An initial analysis of an allele-specific quantitative PCR (Q-PCR) assay which detects minimal residual disease (MRD) in the peripheral blood of follicular lymphoma (FL) patients

Abstract

17541 Background: MyVax Personalized Immunotherapy is an active immunotherapy that targets the idiotype of surface immunoglobulin (Ig) on malignant B cells and is currently in a pivotal Phase 3 trial. Idiotype vaccines have shown promising results in early phase clinical trials. The monitoring of circulating tumor cells in peripheral blood lymphocytes (PBLs) may provide important information regarding disease status and response to therapy. A standard method of assessing MRD in FL is by PCR amplification of the t(14:18) translocation at the major breakpoint region (MBR) locus. Studies have shown that 35–50% of FL tumors are not detected by the MBR assay. Circulating non-malignant cells that carry the t(14:18) translocation may complicate MBR analysis. We have developed an allele-specific oligonucleotide (ASO) Q-PCR assay that detects unique Ig heavy chain sequence. This patient-specific ASO assay has the ability to monitor tumor in PBLs of all patients. Methods: Three TaqMan assays were performed on each sample analyzed: 1) ASO; 2) MBR; 3) albumin. The MBR and albumin assays were standard for all patients. The ASO assay was developed for each patient based on amplification using patient-specific primers and TaqMan probes that span the CDR2/CDR3 region of each patient Ig heavy chain. The Ig heavy chain sequences identified during the production of MyVax Personalized Immunotherapy were used to develop patient-specific primers and probes. MBR and ASO counts were normalized using the results of the albumin assay. Assays were performed on DNA samples isolated from PBLs collected before and 6 months after chemotherapy and following MyVax Personalized Immunotherapy. Results: Q-PCR data were grouped into MBR positive and MBR negative sets. We were able to monitor tumor-specific cells present in peripheral blood by the ASO assay in all patients. ASO values appear to correlate with MBR values. Both the ASO and MBR assays allowed detection of 1 tumor cell in 104 to 105 PBLs with reaction efficiencies of 1.80 or higher. Conclusions: The ASO assay is broadly applicable for detecting MRD in both MBR positive and MBR negative FL patients. [Table: see text]