Quantification of the spliced ΔCD20 mRNA in activated B cells and hematologic malignancies, and illustration of clinical relevance through 2 cases. (A) Design of the RT-qPCR with schematic localization of primers and bifluorescent FAM/TAMRA TaqMan probe. Both PCRs (ΔCD20- and wtCD20-specific) were performed using an iCycler thermocycler (Bio-Rad) under standard TaqMan PCR conditions with the TaqMan Universal PCR Master Mix (Applied Biosystems). Copy number of both forms of CD20 mRNA was assessed by comparison against serial plasmid dilutions carrying either the flCD20 or the ΔCD20 cloned cDNA. Representative qPCR curves confirming absence of cross-amplification between wtCD20- and ΔCD20-qPCR allowing, respectively, detection of both wtCD20 and ΔCD20mRNA. (B) ΔCD20 mRNA quantification in normal PBMCs or BMMCs from healthy donors as well as in in vitro EBV-transformed B-cell lines, in vitro–generated B blasts, or in CD19⁺ cell-sorted cells from tonsillectomy samples and CD138⁺ plasmocytes from multiple myeloma. Quantification of ΔCD20 mRNA, performed in duplicate, in different normal (PBMCs, BMMCs) or hematologic B malignancies or tumor samples from lymph node (LN), spleen (SP), or pleural effusion (PL) was also reported. Number of cases analyzed for each normal or neoplasia cases are given in brackets in the sample's names of the x-axis. (C) RT-qPCR of CD20 transcripts on pre-rituximab (sensitive) and post-rituximab (resistant) primary cells. Left panel shows human samples (LN or PL) of patients (n = 3) with FL and treated with 3 or 4 courses of RTX. Among these 3 representative cases, 1 is a nonresponse to RTX, whereas the other 2 are early (< 12 months) or late (> 12 months) relapses. Right panel shows PB quantification on 3 patients with MCL (n = 3) from a clinical trial of the French GOELAMS group and treated with 4 courses of RTX (375 mg/m² of RTX). Fold changes (× FC) are indicated. ΔCD20 transcript quantification is reported as relative percentage of ΔCD20: R = (ΔCD20 / wtCD20 + ΔCD20) × 100. Error bars in panels B and C represent SE of RT-qPCR replicates.