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.

Quantification of TEL–AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic leukaemia

“…MRD had previously been assessed in nine of these samples using a clono-specific PCR assay based on the detection of T-cell receptor junctional sequences (Cave  et al, 1994b). MRD levels assessed using TEL±AML1 transcript measurement were in agreement with those determined using clono-specific markers (Table IV).…”

“…The prognostic value of minimal residual disease (MRD) is well established in childhood acute lymphoblastic leukaemia (ALL), and an increasing number of protocols now take this parameter into account to tailor treatment (Brisco et al, 1994;Cave  et al, 1998;Coustan-Smith et al, 1998;van Dongen et al, 1998). Because it permits MRD monitoring in up to 90% of children with ALL, detection of clono-specific immunoglobulin (Ig) or T-cell receptor gene (TcR) rearrangements using polymerase chain reaction (PCR) amplification has been widely used, and large clinical trials assessing the value of MRD rely on this approach (Brisco et al, 1994;Cave  et al, 1998;van Dongen et al, 1998). When leukaemia cells have a translocation, another possibility is to monitor MRD by reverse transcription (RT) and PCR amplification of the fusion transcript associated with the translocation (Foroni et al, 1999).…”

“…Quantitative MRD evaluation has been shown to be necessary for prognosis evaluation in ALL (Brisco et al, 1994;Cave  et al, 1998;Coustan-Smith et al, 1998;van Dongen et al, 1998), and quantitative PCR assays are required for this purpose. Competitive PCR and real-time PCR are two main approaches that have been used to quantify specific RNA messengers (Freeman et al, 1999).…”

Minimal residual disease analysis for the prediction of relapse in children with standard‐risk acute lymphoblastic leukaemia

“…At the end of induction therapy we found a highly significant difference in the proportion of patients MRD positive between the relapse group and those remaining in CCR (82% v 32%, P ¼ 0·0003). This divergence has been described previously and has led to great interest in accurate quantitation of disease at this time in an attempt to improve yet further the discrimination of high-risk patients (Brisco et al, 1994;Cavé et al, 1994). For example, those with persistent clonal bands (indicative of MRD at or above 1 in 10 3 ) or quantified MRD in excess of 1 in 10 3 at this time have been shown to have a high propensity to relapse.…”

“…This is in direct contradiction to animal studies which suggest a patchy distribution of MRD (Martens et al, 1987). Secondly, accurate quantitation requires either limiting dilution experiments (Brisco et al, 1994;Ouspenskaia et al, 1995) or co-amplification of a competitor molecule (Cavé et al, 1994). Such approaches increase complexity, significantly complicating the conduct of large-scale clinical studies.…”