Thermostable DNA polymerase chain amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease.

TLDR

Detecting minimal numbers of neoplastic cells is a major challenge, and the t(14;18)(q32;q21) translocation offers a tumor‑specific marker with breakpoints at specific JH regions and the BCL2 MBR. Using universal primers flanking the BCL2 MBR and JH segments, a thermostable DNA polymerase chain reaction amplifies the BCL2‑JH junctions, enabling a highly sensitive assay that detects one lymphoma cell per million normal cells, evaluates marrow purging protocols, and allows rapid breakpoint sequencing without cloning. The assay detected subclinical leukemic cells in all seven patients, including those in remission, and its rapid sequencing capability refines minimal residual disease detection, aiding disease staging, treatment stratification, and therapy evaluation.

Abstract

Achieving the capacity to detect minimal numbers of neoplastic cells is a major cancer diagnostic challenge. Chromosomal translocations such as the t(14;18)(q32;q21) found in follicular and some nonfollicular lymphomas provide a tumor-specific molecular marker. The 14;18 breakpoints are focused at one of six immunoglobulin heavy chain joining (JH) regions on chromosome 14 and a small major breakpoint region (MBR) of the BCL2 gene on chromosome 18. We utilized universal oligonucleotide primers of a region 5' to the BCL2 MBR and at the 3' end of JH segments to initiate a DNA polymerase chain reaction that amplified these BCL2-JH junctures. Use of thermostable DNA polymerase enabled annealing and synthesis steps at temperatures approaching the melting point of the primers, providing a sensitive and specific assay capable of detecting 1 lymphoma cell in 10(6) normal cells. This technique identified the subclinical presence of leukemic cells in all seven patients examined, including two in clinical remission. It also assessed the effectiveness of protocols designed to purge malignant cells from marrow. Moreover, this approach enabled the rapid DNA sequencing of chromosomal breakpoints without their molecular cloning. This assay markedly refines the capacity to detect minimal residual disease and should improve the ability to determine the stage of disease, stratify treatment, and evaluate therapy.