Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia

Abstract

Clonal rearrangements of the Ig heavy chain (IGH) locus occur in nearly all cases of B-cell precursor acute leukemia (BCP-ALL). Some of these rearrangements may be detected by polymerase chain reaction (PCR) using V(H) gene framework III (FRIII) and J(H) consensus primers. However, about 20% of BCP-ALLs fail to amplify with this technique. To determine the causes of these PCR failures and to investigate any possible association with specific subgroups of disease, we analyzed 72 acute leukemias of defined immunophenotype and cytogenetics, comparing FRIII with V(H)-family leader- specific PCR methods and Southern blotting. Of 37 BCP-ALL cases, 6 (16.2%) failed totally to amplify with FRIII and J(H) primers. None of these cases amplified with V(H) leader primers. Additionally, all cases retained germline V(H)6 genes and 5 of 11 rearranged alleles amplified with a consensus D(H) primer, indicating that these rearrangements represented biallelic D(H)-J(H) recombinations. Among the 6 FRIII and V(H) leader PCR-negative BCP-ALL cases, there was no common immunophenotype or consistent cytogenetic abnormality, although all showed structural chromosomal abnormalities and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;q11) (Philadelphia chromosome-positive [Ph+]) and IGH rearrangements (9 BCP-ALL and 4 biphenotypic cases) were also analyzed. Of 23 rearranged IGH alleles, 19 (82%) were positive by FRIII PCR, and all 4 remaining alleles were amplified by V(H) leader primers. Use of the leader primers in these Ph+ cases also detected 3 additional clonal rearrangements that were not anticipated from Southern blotting; such unexpected bands were not observed in 21 other Ph- cases. The additional bands represented 'new' and unrelated V(H) rearrangements rather than V(H)-V(H) replacement events. We conclude that hiallelic D(H)J(H) rearrangements occur in a subgroup of BCP-ALL; in these cases, the activation of the full V(H)D(H)J(H) recombination mechanism had not occurred. Therefore, these cases of BCP-ALL were arrested at an early stage of B-cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute leukemias, which may represent the transformation of multipotent hemopoietic stem cells, had undergone V(H)D(H)J(H) recombination. Of 9 Ph+ BCP-ALL cases, 3 also showed ongoing V(H)D(H)J(H) rearrangement, reflecting the persistent expression of the V(H)D(H)J(H) recombinase. Finally, sequence analysis of 33 rearranged V(H)D(H)J(H) genes showed that only 3 including 2 Ph+ BCP-ALL maintained an intact open-reading frame. Loss of the open-reading frame occurred not only because of out-of-frame V(H)D(H) and D(H)J(H) joining, but also because of V(H) gene mutation and deletion. These data show that most BCP-ALLs may represent the neoplastic transformation of SOPs destined to die in the bone marrow.