Minimal residual disease quantification in childhood acute lymphoblastic leukemia by real-time polymerase chain reaction using the SYBR green dye.
OBJECTIVE
Clone specific immunoglobulin (Ig) and T-cell receptor (TCR) gene sequences can be used as molecular targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Real-time quantitative PCR (RQ-PCR) with no need for post-PCR processing is an attractive approach for detection and quantification of specific DNA or RNA sequences. In the present study we evaluated a real-time PCR-based technology for MRD quantification in children with precursor-B ALL.


METHODS
DNA samples from 36 children with newly diagnosed precursor-B ALL were available for molecular analysis. All patients were uniformly treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocols from 1992. A real-time PCR assay was applied for MRD quantification using LightCycler technology and the SYBR green fluorescent dye for detection of clone-specific Ig and TCR gene rearrangements as target sequences. The specificity of the PCR products was verified by melting curve analysis.


RESULTS
Thirty-four of the 36 children with precursor-B ALL (94%) displayed at least one clonal Ig heavy chain (IgH) or TCR gene sequence useful as a molecular target. These clone-specific targets were successfully applied for real-time PCR quantification in all but one patient. Melting curve analysis was important for identifying all specific PCR products. In 32 pediatric precursor-B-ALL patients an MRD level >/=10(-3) at day 29 during induction treatment was significantly correlated with later bone marrow relapse (p = 0.0025).


CONCLUSIONS
Real-time PCR using clone-specific primers and the SYBR green dye for detection is a feasible technique for identifying patients at risk for relapse. This approach provides an easily applicable tool for detection of IgH/TCR gene rearrangements in the routine setting. Melting curve analysis allowed clear distinction between specific rearrangements and unspecific background signals.
Li
Ai-Hong
AH
Department of Medical Biosciences and Pathology, Umeå University, Umeå, Sweden.
Forestier
Erik
E
Rosenquist
Richard
R
Roos
Göran
G
D000293:Adolescent; D052160:Benzothiazoles; D002648:Child; D002675:Child, Preschool; D004273:DNA, Neoplasm / Q000235:genetics; D003959:Diamines; D018572:Disease-Free Survival; D005260:Female; D005456:Fluorescent Dyes; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D008297:Male; D018365:Neoplasm, Residual / Q000235:genetics / Q000276:immunology / Q000473:pathology*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000473:pathology*; D011804:Quinolines; D012334:RNA, Neoplasm / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D013997:Time Factors
D052160:Benzothiazoles; D004273:DNA, Neoplasm; D003959:Diamines; D005456:Fluorescent Dyes; D007143:Immunoglobulin Heavy Chains; D009930:Organic Chemicals; D011804:Quinolines; D012334:RNA, Neoplasm; D011948:Receptors, Antigen, T-Cell; C098022:SYBR Green I