| BackgroundAcute Leukemia is one of the most common cancers among children, of which ALL takes up 85% of all children cancer cases. Despite the high cute rate and the remarkable progress in the development of effective treatment strategies, complete remission have been obtained in more than 90% of ALL patients. However, at least 20% of childhood ALL patients in the developed countries still suffer from disease recurrence, which is highly correlated with minimal residual diseases (MRD). As studies show, MRD reflects the effect of individual therapy and information of, and is also the main cause of relapse of leukemia. Thus, to accurately determine and analyze MRD during the therapy of leukemia, may be significant for clinical track of progress, estimation of prognosis, and further determination of specific individual therapy strategies.The Ig and TCR gene loci contain many different variable (V), diversity (D), and joining (J) gene segments, which are subject to rearrangement processes during early lymphoid differentiation. During rearrangement, only one V, D or J segment randomly binds together, and deletion of some base pairs, insertion of various nucleotides and mutation occur in the juncture of V-D and D-J, which creates an enormous junctional diversity. In principle, all cells of a lymphoid malignancy have a common clonal origin with identically rearranged Ig and/or TCR genes. Consequently, the junctional regions of these Ig/TCR gene rearrangements can be considered as specific molecular symbol of the malignant cells, which can be used as tumor-specific PCR targets for MRD detection. Moreover, Ig/TCR rearrangement has high detection rate in both children and adult ALL, and using the primers system of European BIOMED-2, the detection rate can reach 100%. Ig/TCR rearrangement shows particular advantages in ALL-MRD detection due to its high specificity and occurrence.Real-time quantitative PCR (RQ-PCR) has advantages on sensitivity (reach 10-4-10-5), specificity, rapidity, easiness and ability for quantification. Currently, detection of Ig/TCR gene rearrangement and surveillance of MRD using RQ-PCR method is the most reliable quantification of MRD, which ensures comparable MRD results between different phases of diseases and laboratories. RQ-PCR detection of Ig/TCR rearrangement involve first PCR amplification and sequencing of Ig/TCR rearrangements of patients, followed by primer and probe design based on specific sequencing results and subsequent surveillance of MRD levels during different therapy phases. After more than 1400 assays of RQ-PCR, the European Study Group (ESG) established a standard detection assay for quantification of ALL-MRD. This assay standardized the range, sensitivity, definition of positive and negative, and Ct value of quantification of Ig/TCR rearrangement by RQ-PCR, which aid in controlling of false positive and negative, as well as comparison and evaluation of MRD detection between different laboratories. Based on the Ig/TCR rearrangement detection results by RQ-PCR, the Berlin-Frankfurt-Münster (BFM) establish the new classification standardization of risk based on MRD level after induction and consolidation therapy, and offers further direction and guidance on subsequent therapy.In China, lack of related system investigation and insufficient application of RQ-PCR in most medical facilities leads to a gap on MRD detection comparing with developed countries. Moreover, the difference in PCR primers, reaction conditions, sample types and quantification definition, as well as the lack of clonality analysis and tracking cases, lead to results with no comparability. In the mean time, issues including cost and technique demanding for MRD detection on Ig/TCR rearrangement, pose obstacles for its domestic development and clinical application. As consequence, establishment of MRD detection assays and related diagnosis standardization according to the condition of China, may be of utmost importance and urgent necessity for ALL investigation.ObjectiveBased on RQ-PCR quantification technique, targeted on Ig/TCR rearrangement for MRD detection, via tracking investigation of ALL children, to establish the RQ-PCR detection on Ig/TCR rearrangement and the subsequent MRD analysis, and further to evaluate the application on ALL therapy.Contents1.Multiplex- PCR detection on Ig/TCR rearrangement of ALL children and analysis of Ig/TCR rearrangement.2.Analysis of the clonal characteristics of Ig/TCR rearrangement by gene scanning.3.Detection of Ig/TCR at different therapy phases by RQ-PCR quantification on Ig/TCR rearrangement of ALL children, and further analysis on the progress model of Ig/TCR expression during ALL therapy.4.Analysis of the correlation of Ig/TCR rearrangement and various clinical characteristics based on the changing tendency of Ig/TCR rearrangement detection rate, clonal characteristics and relative gene expression.Methods1.Samples selection and process: ALL cases identified by marrow cell morphology and FCM were selected, and for each case, marrow samples had been collected in time points including:①initial diagnosis,②after induced remission,③before consolidation therapy,④before early intensive therapy,⑤before consistence therapy,⑥during consistence therapy (sample collected every 90 days after consistence therapy). Mononuclear cell DNA extraction had been performed for each sample.2.Detection of Ig/TCR rearrangement from children at diagnosis: The procedure include selection of 14 pairs from Biomed-1 protocol, PCR amplification of IgH, Igκ, TCRγand TCRδfrom initial therapy samples, identification of PCR products by electrophoresis on 1.5% agarose gel, analysis of clonality of Ig/TCR rearrangement by gene scanning and subsequent sequencing of monoclonal PCR products, followed by design of ASO primers and probes, and determination of MRD targets.3.Detection of MRD from children of follow-up: The procedure include establishment of standard curve by both serial dilution of DNA templates from children undertaking initial therapy to 10-1 to 10-6 targeted on specific Ig/TCR rearrangement of children and serial dilution of DNA templates from normal people to 10-1 to 10-4 targeted on inner control gene albumin, RQ-PCR using TaqMan probe detection of both genes on different time points during ALL therapy, and further quantification based on the expression and proportion of both genes, which reflects the MRD level.4.Analysis of experimental data: The correlation between various clinical characteristics and experimental results including Ig/TCR rearrangement detection rate and proportion from PCR, clonality and occurrence of Ig/TCR rearrangement from gene scanning, and change model of MRD level from RQ-PCR, were studies and analyzed.Results1.Ig/TCR rearrangement detection rate: Among 86 cases of initial diagnostic ALL children, at least one type of Ig/TCR rearrangement were found in 83 (96.51%), and an average of 2.52 Ig/TCR rearrangement for each case were also detected.2.Proportion of each Ig/TCR rearrangement: Among 83 cases with Ig/TCR rearrangement, highest proportion was observed in IgH gene, with 80.72% (67/83), followed by TCRδ(67.47%, 56/83), Igκ(55.42%, 46/83) and TCRγ(49.40%, 41/83).3.Clonality of Ig/TCR rearrangement: Among 61 cases with 172 Ig/TCR rearrangements, 56 (91.80%) cases were detected to show at least one monoclonal and/or oligoclonal Ig/TCR rearrangement. The detection rate for monoclonal, oligoclonal and polyclonal Ig/TCR rearrangement were 58.14%, 30.81% and 11.05%, with significant difference obtained and monoclonal Ig/TCR rearrangement was prevalent. Of monoclonal and oligoclonal gene rearrangement, no significant difference was observed among 4 types of Ig/TCR genes; nevertheless, for polyclonal gene rearrangement, significant difference was found among IgH, TCRγand TCRδ. Statistical analysis had been performed on the number of positive cases, the number of positive genes and clonality at diagnosis, with no significant difference observed between CCR patients and patients of follow-up.4.Quantification of Ig/TCR rearrangement by RQ-PCR:①For 22 cases of CCR patients, the relative expression of Ig/TCR rearrangement decrease dramatically from initial diagnosis to after induced remission, with MRD negative cases also detected. Before consolidation therapy, the number of MRD negative cases increased gradually, which revealed significant difference comparing with after induced remission. As therapy continues, the relative Ig/TCR rearrangement expression consistently decreased, and before consistent therapy, all tested MRD cases were detected to be negative.②For 4 relapse cases, all tested MRD cases were negative from beginning of induced remission to relapse, and the clinically average relapse time was 3.25 months (ranging from 2-8 months) since the relative expression of Ig/TCR rearrangement started increasing.5.Analysis of correlation between Ig/TCR rearrangement and clinical characteristics:①Significant difference in IgH detection rate had been obtained among different gross number of white blood cell at diagnosis, cell morphological type and immunophenotype.②No significant was observed between various clinical characteristics and clonality of Ig/TCR rearrangement.③The relative expression of Ig/TCR rearrangement showed significant difference in different groups including immunophenotype and 15 days induced therapy.④At the end of induced remission and beginning of consolidation therapy, the relative expression of Ig/TCR rearrangement for PPR group in 7 days prednisone reaction was higher than PGR group, with significant difference.⑤Before consistent therapy, different groups of bone marrow blast cells at diagnosis showed significant difference in the Ig/TCR relative expression.⑥Comparing risk group based on relative Ig/TCR rearrangement expression at the end of induced remission to both time points of initial diagnosis and after induced remission, no significant difference had been observed. For 2 out of 4 relapse cases, the risk rose after induced remission.Conclusions1.High detection rate of Ig/TCR rearrangement had been obtained from ALL cases. Combination of several targets for Ig/TCR gene rearrangement leads to high detection rate, even with relative fewer primers pairs.2.Monoclonal Ig/TCR rearrangement were prevalent in ALL cases, while for some ALL cases, oligoclonal Ig/TCR rearrangement also existed. Thus, gene scanning had been demonstrated to be a useful tool for simple, rapid and accurate analysis of the Ig/TCR rearrangement clonality and further selection of Ig/TCR rearrangement with different clonal characteristics.3.Surveillance of ALL children's MRD level by RQ-PCR with Ig/TCR gene rearrangement as targets, is a potentially useful assay for evaluation and assessment of therapy effect and relapse prediction for both clinical and application sakes.This study had demonstrated the important role of RQ-PCR detection on Ig/TCR rearrangement for ALL cases, and the applicability of this method on MRD surveillance. However, this study had been limited by the extent, scale and time span. To apply MRD surveillance in China, and ensure the accuracy of RQ-PCR detection of ALL-MRD still requires further large scale, perspective and long-term tracking investigation. The establishment and standardization of MRD detection, as well as the proper use in the whole country, should be as important as the evaluation of national therapy strategy for leukemia. |
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