Prdi-BF-1 Strongly Detected in Active Multiple Myeloma By Droplet Digital PCR: A Comparison with Real-Time PCR

Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that has been demonstrated to act as a master regulator required and sufficient for the generation and for the prolonged maintenance of plasma cells (PC). PRDI-BF1 essentially functions as a repressor, causing exit from cell cycling and the extinction of the expression of several genes critical for B-cell development at earlier stages. The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of generating a PRDI-BF1 deleted protein (called PRDI-BF1β), which lacks 101 amino acids comprising most of the regulatory domain. The potential role of Blimp-1/PRDM1 in multiple myeloma, may help in understanding the progression of the disease and the potential specific function of a new drug as elotuzumab that selectively blocks CS1. To the best of our knowledge this is the first report that uses two new molecular techniques to identify and perform a quantitative levels of PRD-BF1. We measured, using real time polymerase chain reaction (RT- PCR) and Droplet digital PCR (ddPCR), the levels of PRDI-BF1 in Myeloma patients`(n=15) and normal human bone marrow (n=12). qPCR represents the most widely used method for molecular analysis. However, it has a major limitation from being a relative quantification approach. This results in the need of a reference standard curve usually built by dilutions of the tumor-specific target obtained from diagnostic DNA, plasmids, or cell lines that contain the rearrangement of interest. ddPCR is based on sample compartmentalization in single oil droplets that represent independent PCR reactions and on end point amplification and Poisson statistics. ddPCR has several theoretical advantages compared with qPCR. most notably allowing for absolute quantification of target DNA molecules and avoiding the need for a reference standard curve. dd-PCR was performed using the QX100 platform (BioRad®, Hercules, CA, USA), consisting of two instruments: the droplet generator and the droplet reader. The droplet generator divides the sample by creating about 20,000 partitions (droplets). The droplets are then transferred into PCR plates and, at the end of the amplification cycles, placed into the droplet reader, where each droplet is read as mutated or wild-type by issuing specific fluorescence signals (FAM for BLIMP and Hex for GAPDH). These signals, after being counted, are redistributed according to the Poisson's algorithm. In our samples, total RNA was extracted and reverse transcribed from bone marrow blood using RNA isolation and transcriptor kits from Roche (Barcelona, Spain). Primer mixes and TaqMan probes, assay references Hs00153357_m1 for PRDIBF1 and s99999903_m1 for β-actin (used as an internal refer-ence), were obtained from Applied Biosystems (Foster City, CA, USA). The relative quantitative values were calculated using the 2-∆∆CT method. In all MM patients the mean expression of PRDI-BF1 was 5.92 (range 1.00-21.75) fold higher than in healthy controls with RT PCR. Using ddPCR, PRDI-BF1 was also expressed at considerable levels in all samples tested and the mean expression of PRDI-BF1 was 10.10 (range 0.63-52.52) fold higher than in healthy controls.In particular in 4 patients with very aggressive disease the PRDI-BF had a marked higher transcript level (mean 28.52 range 15.12-52.52), Similar results were obtained with RT-PCR higher transcript level (mean 13.5 range 7.41-21.75) Our data suggest that the total level of PRDI-BF is clearly detectable in MM patients . The expression of these gene could be a feature for identifying active disease of MM with particular interest in very aggressive disease.