Abstract
Rearrangements of the immunoglobulin and T-cell receptor genes provide molecular markers for clones in acute lymphoblastic leukemia (ALL). Determination of the repertoire of gene rearrangements in ALL aids in understanding the clonal biology of the disease and provides molecular markers which can be used to quantify minimal residual disease (MRD).
We have developed a sensitive PCR-based method for analysing the repertoire of immunoglobulin heavy chain (IgH) rearrangements in ALL. Multiple parallel quantitative PCR’s are performed in microplates using different segment-specific primers in different wells in order to determine the individual V (D) and J segments utilised by each rearrangement. The number of rearrangements detected in 18 children and 10 adults with ALL is shown in the table:
| . | VDJ rearrangements . | DJ rearrangements . | ||||
|---|---|---|---|---|---|---|
| No. of rearrangements | 1 | 2 | 3 | 4 | 1 | 2 |
| Childhood ALL | 0 | 11 | 3 | 2 | 1 | 1 |
| Adult ALL | 7 | 2 | 0 | 1 | ||
| . | VDJ rearrangements . | DJ rearrangements . | ||||
|---|---|---|---|---|---|---|
| No. of rearrangements | 1 | 2 | 3 | 4 | 1 | 2 |
| Childhood ALL | 0 | 11 | 3 | 2 | 1 | 1 |
| Adult ALL | 7 | 2 | 0 | 1 | ||
Since each PCR well contained only 2 ng of DNA, more sensitive repertoire analysis was also performed in samples from 10 of the children and 4 of the adults by using 100 ng of DNA in an initial preamplification, which involved a multiplexed PCR containing primers for all leader and J sequences of the IgH gene and which thus amplified all immunoglobulin sequences. The IgH repertoire of the amplified material was then analysed. This two-step approach should theoretically enable detection of clones which comprise down to approximately 10−4 the leukemic population. It detected all rearrangements previously detected by one-step repertoire analysis and, in addition, it detected 0–5 (mean 1.2) rearrangements marking small clones in childhood ALL and 0–3 (mean 1.0) rearrangements marking small clones in adult ALL. Sequencing showed that most, but not all, small clones had a lineage relationship to the dominant clone present in the leukemic population.
Repertoire analysis of IgH rearrangements is a promising technique for identifying molecular markers for measurement of MRD in B-ALL, particularly childhood ALL, since:
–it is conceptually simple and relatively quick
–it detects IgH rearrangements with high efficiency, probably higher than that of current techniques.
–IgH rearrangements are the best markers to use for measurement of MRD owing to the specificity and sensitivity that they provide
–an enhanced ability to identify markers for both large and small leukemic clones may improve the identification of patients prone to relapse.
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