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Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia
Published on: March 5, 2018
Real-time quantitative PCR: standardized detection of minimal residual disease in pediatric acute lymphoblastic
Sharon R Pine1, Fred H Moy, Joseph L Wiemels
1Department of Pediatric Hematology/Oncology, New York Medical College, Valhalla 10595, USA. sharon_mayer@nymc.edu
Purpose:
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).
Patients And 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.
