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
Jjm. Van Dongen et al., 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, LEUKEMIA, 13(12), 1999, pp. 1901-1928
Prospective studies on the detection of minimal residual disease (MRD) in a
cute leukemia patients have shown that large-scale MRD studies are feasible
and that clinically relevant MRD-based risk group classification can be ac
hieved 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 t
echniques. This was the aim of the European BIOMED-1 Concerted Action 'Inve
stigation of minimal residual disease in acute leukemia: international stan
dardization and clinical evaluation' with participants of 14 laboratories i
n eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardiza
tion and quality control was performed for the three main types of MRD tech
niques, ie flow cytometric immunophenotyping, PCR analysis of antigen recep
tor genes, and RT-PCR analysis of well-defined chromosomal aberrations. Thi
s 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 BC
R-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 prime
rs 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 p
erforming an independent PCR together with one of the internal primers for
confirmation (or exclusion) of positive results. Various local RT and PCR p
rotocols were compared and subsequently a common protocol was designed, tes
ted 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, u
sing 17 cell lines and patient samples as positive controls. This testing i
ncluded comparison with local protocols and primers as well as sensitivity
testing via dilution experiments. The collaborative efforts resulted in sta
ndardized primer sets with a minimal target sensitivity of 10(-2) for virtu
ally all single PCR analyses, whereas the nested PCR analyses generally rea
ched the minimal target sensitivity of 10(-4). The standardized RT-PCR prot
ocol and primer sets can now be used for molecular classification of acute
leukemia at diagnosis and for MRD detection during follow-up to evaluate tr
eatment effectiveness.