Improving individualized monitoring strategy for Rituximab treatment in patients with Neuromyelitis Optica Spectrum Disorders: development of a high sensitive ddPCR assay for CD19 mRNA quantification

Objective: To develop and validate a droplet digital (dd) PCR assay for the quantification of CD19 mRNA in blood of patients with Neuromyelitis Optica Spectrum Disorders (NMOSD) treated with Rituximab (RTX), and to evaluate its clinical performance compared to Flow Citometry (FC). Background: Current RTX monitoring strategy in NMOSD is based on the evaluation of CD19+ B cells by FC. Evidence of depletion relapses suggests a low sensitivity of the method. Design/Methods: 14 patients followed at CRESM and monthly monitored for CD19+ B cells by FC were included. 143 whole blood samples were used to set up and validate the CD19 mRNA-ddPCR assay. CD19 mRNA expression was expressed as a “Ratio” between CD19 mRNA concentration and TATA-binding protein (TBP). High positive, low positive and negative controls were generated and analyzed together with the “no template control” (NTC) to evaluate the analytical performance of the assay. The comparison between FC and dd-PCR was performed using 161 available paired samples. Results: Acceptability criteria to pass the quality control of each plate were established for NTC, control samples and unknown samples. DDPCR assay quantified CD19 mRNA in all samples, thus a positivity threshold was calculated = 0,00235. Analyses showed a good analytical performance of the CD19 mRNA-ddPCR assay. In all samples where CD19+ B cells were above FC threshold (n=34), they resulted above the ddPCR threshold as well. CD19 mRNA expression was above threshold in 32 other samples, resulted negative by FC. In 16/21 analyzed RTX infusions, mRNA-ddPCR assay anticipated FC of at least 1 month in detecting CD19+ B cells, showing higher sensitivity. In two cases the ddPCR detected an elevation followed by a relapse. Conclusions: DD-PCR assay is a reliable and high sensitive molecular method to quantify CD19 mRNA in whole blood and could be introduced in clinical practice to improve personalized monitoring of anti-CD20 treatments. Disclosure: Dr. Bertolotto has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Biogen, Merck, Novartis, Roche, Sanofi, and Teva.Dr. Valentino has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Biogen Idec, Novartis. Dr. Mirabile has nothing to disclose. Dr. Bertolo has nothing to disclose. Dr. Mussolin has nothing to disclose. Dr. Siravegna has nothing to disclose. Dr. Martire has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Biogen Idec. Dr. Marnetto has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Biogen Idec.