Hélène Cavé
Laboratoire de Biochimie Génétique,
Hôpital Robert Debré, 48 Boulevard Sérurier, 75019Paris, France

Minimal residual disease

When acute lymphoblastic leukemia is diagnosed in a patient, the total numberof leukemia cells is approximated to 10¹² to 10¹³. A majorityof patients reach complete remission (CR) after about 4 weeks of chemotherapy.Complete remission does not mean that leukemia cells are totally eradicated fromthe body but that their level is beyond the sensitivity level of classical cytomorphologicmethods (e.g. 1 to 5%). At this time, up to 10¹⁰ malignant cells canstill remain in the patient. They represent the minimal residual disease (MRD).Techniques which are more sensitive than cytomorphology are now available thatpermit to detect these residual blasts (reviewed in (Campana and Pui, 1995; Foroniet al., 1999)).
Detection of residual cells allows a longer follow-up of the tumor burdenduring chemotherapy and thus, permits to better appreciate the sensitivity ofleukemia cells to treatment. It is now established that the level of MRDrepresents a powerful prognostic factor. Besides, the detection of an increaseof the MRD level enables to anticipate impending relapse.

Techniques for the follow-up of the residual disease

Techniques aimed at studying MRD rely on the detection of a leukemiacell specific marker which enable to distinguish blasts from normal marrowcells. Such markers have to be detected with high sensitivity, to be present inall leukemia cells and to be stable during disease evolution. Two kinds ofmarkers are currently used: genetic markers, which can be detected by PCR, andimmunophenotypic markers, which can be detected by flow cytometry.

PCR-bases techniques

PCR based strategies can be directed to 2 types of genetic targets: breakpointsof leukemia-related chromosome aberrations, and antigen-receptor generearrangements. The extreme sensitivity of polymerase chain reaction (PCR)makes possible the consistent detection of 1 leukemia cell among 10⁵ normalcells (10^-5).

Chromosome aberrations

Recurrent chromosome translocations are found in 30 to 40% of ALL (figure 1) (Pui.and Evans, 1998). The molecular counterpart of these translocations can be detectedby PCR using primers located on each side of the breakpoint (Pui. and Evans, 1998).DNA amplification can only be used for chromosome aberrations in which breakpointscluster in a small area, such as Tal-1 deletions or Ig-cMyc fusions. In most cases,breakpoints spread over large intronic regions, but translocations give rise tofusion transcripts suitable for PCR amplification after a reverse transcriptionstep (RT-PCR). All fusion transcripts can be used as markers for MRD follow-up.Their detection in remission samples is achieved, as for diagnosis, by RT-PCR.The detection sensitivity depends upon both the amount of RNA studied and thelevel of expression of the transcript. When 1-2 ug of RNA is studied, fusion transcriptsassociated to ALL permit to detect residual blasts with a sensitivity of 10^-4to 10^-6.
The main advantage of such markers is to be directly involved inleukemogenesis. Accordingly, their presence is constant all over diseaseevolution. However, variations in their expression level during the disease andparticularly during chemotherapy cannot be excluded. This makes difficult tocorrelate the level of detection of a transcript to the amount of leukemiacells.
Given the frequency of each translocation, each fusion transcript permits tostudy only a subset of patients (figure 1). Another inconvenient is that,because they are not patient specific, these PCR targets are much more prone tofalse positive results due to carry-over than antigen-receptor genes.

Figure 1: Frequencies of the main fusion genes which can be used as PCRtargets for MRD monitoring in ALL

Antigen-receptor gene rearrangements

The use of antigen-receptor gene rearrangements for MRD detection has beendeveloped in order to overcome the lack of recurrent chromosomal abnormalitiesin most of the patients with lymphoid malignancies. In ALL, T-cell receptor(TcR) and immunoglobulin (Ig) loci undergo somatic rearrangement by V(D)Jrecombination without strict lineage specificity. Provided the extremediversity created by V(D)J rearrangements, each malignant clone will present aspecific configuration and the sequence of the junctional region(s) (N-region)is highly clone-specific. For technical convenience, rearrangements studiedfor MRD follow up are those of TcRgamma-delta, Ig heavy chain (IgH) and Igkappa. The frequencyof these rearrangements in ALL is indicated in table 2. A combined study ofthese 4 loci permits to identify one or more rearrangement in virtually allcases of ALL.


Table 1: Frequency of TcR and Ig recombination in childhood ALL (For somerearrangements, frequencies are slightly different in adult ALL)