Jacques van Dongen article
"Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood." (6)
Jacques van Dongen's group is based at the Department of Immunology, Erasmus University Rotterdam, Netherlands. The study included patients accrued from March 1991 to May 1995 at several I-BFM-SG centers. The children were treated according to protocols of the Austrian BFM group, the German BFM group, the Associazione Italiana group, or the Dutch Childhood Leukemia SG.
Bone marrow samples were taken at diagnosis from 625 children, and then at 9 time points during treatment. Of these, only 240 were studied for MRD because many of the bone marrow samples time points were missing, some patients did not go into remission, or because a PCR target was not found in the child’s leukemia. Of the 240 children, 210 were pre-B and 30 were T-cell. 60 relapsed: 4 isolated extramedullary, 11 extramedullary and bone marrow, and 45 only in the bone marrow.
Mononuclear cells isolated from the bone marrow samples obtained at diagnosis were studied for rearrangements in the TCR coding region. The authors chose the TCR coding region to study instead of the IgH region because of "the high frequency of oligoclonality and continuing rearrangements in the IgH genes, and the assumed lower frequency of such rearrangements in TCR-gamma and TCR-delta genes." They also used TAL1 deletions in T-ALL and kappa deleting element in pre B ALL.
The DNA of each patient was studied by Southern blot analysis. If a rearrangement was found (e.g., a band other than the parent germline band), a rearrangement was confirmed by PCR analysis and direct sequencing of the junctional regions with standardized sets of oligonucleotide primers. Once the sequence of each patient's rearranged junctional region was known, patient-specific oligonucleotides were designed. They were able to study about 90% of the patients for MRD. All samples were sent to a central lab for MRD analysis.
During treatment, 9 bone marrow samples from different time points were studied. They took 1 mg of DNA (corresponding to 105-106 cells) and mixed with standard primers to make PCR product, then dot blot hybridized with 32P labeled patient specific oligomers. The sensitivity of the method was established by dilution experiments. The sensitivity was usually 1 in 104, although sometimes 1 in 105 or 106 (these were lumped in with the 1 in 104 data). If a sensitivity of 104 was not reached on the first try for a particular sample, they used a "nested PCR" strategy until the desired sensitivity was reached.
The table linked below summarizes the correlation of degree of MRD and relapses. The total number of patients at different time points is not always the same because of protocol differences and missing samples. In the MRD degree column, "positive" and "negative" indicate presence or absence of a hybridization signal or ethidium bromide band signal irrespective of the PCR target sensitivity, while the low, intermediate, and high values indicate the amount of MRD detected.
The authors found that reliable PCR-based MRD detection needs two Ig or TCR gene targets per patient to prevent false-negative results because of ongoing or secondary rearrangements; 90-95% of the patients can be followed if IGH gene targets are used in addition to IGK, TCR G and D, and T Al1.
According to the authors, "this large PCR-based study of MRD in childhood ALL unequivocally demonstrates that monitoring patients serially gives clinically relevant insight into the effectiveness of treatment." If MRD is not detected during treatment, the relapse rate was 3-15%; if MRD was detected, it was 39-86%. The higher the degree of MRD positivity, the higher the relapse rate. Van Dongen and colleagues believe that their data indicate analysis of MRD at a single time point is not sufficient to predict relapse, rather, several time points should be studied.
The authors compared the MRD group with another, larger group not enrolled in the study, and applied multivariate analysis. According to the authors, MRD degree in the first year is a valid prognostic factor - independent of age, sex, immunophenotype, white blood count at diagnosis, country, or treatment group.
Also according to the authors, the patients in the MRD-based low risk designation make up a group of substantial size (about 43%), comparable with the frequency of survivors of childhood ALL in the early seventies, before treatment intensification was introduced. This group might profit from treatment reduction. On the other hand, the group of patients in the MRD-based high risk group is larger than any previously identified high-risk group (about 15%) and has an unprecedented high 4 year relapse rate of about 80%. This group might benefit from more intensive treatment protocols (including bone marrow transplant or an innovative treatment).
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