Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma

Abstract

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

Figure 1

Patient rearrangements and oligonucleotide sequences. Junction regions VDH, DHJ, or both are indicated with vertical arrows. N nucleotide insertions and somatic mutations are represented in lowercase letters. Allele-specific and JH intronic primer are underlined with dotted lines, probe sequences are underlined with black lines, and the self-quenched sequence is inside the rectangle. All cases have been sequenced using the JH consensus primer. Cases 10160, 9985, and 4526 (cases that did not work) have also been sequenced using the next JH intronic primer just downstream to study the mutations present in the probe and primer binding sites.

Figure 2

Scheme and standard curves of two methodologies. A: Example of TaqMan PCR. Amplification curves of one patient’s diagnostic sample, several dilutions to calculate standard curve, the sample after treatment (MRD), and the sample at the time of the relapse. NTC, nontemplate control. B: Example of self-quenched PCR. C: Melting curves in one self-quenched method case. There are two different melting temperatures: 84°C for the specific fragments (B) and 77°C for the nonspecific fluorescence (A).