Minimal Residual Disease in Acute Lymphoblastic Leukaemia—PCR Analysis of Immunoglobulin Gene Rearrangements

Abstract

Rearrangement of the immunoglobulin heavy chain (IgH) gene can be utilized as a marker of clonality in a number of B-lineage lymphoproliferative disorders including acute lymphoblastic leukaemia (ALL). We have used a PCR technique involving a panel of amplimers for the 6 different Variable (VH) region families and for a consensus sequence of the Joining (JH) segment to detect clonal IgH rearrangements in the peripheral blood (PB) and/or bone marrow (BM) of 28 patients (17 children and 11 adults) with B-lineage ALL at presentation (20 patients) or with overt relapse (8 patients). The age range of the patients was 2–65 years (mean 15.7 years). Follow up remission BM samples were analysed in 22 patients during and after therapy (2–7 samples per patient), 1–50 months after presentation or relapse. In 1 relapsed case, previously stored complete remission (CR) samples were analysed retrospectively.

Clonal IgH chain rearrangements were detected by PCR in 90% of patients studied initially. The 2 VH region families most commonly used were the large VH3 family (65%) and the smaller more JH-proximal VH4 family (22%). More than one VH clone was detectable in 25% of the cases. A gene “fingerprinting” modification of a previously described method was applied to the detection of minimal residual disease (MRD) in follow up BM samples with a sensitivity of 10⁻³ to 10⁻⁴. In 8 of 14 patients remaining in complete remission (CR) during the time of study, all PCR analyses on BM samples in the first 6 months were negative, in some cases as early as 2 weeks post-induction therapy, and a further patient reverted from being PCR positive in the first month after the commencement of therapy to sustained PCR negativity. One adult remains in CR at 50 months after presentation and has been PCR negative at 2 time points after cessation of maintenance therapy (30 and 50 months).

Eight patients relapsed in the study period comprising 6 BM and 2 isolated CNS relapses. In 4 cases of BM relapse occurring within 7 months of the start of therapy, all BM remission samples tested in this period were PCR positive. In 2 other patients BM samples tested 7 and 2 months respectively prior to relapse were PCR negative. In the 2 patients with isolated CNS relapse, PCR of BM samples from 2 and 10 months before the relapse were negative. Results thus far suggest that early and sustained loss of PCR positivity after commencement of therapy, particularly in children may be associated with a low risk of early relapse and this early negative PCR result may prove to be predictive for a better prognosis in this group of ALL patients. Further long term analyses of a larger number of appropriately stratified patients will be required to determine the ultimate significance of these studies, the frequency of clonal evolution and the relevant contributions of various treatment phases including transplantation at eliminating the disease.

Key Words:

• Acute lymphoblastic leukaemia (ALL)
• minimal residual disease (MRD)
• gene “fingerprinting”