Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes

Abstract

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B- lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects

“…Quantitation of the MRD-PCR target can be performed by comparing the PCR signal (often after blotting and hybridization) with serial dilutions of a standard with known amounts of target DNA or RNA, 5,35 by limiting dilution experiments until negative PCR results are obtained, 36,37 and by competitive PCR. 28,29,38,39 However, 'real-time' quantitative PCR (RQ-PCR) methods have recently been developed and, together with recent GeneScan technology, [40][41][42][43] may replace the complex and time-consuming (semi)quantitative PCR analyses. Here, we will discuss the recent technical developments in RQ-PCR analysis, with emphasis on detection of MRD in hematologic malignancies.…”

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

“…Accordingly, new methods for early diagnosis and treatment need to be developed (Campana & Pui, 1995; Baer, 1998). In haematological malignancies, minimal residual disease (MRD) after treatment has been successfully monitored in some patients using leukaemia‐specific gene rearrangements, including t(9;22)(q34;q11) (Cross et al , 1993; Lion et al , 1995; Lin et al , 1996; Preudhomme et al , 1997; Radich et al , 1997; Drobyski & Hessner, 1998; Moravcova et al , 1998), t(8;21)(q22;q22) (Muto et al , 1996; Tobal & Yin, 1996), and rearrangement of immunogloblin (Ig) (Roberts et al , 1997) and T‐cell receptor (TCR) (Beishuizen et al , 1994; Cave et al , 1994; Seriu et al , 1995). Leukaemia specific markers, however, are not yet available for many patients, although the WT1 gene has been reported to be a useful leukaemia marker for these patients (Inoue et al , 1994, 1996).…”

Molecular characterization of paediatric idiopathic hypereosinophilia

“…In order to assess the clonality of IGH and TCR repertoires, BM genomic DNA was extracted from bone marrow mononuclear cells. TCR clonality was performed by denaturing high performance liquid chromatography (DHPLC) sequencing of the TRB@ genomic region (Cave et al , 1994). IGH clonality analysis was performed on BM genomic DNA by amplifying CDRI, CDRII and CDRIII regions of the heavy chain immunoglobulin genes ( IGH ‐PCR).…”