acute Leukemias. Am J Hematol,2006, 81(3):162-170.
6 Krajinovic M, Sinnett H,Richer C,et al. Role of NQO1, MPO and CYP2E1 genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia. Int J Cancer,2002, 97(2):230-236
7 Davies SM,Bhatia S,Ross JA,et al.Glutathione S-transferase genotypes,genetic susceptibility,and outcome of therapy in childhood acute lymphoblastic leukemia. Blood, 2002, 100(1):67-71.
8 Zheng Ye,Honglin Song. Glutathione s-transferase polymorphisms (GSTM1, GSTP1 and GSTT1) and the risk of acute leukaemia: A systematic review and meta-analysis. Eur J Cancer. 2005, 41(7):980-989
9 Infante-Rivard C, Vermunt JK, Weinberg CR.Excess Transmission of the NAD(P)H:Quinone Oxidoreductase 1 (NQO1) C609T Polymorphism in Families of Children with Acute Lymphoblastic Leukemia. Am J Epidemiol,2007,165(11):1248-1254
10 Lanciotti M, Dufour C, Corral L,et al.Genetic polymorphism of NAD(P)H:quinone oxidoreductase is associated with an increased risk of infant acute lymphoblastic leukemia without MLL gene rearrangements. Leukemia,2005,19(2):214-216
11 Krajinovic M, Lamothe S, Labuda D, et al. Role of MTHFR genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia. Blood,2004,103(1):252-257.
12 Wiemels JL,Smith RN,Taylor GM,et al.Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia.Proc Natl Acad Sci USA,2001,98(7):4004-4009.
13 Joseph T, Kusumakumary P, Chacko P,et al.DNA repair gene XRCC1 polymorphisms in childhood acute lymphoblastic leukemia. Cancer Lett, 2005,217(1):17-24.
14 Pakakasama S, Sirirat T,Kanchanachumpol S,et al. Genetic Polymorphisms and Haplotypes of DNA Repair Genes in Childhood Acute Lymphoblastic Leukemia. Pediatr Blood Cancer, 2007,48(1):16-20.
15 Badham HJ, Winn LM.Investigating the role of the aryl hydrocarbon receptor in benzene-initiated toxicity in vitro. Toxicology.2007, 229(3): 177-185
16 Giovannetti E, Ugrasena DG, Supriyadi E,et al. Methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase promoter (TSER) polymorphisms in Indonesian children with and without leukemia. Leuk Res. 2008, 32(1):19-24
17 Vogel CF, Li W, Sciullo E,et al. Pathogenesis of aryl hydrocarbon receptor-mediated development of lymphoma is associated with increased cyclooxygenase-2 expression. Am J Pathol.2007, 171(5):1538-1548
18 Yu Z, Loehr CV, Fischer KA, et al. In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor. Cancer Res. 2006, 66(2):755-762
2.V.H.J.Vander Velden, N. Boeckx, E.R.Van Wering,et al. Detection of minimal residual disease in acute leukemia. J Biol Regul Homeost Agents. 2004, 18: 146-54.
3.J Moppett,G A A Bruke,C G Steward et al.The clinical relevance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia.J Clin Pathol .2003,56:249-253.
4.A Hochhaus. A further milestone towards comprehensive standardization of quantitative RT-PCR protocols for leukemic fusion gene transcripts has been reached. Leukemia. 2003, 17: 2383–2384.
5.VHJ van der Velden, A Hochhaus, G Cazzaniga.et al. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia. 2003, 17: 1013–1034.
6.何映谊,叶铁真.PCR检测儿童微小残留白血病的研究进展.中国实验血液学杂志.2007,15(3):652-656.
7.Gabert J, Beillard E, van der Velden VH,et al.Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.Leukemia.2003,17(12):2318-57.
8.钟南山.医学科研设计.广州:中山大学出版社,2000.
9.中华医学会儿科分会血液学组.儿童急性淋巴细胞白血病诊疗建议(第三次修订草案).中华儿科杂志. 2006,44(5):392-395.
10.H. C. Schouten. Diagnosis, staging and prognostic factors. Annals of Oncology. 2007, 18 (Supplement 1): i22–i28.
11. M Bruggemann, H White, P Gaulard,et al. Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936. Leukemia. 2007, 21:215–221.
12 . Van Dongen JJM, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations:Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia, 2003,17:2257-2317.
13.Beillard E, Pallisgaard N, van der Velden VH, et al.Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program.Leukemia.2003,17(12):2474-86.
14.黄鹏,陈建昌.目前临床检验室内质量控制方法的选择.中国现代实用医学杂志.2004,3(7):62-65.
15.李金明.临床分子诊断质量保证的重要性.中华检验医学杂志.2003,26(11):652-653.
16.临床实验室定量检测室内质量控制工作指南(讨论稿3).卫生部临床检验中心.2001.
17.王胜蓝,黄静.“即刻法”质控中出现“告警”时应该删去最大偏离值.中国输血杂志,2005,18(3):230-231.
18.Birgitte S. Preissa, Gitte B. Kerndrupa,*, Rikke K. Pedersen.et al. Contribution of multiparameter genetic analysis to the detection of genetic alterations in hematologic neoplasia. An evaluation of combining G-band analysis, spectral karyotyping, and multiplex reverse-transcription polymerase chain reaction (multiplex RT-PCR). Cancer Genetics and Cytogenetics. 2006, 165:1–8.
19.Wolfgang Kern, Claudia Haferlach, Torsten Haferlach,et al. Monitoring of Minimal Residual Disease in Acute Myeloid Leukemia. Cancer. 2008, 112(1): 4-16
20.Grimwade D. The significance of minimal residual disease in patients with t(15;17). Best practice & research. Clinical haematology. 2002, 15: 137-158.
21.Carlos Santamaría, Maria Carmen Chillón, Carina Fernández,et al. Using quantification of the PML-RARa transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia. Haematologica 2007; 92:315-322.
22.Yokota H, Tsuno NH, Tanaka Y,et al.Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using areal-time RT-PCR assay.Leukemia.2002,16(6):1167-75.
23.林丽丹,叶铁真.BCR-ABL阳性的儿童急性淋巴细胞性白血病的研究进展.国际输血及学业学杂志.2006,29(6):509-512.
24.V Asnafi, M-T Rubio, E Delabesse,et al. Prediction of relapse by day 100 BCR-ABL quantification after allogeneic stem cell transplantation for chronic myeloid leukemia. Leukemia.2006;20: 793–799.
25.Hughes TP, Deininger MW, Hochhaus A, Branford S, Radich JP, Kaeda J et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors– review and recommendations for‘harmonizing’current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37.
26.Miguel A. Sanz, David Grimwade, Martin S. Tallman, et al. Management of acute promyelocytic leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood, 2009;113:1875-1891.
1 Shaham J, Bomstein Y, Gurvich R, et al. DNA-protein crosslinks and p53 protein expression in relation to occupational exposure to formaldehyde. Occup Environ Med, 2003,60(6):403-409.
2 Bolufer P, Barragan E,Collado M,et al.Influence of genetic polymorphisms on the risk of developing leukemia and on disease progression. Leuk Res,2006,30(12):1471-1491
3 Krajinovic M,Labuda D,Richer C,et al.Susceptibility to childhood acute lymphoblastic leukemia: influence of CYP1A1,CYP2D6,GSTM1,and GSTT1 genetic polymorphisms. Blood, 1999,93(5):1496-1501
4 Canalle R,.Burim RV, Tone LG, et al. Genetic Polymorphisms and Susceptibility to Childhood Acute Lymphoblastic Leukemia. Environ Mol Mutagen,2004,43(2):100-109.
5 Aydin-Sayitoglu M, Hatirnaz O, ErensoyN,et al.Role of CYP2D6, CYP1A1, CYP2E1, GSTT1, and GSTM1 Genes in the Susceptibility to Acute Leukemias. Am J Hematol,2006, 81(3):162-170.
6 Krajinovic M, Sinnett H,Richer C,et al. Role of NQO1, MPO and CYP2E1 genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia. Int J Cancer,2002, 97(2):230-236
7 Davies SM,Bhatia S,Ross JA,et al.Glutathione S-transferase genotypes,genetic susceptibility,and outcome of therapy in childhood acute lymphoblastic leukemia. Blood, 2002, 100(1):67-71.
8 Zheng Ye,Honglin Song. Glutathione s-transferase polymorphisms (GSTM1, GSTP1 and GSTT1) and the risk of acute leukaemia: A systematic review and meta-analysis. Eur J Cancer. 2005, 41(7):980-989
9 Infante-Rivard C, Vermunt JK, Weinberg CR.Excess Transmission of the NAD(P)H:Quinone Oxidoreductase 1 (NQO1) C609T Polymorphism in Families of Children with Acute Lymphoblastic Leukemia. Am J Epidemiol,2007,165(11):1248-1254
10 Lanciotti M, Dufour C, Corral L,et al.Genetic polymorphism of NAD(P)H:quinone oxidoreductase is associated with an increased risk of infant acute lymphoblastic leukemia without MLL gene rearrangements. Leukemia,2005,19(2):214-216
11 Krajinovic M, Lamothe S, Labuda D, et al. Role of MTHFR genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia. Blood,2004,103(1):252-257.
12 Wiemels JL,Smith RN,Taylor GM,et al.Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia.Proc Natl Acad Sci USA,2001,98(7):4004-4009.
13 Joseph T, Kusumakumary P, Chacko P,et al.DNA repair gene XRCC1 polymorphisms in childhood acute lymphoblastic leukemia. Cancer Lett, 2005,217(1):17-24.
14 Pakakasama S, Sirirat T,Kanchanachumpol S,et al. Genetic Polymorphisms and Haplotypes of DNA Repair Genes in Childhood Acute Lymphoblastic Leukemia. Pediatr Blood Cancer, 2007,48(1):16-20.
15 Badham HJ, Winn LM.Investigating the role of the aryl hydrocarbon receptor in benzene-initiated toxicity in vitro. Toxicology.2007, 229(3): 177-185
16 Giovannetti E, Ugrasena DG, Supriyadi E,et al. Methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase promoter (TSER) polymorphisms in Indonesian children with and without leukemia. Leuk Res. 2008, 32(1):19-24
17 Vogel CF, Li W, Sciullo E,et al. Pathogenesis of aryl hydrocarbon receptor-mediated development of lymphoma is associated with increased cyclooxygenase-2 expression. Am J Pathol.2007, 171(5):1538-1548
18 Yu Z, Loehr CV, Fischer KA, et al. In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor. Cancer Res. 2006, 66(2):755-762