Screening for residual disease in pediatric burkitt lymphoma using consensus primer pools

Abstract

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgV(H)) genes, IgV(H) primer pools from IgV(H1)-IgV(H7) regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgV(H) primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgV(H) primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.

Figure 1

Examples of melt curve analyses with corresponding ethidium-stained amplified products. Melt curves displayed as −dF/dT plots: (a) defined peak with minimal to no shoulder indicates a positive MRD result; (b) a broad peak indicates a negative result; (c) a defined peak with significant shoulders indicates equivocal results. Representative 2.0% agarose gels are at the left of each of graph. Melt curves of negative controls: (d) PBMC from normal healthy individual shows a broad undefined peak; (e) water (no template) shows no peak; (f) melt curve assessment of PCR products: melt curve peak shift disparities are noted between diagnostic and followup specimens, consistent with possible different clonal differentiation over time.

Figure 2

MRD sensitivity with patient primary tumor DNA. Primary diagnostic tumor DNA (100% infiltrated with malignant cells) was diluted with PBMC DNA to represent the equivalent of 1 malignant cell in 10³, 10⁵, 10⁷, 10⁸, and 10⁹ PBMC. Each representative melt curve has a distinct peak corresponding to tumor DNA or PBMC DNA, which are designated by the respective arrows.