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

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 106 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.

Mitochondria and cancer: is there a morphological connection?

“…In the following years, the antiapoptotic protein Bcl2 was shown to be a tumour-specific marker which expression was enhanced in many lymphoma (Tsujimoto et al, 1984;Bakhshi et al, 1985;Crescenzi et al, 1988;Vaux et al, 1988;Nunez et al, 1990), opening the investigation of apoptosis in cancer at the molecular level. Suppression of apoptosis is among the minimal requirements for a cell to become cancerous, and a hallmark of most, if not all, types of cancer (Hanahan and Weinberg, 2000;Green and Evan, 2002).…”

PCR Analysis of Immunoglobulin Heavy Chain (IgH) and TcR-γ Chain Gene Rearrangements in the Diagnosis of Lymphoproliferative Disorders: Results of a Study of 525 Cases

“…Amplifications of bcl-2/J H PCR fragments were confirmed by hybridization with an internal oligonucleotide specific for bcl-2-MBR (MBR-Pϩ probe) or the bcl-2-mcr (MC12-Fϩ probe) of the t(14;18), obtained from the sequences reported by Crescenzi et al (32) and Ngan et al (30), at 63°C after alkaline transfer of PCR products to Hybond-Nϩ membranes (Amersham Pharmacia Biotech Europe GmbH, Saclay, France). These probes were end labeled with digoxigenin-11-dUTP 3Ј before hybridization, and their fixation was revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody, followed by BromoChloroIndoyl Phosphate-nitro blue tetrazolium incubation (Roche Diagnostics, Meylan, France).…”

t(14;18), a journey to eternity

“…7 The PCR technique may detect a single t (14;18), reflecting a single lymphoma cell, in a background of about one hundred thousand normal cells. 8,9 By PCR, it now becomes possible to monitor consecutive patient blood and bone marrow samples for minimal residual disease during treatment. 10-12…”

“…26 The chromosomal breakpoints in the t(14;18) cluster in small regions. The PCR is therefore an excellent method for easy, fast and extremely sensitive detection of residual lymphoma cells within blood or bone marrow samples of follicular NHL patients, 8,9,19 and has been applied in several experimental as well as clinical studies. 7,33,34 Although t(14;18)s have been found at very low frequency in benign hyperplastic lymph nodes and blood samples of normal blood donors (see section 2.3 [35][36][37] ), the exact molecular composition of each t(14;18) is unique and can therefore be used as lymphomaspecific marker in t(14;18)-positive follicular NHL patients.…”