Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease - Report of the BIOMED-1 Concerted Action: Investigation of minimal residual disease in acute leukemia

• Type: Another Publication in an International Scientific Journal
• Date: 1999

Authors

van Dongen, JJM (Author) Other; Macintyre, EA (Author) Other; Gabert, JA (Author) Other; Delabesse, E (Author) Other; Rossi, V (Author) Other; Saglio, G (Author) Other; Gottardi, E (Author) Other; Rambaldi, A (Author) Other; Dotti, G (Author) Other; Griesinger, F (Author) Other; Parreira, A (Author) Other; Gameiro, P (Author) Other; Diaz, MG (Author) Other; Malec, M (Author) Other; Langerak, AW (Author) Other; San Miguel, JF (Author) Other; Biondi, A (Author) Other

Journal

Title: Leukemia

Vol. 13

Pages: 1901-1928

ISSN: 0887-6924

Publisher: Springer Nature

Indexing

ISI Web of Knowledge - 802 Citations

Scopus - 949 Citations

Scientific classification

FOS: Medical and Health sciences > Clinical medicine

Other information

Authenticus ID: P-001-2QY

DOI: 10.1038/sj.leu.2401592

Abstract (EN): Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: 1(1;19) with E2A-PBX1, 1(4;11) with MLL-AF4, 1(8;21) with AML1-ETO, 1(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <-> B) and nested PCR (internal primers C <-> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A - B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10(-2) for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10(-4). The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 28