Simultaneous Detection of Circulating Tumor Antigens in Acute Leukemia after HSCT

Background Real-time quantitative PCR (RTqPCR) detects minimal residual disease (MRD) in few leukemia patients after transplant because of the rarity of available targets. We sought to quantify the commonly expressed tumor-associated antigens (TAA) commonly expressed by high risk leukemias: WT1, PRAME, and BIRC5 (survivin), by meausring circulating mRNA in peripheral blood. We employed droplet digital PCR (ddPCR) as a novel, minimally invasive method that enables the quantitation of target nucleic acids with high precision, accuracy, and low specimen input. Objective Demonstrate that simultaneous quantification of WT1, PRAME, and BIRC5 mRNA levels in peripheral blood by ddPCR correlates with leukemia burden in HSCT recipients. Design/Methods RNA was isolated from PBMCs and bone marrow from HSCT patients on an IRB-approved study, and from healthy controls. RainDance ddPCR was first optimized to simultaneously quantitate levels of the TAAs in tumor cell lines (hepatoblastoma and K-562 myelogenous leukemia) and then used to assay mRNA levels in marrow and peripheral blood of patients with acute leukemia and healthy controls. Expression of WT1, PRAME, BIRC5, and ABL1 (homeostatic gene) were assessed simultaneously in all samples with RNA integrity number (RIN) ≥6. Results/discussion Following assay optimization the median levels in healthy controls were determined for each TAA/ABL1 ratio (n=20). Results were consistent with previously published values for qPCR. Marrow levels of TAA mRNA exceeded peripheral blood (mean fold increase 8.4; range: 1.6-17), consistent with TAA/ABL1 ratio changes reflecting marrow leukemia burden. Disease response directly correlated with blood ddPCR TAA/ABL1 levels (ID#1 Day 21: BIRC5: 0.97 (relapse) and Day 100: BIRC5: 0.096 (remission); ID#2 day 60: BCR-ABL-neg/ BIRC5: 0.19 (CNS relapse) and Day 100: BCR-ABL 0.4%/ BIRC5: 0.77 (progression). Of those patients with active leukemia, 93% (13/14) were positive for at least one TAA. In summary, we have developed a new method for monitoring MRD in leukemia (patent submitted) and present preliminary data that ddPCR measurement of TAA/ABL1 ratio may correlate with circulating leukemia burden in HSCT recipients.