Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.
Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.
Gabert
J
J
Department of Hematology Biology, Institut Paoli Calmettes, France. gabert.j@jean-roche.univ-mrs.fr
Beillard
E
E
van der Velden
V H J
VH
Bi
W
W
Grimwade
D
D
Pallisgaard
N
N
Barbany
G
G
Cazzaniga
G
G
Cayuela
J M
JM
Cavé
H
H
Pane
F
F
Aerts
J L E
JL
De Micheli
D
D
Thirion
X
X
Pradel
V
V
González
M
M
Viehmann
S
S
Malec
M
M
Saglio
G
G
van Dongen
J J M
JJ
D014408:Biomarkers, Tumor / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D005060:Europe; D006801:Humans; D007938:Leukemia / Q000175:diagnosis* / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010957:Plasmids; D011379:Prognosis; D011786:Quality Control; D012333:RNA, Messenger; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*
D014408:Biomarkers, Tumor; D017931:DNA Primers; D018076:DNA, Complementary; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger