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Tailored Digital PCR Follow-Up of Rare Fusion Transcripts after Initial Detection through RNA Sequencing in
Marie-Laure Boulland1, Amyra Aliouat2, Elie Jalaber3
1Hematology Laboratory, Rennes University Hospital, Rennes, France; Inserm U1236, Rennes University, Rennes, France.
Abstract:
Minimal residual disease (MRD) monitoring plays a pivotal role in the management of hematologic malignancies. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-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-sequencing (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 of <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.
Insights
New fusion transcripts detected by RNA-sequencing offer novel biomarkers for minimal residual disease (MRD) monitoring in hematologic malignancies. Tailored digital PCR assays provide sensitive MRD quantification, potentially setting a new standard for patients without conventional targets.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Minimal residual disease (MRD) monitoring is crucial for managing hematologic malignancies.
- Conventional MRD assessment relies on established molecular targets like PML::RARA.
- Transcriptomic analysis has revealed a wider range of fusion transcripts.
Purpose of the Study:
- To compare targeted RNA-sequencing (FusionPlex) with whole-transcriptomic sequencing (Advanta RNA-Seq XT) for detecting fusion transcripts.
- To evaluate the utility of newly discovered fusion transcripts as novel MRD markers.
- To validate tailored digital PCR assays for sensitive MRD quantification.
Main Methods:
- Comparative analysis of targeted vs. whole-transcriptomic RNA-sequencing in 21 training samples.
- Prospective evaluation of targeted RNA-sequencing in 126 hematological malignancy patients.
- Development and validation of digital PCR assays for rare fusion transcripts.
Main Results:
- 100% concordance in detecting known fusions between targeted and whole-transcriptomic RNA-seq.
- Good correlation (Spearman r=0.77) in gene expression quantification.
- Fusion transcripts detected in 48% (60/126) of prospectively evaluated patients.
- Digital PCR assays validated for MRD quantification with a limit of detection <0.01%.
Conclusions:
- Targeted RNA-sequencing effectively detects known and novel fusion transcripts.
- Newly identified fusion transcripts are promising biomarkers for MRD monitoring.
- The combination of RNA-seq and digital PCR offers a powerful approach for MRD evaluation, especially for patients lacking conventional targets.
