1. ¹Department of Immunology, Erasmus MC, Rotterdam, The Netherlands
2. ²Children's Cancer Research Institute and St Anna Kinderspital, Vienna, Austria
3. ³M Tettamanti Research Center, Pediatric Clinic, San Gerardo Hospital, University of Milan Bicocca, Monza, Italy
4. ⁴Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany
5. ⁵Children's Cancer Institute Australia for Medical Research, University of NSW, Sydney, Australia
6. ⁶Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Germany
7. ⁷Hemato-Oncology Laboratory, Department of Pediatrics, University of Padova, Padua, Italy

Correspondence: Prof Dr JJM van Dongen, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: j.j.m.vandongen@erasmusmc.nl

Received 10 May 2006; Revised 6 September 2006; Accepted 15 November 2006; Published online 8 February 2007.

Abstract

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (<10^-4). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r²=0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.