Real-time quantification of different types of bcr-abl transcript in chronic myeloid leukemia.
OBJECTIVE
The most common translocation in chronic myeloid leukemia (CML) t(9;22) (q34;q22) produces the BCR/ABL fusion gene. We set up and evaluated a rapid and reliable real-time reverse-transcription-polymerase chain reaction (RT-PCR) approach using TaqMan technology for detection and quantification of bcr-abl transcripts in CML patients at diagnosis and during therapy.


METHODS
A pair of primers and probe complementary to ABL exon 2 were designed, enabling detection of the most frequent bcr-abl transcripts, and also of the normal ABL-Ia transcript as an internal control. Conditions were established to amplify less than 1(-10) target molecules/reaction and detect one CML cell in 10(6) cells from healthy donors. To determine the utility of the assay, we quantified the bcr-abl/ABL-Ia ratio in 59 bone marrow samples (45 samples with evidence of different Ph+ chromosome percentages and 14 samples in complete cytogenetic remission) from 48 CML patients, 34 of them at diagnosis and 14 in clinical remission (CR). In 14 cases, this ratio was compared with results obtained by a competitive-quantitative RT-PCR/capillary electrophoresis method from contemporary specimens.


RESULTS
By real-time RT-PCR, the median value of bcr-abl/ABL-Ia ratio at diagnosis was 15.334 (range 3.3-28.81) and fell to 0.9 (range 0.003-26.1) in CR. The median value of bcr-abl/ABL-Ia ratio at cytogenetic remission was 0.7 (range 0.003-2.83). The real-time bcr-abl/ABL-Ia ratios correlated with those obtained by competitive RT-PCR (p < 0.0001) and the percentage of Ph+ metaphases (p < 0.0001). The high sensitivity and specificity of the real-time RT-PCR procedure was confirmed in all 14 patients with minimal residual disease. INTERPRETATION AND CONCLUSIONS. We conclude that this real-time RT-PCR procedure is a reliable and sensitive method of monitoring CML patients after therapy, and that the bcr-abl/ABL-Ia ratio correlates strongly with cytogenetic analysis.
Amabile
M
M
Institute of Hematology and Medical Oncology "Seràgnoli", University of Bologna, Italy.
Giannini
B
B
Testoni
N
N
Montefusco
V
V
Rosti
G
G
Zardini
C
C
Terragna
C
C
Buonamici
S
S
Ottaviani
E
E
Soverini
S
S
Fiacchini
M
M
Bassi
S
S
de Vivo
A
A
Trabacchi
E
E
Saglio
G
G
Pane
F
F
Baccarani
M
M
Tura
S
S
Martinelli
G
G
D001853:Bone Marrow; D005260:Female; D016313:Genes, abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D008297:Male; D008722:Methods; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D012680:Sensitivity and Specificity
D012333:RNA, Messenger