Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.
We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.
Slack
J L
JL
Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. james.slsck@roswellpark.org
Bi
W
W
Livak
K J
KJ
Beaubier
N
N
Yu
M
M
Clark
M
M
Kim
S H
SH
Gallagher
R E
RE
Willman
C L
CL
D014408:Biomarkers, Tumor; D003199:Computer Systems; D004274:DNA, Recombinant / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis*; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D004274:DNA, Recombinant; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases