ORIGINAL ARTICLE Optimization of PCR-Based Minimal Residual Disease Diagnostics for Childhood Acute Lymphoblastic Leukemia in A
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Leukemia (2007) 21, 706–713 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting VHJ van der Velden1, ER Panzer-Gru¨mayer2, G Cazzaniga3, T Flohr4, R Sutton5, A Schrauder6, G Basso7, M Schrappe6, JM Wijkhuijs1, M Konrad2, CR Bartram4, G Masera3, A Biondi3, JJM van Dongen1 1Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; 2Children’s Cancer Research Institute and St Anna Kinderspital, Vienna, Austria; 3M Tettamanti Research Center, Pediatric Clinic, San Gerardo Hospital, University of Milan Bicocca, Monza, Italy; 4Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany; 5Children’s Cancer Institute Australia for Medical Research, University of NSW, Sydney, Australia; 6Department of Pediatrics, University Hospital Schleswig- Holstein, Campus Kiel, Germany; 7Hemato-Oncology Laboratory, Department of Pediatrics, University of Padova, Padua, Italy Minimal residual disease (MRD) diagnostics is used for patients at intermediate risk (IR; 5-year relapse rate of 22%).3,9 Of treatment stratification in childhood acute lymphoblastic leu- note, for recognition of LR patients, the MRD assay had to reach a kemia. We aimed to identify and solve potential problems in À4 3,9 multicenter MRD studies to achieve and maintain consistent sensitivity of at least 10 . On the basis of these results, MRD results between the AIEOP/BFM ALL-2000 MRD laboratories. As diagnostics for treatment stratification is currently applied in the dot-blot hybridization method was replaced by the real-time many childhood ALL treatment protocols, including the ongoing quantitative polymerase chain reaction (RQ-PCR) method AIEOP/BFM ALL-2000 and DCOG-ALL10 protocols. during the treatment protocol, special attention was given to Analysis of MRD is mostly performed using polymerase chain the comparison of MRD data obtained by both methods and to reaction (PCR) analysis of immunoglobulin (Ig) and T-cell the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that receptor (TCR) gene rearrangements, as this method is appli- resulted in discordant MRD results. In particular, guidelines for cable in the vast majority of childhood ALL patients and À4 RQ-PCR data interpretation appeared to be crucial for obtaining generally reaches sensitivities of 10 required for identification concordant MRD results. The experimental variation of the RQ- of LR patients.3,9,10 Within the I-BFM-SG, PCR analysis was PCR was generally less than three-fold, but logically became initially followed by dot-blot hybridization using a radio-labeled larger at low MRD levels below the reproducible sensitivity of À4 junctional region-specific probe, resulting in a semi-quantitative the assay (o10 ). Finally, MRD data obtained by dot-blot 3,9 hybridization were comparable to those obtained by RQ-PCR analysis of MRD levels. In the meantime, real-time quantita- analysis (r2 ¼ 0.74). In conclusion, MRD diagnostics using RQ- tive RQ-PCR analysis has become available and offers an easier, 11 PCR analysis of immunoglobulin/T-cell receptor gene rearran- faster and more quantitative method for MRD analysis. gements is feasible in multicenter studies but requires MRD detection by PCR analysis of rearranged Ig/TCR genes is standardization; particularly strict guidelines for interpretation however a complex process, involving many steps (Figure 1). of RQ-PCR data are required. We further recommend regular Identification of pitfalls in this process is of importance in order quality control for laboratories performing MRD diagnostics in international treatment protocols. to ensure comparable MRD results between the MRD-PCR Leukemia (2007) 21, 706–713. doi:10.1038/sj.leu.2404535; laboratories of multicenter national or international treatment published online 8 February 2007 protocols. Furthermore, the move from a laboratory research Keywords: minimal residual disease; real-time quantitative PCR; tool used for retrospective analysis of clinical trials to a quality control; reproducibility; immunoglobulin; T-cell receptor diagnostic tool for stratification of patients necessitates uni- formity in MRD data not only within single treatment protocols but also between different treatment protocols. Introduction Within the MRD Task Force of the I-BFM-SG, we therefore aimed to identify and solve potential problems in multicenter Several studies have shown that detection of minimal residual MRD studies and to achieve and maintain consistent MRD disease (MRD) has prognostic relevance in childhood acute results between the MRD-PCR laboratories participating in the lymphoblastic leukemia (ALL).1–8 On the basis of MRD analysis AIEOP/BFM ALL-2000 protocol. To this end, we evaluated during the early phases of treatment, preferably at two different several steps in PCR-based MRD detection, including detection time points, MRD-based risk groups can be recognized. Within and sequencing of Ig/TCR gene rearrangements, MRD analysis the International BFM Study Group (I-BFM-SG), patients were of follow-up samples, and interpretation of RQ-PCR MRD data. classified according to MRD levels at day 33 and day 78 of As the dot-blot hybridization method was fully replaced by RQ- therapy, and three risk groups could be distinguished: low-risk PCR techniques during the course of the AIEOP-BFM ALL-2000 patients (LR), having MRD negativity at both time points (about protocol, we particularly focused on the comparison of MRD 45% of patients; 5-year relapse rate of 2%); patients at high-risk data obtained by both methods and on the reproducibility of the (HR), having high (X10À3) MRD levels at both time points (about RQ-PCR methods, both experimental variation and variation in 15% of patients; 5-year relapse rate of 80%); and the remaining the interpretation of RQ-PCR data. Correspondence: Prof Dr JJM van Dongen, Department of Immunol- Materials and methods ogy, Erasmus MC, University Medical Center Rotterdam, Dr Molewa- terplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: [email protected] MRD analysis 12 Received 10 May 2006; revised 6 September 2006; accepted 15 DNA was isolated as described previously. The presence of November 2006; published online 8 February 2007 IGK-Kde, TCRG and TCRD rearrangements in diagnostic Improving molecular MRD diagnostics VHJ van der Velden et al 707 Rotterdam and Sydney, the latter performing the MRD analysis for the I-BFM-SG-related Australian ANZCHOG Study VIII clinical trial. First, each of the five participating laboratories repeated the RQ-PCR MRD assays for a number of patient cases (total number of patients: 74). These repetitions were performed using new DNA dilutions but the same oligonucleotides, one to several months after the initial analysis. Second, the newly obtained RQ-PCR data were interpreted by both the executing laboratory and a second laboratory. Data analysis All data were analyzed by the department of Immunology, Rotterdam (VHJvdV). Data were presented non-blinded to facilitate the identification of the underlying causes of dis- crepancies and the discussion of how to overcome the pitfalls and achieve concordance. Results and discussion Figure 1 Overview of all key steps in MRD analysis applying Ig/TCR gene rearrangements. (a) MRD-PVR target identification. (b) Sensitivity MRD diagnostics using PCR analysis of Ig/TCR gene rearrange- testing. (c) MRD analysis of follow-up samples. ments includes three main steps: (1) MRD-PCR target identifica- tion; (2) sensitivity testing; and (3) MRD analysis of follow-up samples (Figure 1). These three main steps were evaluated by comparing the results obtained in the laboratories of the I-BFM- samples was determined using various primer combina- SG MRD task force using centrally provided samples and data tions.3,13,14 Complete IGH rearrangements were detected using files. five VH family primers in combination with one consensus JH primer.15 Sequence analysis was performed as described previously.3 Evaluation of step 1: MRD-PCR target identification MRD levels in follow-up samples were either analyzed by dot-blot hybridization3 or by RQ-PCR analysis. Four laboratories Potential MRD-PCR targets were identified by PCR-heterodu- performed RQ-PCR analysis using the ABI Prism equipment (ABI plex analysis in eight ALL patients. These eight patients were not Prism 7700, 7900 or 7000) and single PCR assay with hydrolysis chosen randomly, but were selected based on the availability of (TaqMan) probes;11,15–17 one laboratory performed a nested sufficient DNA, the presence of particular rearrangements and/ PCR assay in which the second PCR was run on the Light Cycler or the presence of subclonal rearrangements. As shown in using SYBR Green I.18 As these two approaches theoretically Table 1 a total of 40 clonal Ig/T-cell receptor rearrangements differ considerably, the results are shown separately where could be detected by at least one of the four participating relevant. laboratories. Twenty-five out of these 40 rearrangements (63%) were identified in all four laboratories. Discrepancies in target identification between the four laboratories were particularly Exchange of samples and data caused by: 1, lack of detection of clonal Ig/TCR gene Several steps of blinded testing were conducted on DNA rearrangements; 2, sequencing errors; and 3, errors in sequence samples from 18 patients with