Tailored Digital PCR Follow-Up of Rare Fusion Transcripts after Initial Detection through RNA Sequencing in Hematological Malignancies

Abstract

The management of hematologic malignancies has entered a new era in which minimal residual disease (MRD) monitoring plays a pivotal role. Well-established molecular targets such as PML::RARA, CBFB::MYH11 or RUNX1::RUNX1T1 are conventionally tracked by quantitative reverse-transcription PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-seq approach (FusionPlex) with a whole transcriptomic strategy (Advanta RNA-seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r=0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection below 0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets.