IgH/TCR delta PCR oligonucleotide liquid hybridization, a fast and sensitive assay for monitoring minimal residual disease in childhood B-precursor ALL.
The detection of minimal residual disease (MRD) in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR) may turn out to be a powerful tool in the evaluation and guidance of therapy. Most previously described techniques are highly sensitive but also too laborious for application in a routine setting. Here we describe a technique based on the determination of IgH VHDJH and TCR V delta 2D delta 3 junctional regions by PCR/cycle sequencing analysis and hybridization of junctional region oligonucleotide probes in a standard liquid hybridization (LH) assay. We systematically analyzed the applicability of this simplified approach for the monitoring of MRD in a large patient group. IgH VHDJH and TCR V delta 2D delta 3 junctional regions were amplified from presentation bone marrow samples obtained from 53 childhood B-precursor ALL patients. The combined approach allowed the identification of at least one tumor marker for 49/53 (92.5%) of patients. A total of 75 oligonucleotide probes (54 DJH, 21 V delta 2D delta 3) was tested in the LH assay. Sensitivity range was 10(-2)-10(-5) and 10(-4)-10(-5) for DJH and V delta 2D delta 3 junctional region probes, respectively. A sensitivity of at least one malignant cell in 10(4) normal cells was obtained for 84.8% of evaluable patients, applying on average 1.1 IgH and 0.47 TCR delta probes per patient. Comparison to a method based on the use of initial PCR product as clone-specific probe showed that oligonucleotide LH was one log more sensitive in six of nine patients tested. The presented technique allows the monitoring of MRD with acceptable sensitivities in over 90% of childhood B-precursor ALL patients. Moreover, the technique is suitable for prospective patient studies in a routine setting as it is fast, reproducible and makes use of a standard hybridization protocol for different oligonucleotide probes.
Steenbergen
E J
EJ
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Verhagen
O J
OJ
van Leeuwen
E F
EF
van den Berg
H
H
Behrendt
H
H
von dem Borne
A E
AE
van der Schoot
C E
CE
D001483:Base Sequence; D002648:Child; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D018365:Neoplasm, Residual / Q000175:diagnosis; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*
D007143:Immunoglobulin Heavy Chains; D016692:Receptors, Antigen, T-Cell, gamma-delta