Study Of Minimal Residual Disease Detection In Leukemia

BackgroundAs 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.Objective1.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.Contents1.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 Strategy1.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.Results1.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. Conclusions1.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.