Assessment of clonal evolution at Ig/TCR loci in acute lymphoblastic leukaemia by single-strand conformation polymorphism studies and highly resolutive PCR derived methods: implication for a general strategy of minimal residual disease detection.

Junctional sequences of immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements are used as patient-specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high-resolution PCR-based analyses (including denaturing gel electrophoresis and single-stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V gamma-J gamma and V delta 1-J delta 1 rearrangements were analysed in 12 T-ALLs. VH-JH, V gamma-J gamma, V delta 2-D delta 3 and, in selected cases, DH-JH rearrangements were studied in 14 B-lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T-ALLs and in 5/14 B-lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient-specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.

In vitro amplification of T cell gamma gene rearrangements: a new tool for the assessment of minimal residual disease in acute lymphoblastic leukemias.

In order to improve the level of sensitivity and specificity of detection of bone marrow minimal residual disease in the acute lymphoid leukemias, we have performed amplification by the polymerase chain reaction of T cell receptor gamma chain gene rearrangements. Cloning and sequencing of amplified leukemic DNA allowed the construction of a clone-specific anti-junctional oligonucleotide to be used for subsequent detection of minimal infiltration by this clone. Using such an oligonucleotide, it was possible to distinguish clonal DNA from polyclonal T lymphocytes and to detect infiltration by this clone at 10(-6) dilution into germline DNA. We therefore describe a generally applicable method for the detection of minimal residual disease in both T-ALL and the majority of B lineage ALLs.