Digoxigenin-end labeled oligonucleotides facilitate the detection of minimal residual disease in acute lymphoblastic leukemia

Abstract

TO THE EDITORRecently, three large prospective trials unequivocally demonstrated that the evaluation of minimal residual disease (MRD) plays a pivotal role as a prognostic parameter in acute lymphoblastic leukemia (ALL). [1][2][3] Individualized therapeutic adaptations based on the MRD status might be of considerable benefit for ALL patients. Several methodologies are available for MRD analyses in ALL patients. [1][2][3][4] Immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements have been utilized as PCR targets. An important PCR strategy for MRD analysis is the amplification of the rearranged Ig or TCR genes and hybridization with 32 P-labeled clone-specific probes in dot blot hybridization. Now that the relevance of MRD monitoring in ALL has been recognized, modification of current techniques or development of new techniques enabling rapid and reliable MRD analysis is relevant. We report on a non-radioactive dot blot analysis taking advantage of digoxigenin (DIG)-labeled oligonucleotides for the rapid detection of MRD. DIG is a steroid hapten which is linked via a spacer arm to a nucleotide. DIG-ddUTP is incorporated at the 3′-end of an oligonucleotide by terminal transferase. After hybridization, the target DNA is detectable by an enzyme-linked immunoassay through an anti-DIG-AP Fab fragment.We applied this technique on 10 adult ALL patients with IGK and/or TCRD gene rearrangements. The patients were treated according to the German multicenter trial of ALL in adults 05/99. 5 High molecular weight DNA was prepared from bone marrow samples obtained at initial diagnosis using standard techniques. IGK and TCRD gene rearrangements were identified and characterized by PCR and sequencing as described. 6,7 Oligonucleotides corresponding to the clonespecific junctional regions of the respective IGK/TCRD genes are shown in Table 1.Ten-fold serial dilutions of initial leukemic DNA into normal buffy coat (BC) DNA were prepared in a range from 10 −1 to 10 −6 . One g of each DNA preparation as well as BC DNA were amplified using a set of outer primers. 6 A negative control (H 2 O) was included in each PCR reaction. PCR was performed using the touch down program with 40 cycles. 7 Five l of the products were spotted in duplicate on to nylon membrane (NYTRAN 0.45, Schleicher and Schuell, Dassel, Germany). The membranes were soaked with denaturing buffer (0.5 M NaOH, 1.5 M NaCl) and neutralizing buffer (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.0) for 2 min each and cross-linked by UV. 100 pmol of each oligonucleotide was 3′-end labeled with DIGddUTP using a DIG oligonucleotide 3′-end labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Non-incorporated DIG-ddUTPs were removed by ethanol precipitation and the labeled probe was dissolved into 20 l of distilled water (final concentration 5 pmol/ l). One pmol of the DIG-labeled oligonucleotide was spotted on to a nylon membrane, which was subsequently processed in the immunological detection step to verify the labeling eff...