The assessment of minimal residual disease (MRD) during first-line therapy is regarded as most important prognostic factor in adult and childhood acute lymphoblastic leukemia (ALL). However, current methods used for MRD assessment have major drawbacks. Multiparameter flow cytometry is not fully standardized and requires expert skills. Allele specific real-time quantitative PCR of clonal immunoglobuline (IG) and T-cell receptor (TR) gene rearrangements is time consuming and needs patient-specific reagents, thus making it ineligible for IVD-guided analytical validation. In this context amplicon based IG/TR NGS was introduced, which allows for the characterization of millions of IG/TR rearrangements in parallel without the need for patient specific reagents, thus allowing IVD-guided analytical validation. The downside of this method is that it is prone to amplification biases that hamper correct quantification. Also, contamination of libraries leading to false positive results constitutes a severe problem in diagnostic routine. The goal and hypothesis of Quant-ALL is to eliminate these disadvantages by a combination of innovative high throughput molecular tools.
Aims and methods: (1) Establishment of an amplicon based IG/TR NGS approach using single DNA copy barcoding and droplet microfluidics for IG/TR based MRD quantification in ALL; (2) optimization of existing bioinformatics tools used for IG/TR NGS to quantify index IG/TR sequences; (3) Transfer of this technology to a pre-existing centrifugal microfluidic platform for full automation and standardization; (4) Validation of technology for childhood and adult ALL using biobanked follow-up samples that are MRD quantified with standard technologies.
The outcome of Quant-All is three-fold:
1) Development and validation of a bioinformatic workflow called UMIC for the execution of barcode demultiplexing. This workflow includes approaches for clonal evolution and identification of sequencing errors that may have an impact in MRD quantification, the calculation of the initial number of cells harbouring each particular rearrangement using Unique Molecular Identifiers (UMIs).
2) Development of a new centrifugal microfluidic unit operation for hydrogel bead emulsification. These hydrogel beads transport primers with UMIs thus allowing a barcoding of target DNA before amplicon PCR.
3) Validation of an automated library preparation for IG/TR NGS. The method of automation is centrifugal microfluidics. The microfluidic chip and device were validated by running it with real patient samples in clincial laboratories by laboratory personnel and comparing the results to manually performed library preparations of the same patient samples.