Real-time quantitative PCR: standardized detection of minimal residual disease in pediatric acute lymphoblastic leukemia. Polymerase chain reaction.
OBJECTIVE
To develop a standardized real-time polymerase chain reaction (PCR) method of quantifying minimal residual disease (MRD) in patients with pre-B acute lymphoblastic leukemia (ALL).


METHODS
In a series of 24 follow-up bone marrow (BM) samples in 11 patients (14 clonal markers), we performed real-time PCR assays using one consensus and one clone-specific primer for each marker. The markers analyzed included immunoglobulin heavy chain (IgH), T-cell receptor (TCR) and TEL-AML1 rearrangements.


RESULTS
We achieved a detection limit of 3.3 x 10(-5) +/- 1.2 x 10(-5) and an accurate quantitation (r = -0.99) limit of 2.0 x 10(-4) +/- 8.8 x 10(-5) blasts. Both inter- and intra-assay reproducibility were exceptional. Additionally, we found comparable results to those of a "gold standard" limiting-dilution PCR assay (r = 0.62).


CONCLUSIONS
The IgH, TCR and TEL-AML1 markers can be used as targets by real-time PCR under the same cycling profile, allowing quantitation of MRD in more 95% of patients with pre-B ALL. This standardized, real-time PCR technique should simplify monitoring MRD in clinical trials.
Pine
Sharon R
SR
Department of Pediatric Hematology/Oncology, New York Medical College, Valhalla 10595, USA. sharon_mayer@nymc.edu
Moy
Fred H
FH
Wiemels
Joseph L
JL
Gill
Ramneet K
RK
Levendoglu-Tugal
Oya
O
Ozkaynak
Mehmet F
MF
Sandoval
Claudio
C
Jayabose
Somasundaram
S
D014408:Biomarkers, Tumor / Q000097:blood*; D002648:Child; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D019672:Genes, T-Cell Receptor; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000097:blood; D008297:Male; D018365:Neoplasm, Residual / Q000175:diagnosis; D015514:Oncogene Proteins, Fusion / Q000097:blood; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics*; D015203:Reproducibility of Results; D000097764:ETS Translocation Variant 6 Protein
D014408:Biomarkers, Tumor; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains; D015514:Oncogene Proteins, Fusion; C102421:TEL-AML1 fusion protein; D000097764:ETS Translocation Variant 6 Protein