DNA-Based Adaptome Profiling for Sensitive MRD Quantification
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Solution Overview
Problem
Current methods for detecting minimal residual disease (MRD) in hematological malignancies are limited by a detection threshold of around 1 in 10,000 cells and are primarily restricted to detecting chromosomal translocations, lacking sensitivity and specificity for leukemias and lymphomas with unstable cell immunophenotypes.
Innovation Solution
A method involving multiplex PCR and high-throughput sequencing of T and B cell receptor gene rearrangements, using a designed library of oligonucleotides to amplify specific genomic regions, followed by semi-global alignment and clustering algorithms to correct for PCR and sequencing errors, enabling precise quantification of MRD through multiple PCR reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunological based testing for surface proteins is used for MRD testing, then the detection limit is around 1 in 10,000 cells, but the method can only be used in detecting leukemias with a stable cell immunophenotype
Solution Approach 1:
The patent replaces immunological testing (flow cytometry) with DNA-based molecular testing. Instead of detecting surface proteins on cells, the method sequences DNA rearrangements (TCR and BCR genes) to identify malignant clones. This substitution enables detection in leukemias with unstable immunophenotypes because DNA rearrangements remain stable even when protein expression changes.
Solution Approach 2:
The method changes the detection parameter from protein expression levels (immunophenotype) to DNA sequence characteristics (gene rearrangements). By detecting specific V(D)J rearrangements in T and B cell receptor genes, the system can identify malignant cells regardless of changes in their surface protein expression, thereby expanding applicability to cases with unstable immunophenotypes.
2Reliability
If DNA markers (chromosomal translocations, microsatellites, point mutations) are tested for MRD, then specificity is improved, but the methods are essentially limited to detecting chromosomal translocations
Solution Approach 1:
The patent creates a universal testing platform that can detect multiple types of genetic alterations (chromosomal translocations, microsatellite instability, point mutations, and V(D)J rearrangements) using the same sequencing infrastructure. The system uses universal primers and sequencing protocols that work across different mutation types, making the platform adaptable to various leukemia subtypes and genetic profiles.
Solution Approach 2:
The method segments the detection process into separate analysis modules: (1) PCR amplification of specific genomic regions, (2) High-throughput sequencing, and (3) Bioinformatic analysis for identifying different mutation types. This segmentation allows the system to handle diverse genetic markers through a standardized workflow, improving both specificity and detection scope.
3Measurement precision
If multiple independent PCR reactions are performed for follow-up monitoring, then MRD quantification accuracy is improved, but the complexity and time required for testing increases
Solution Approach 1:
The patent merges multiple PCR reactions into a single multiplex PCR assay that simultaneously amplifies multiple gene regions (TCR alpha/beta/gamma/delta and BCR heavy/light chain genes) in one reaction. This consolidation maintains the ability to perform comprehensive MRD quantification while reducing the number of separate reactions needed, thereby decreasing complexity and time requirements.
Solution Approach 2:
The system transitions from sequential analysis of single markers to parallel analysis of multiple markers simultaneously. By using multiplex PCR with multiple primers targeting different gene regions, the method achieves high-dimensional data collection (multiple rearrangement types) in a single reaction, improving efficiency without sacrificing quantification accuracy.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enhances MRD detection sensitivity and accuracy, allowing for the identification and quantification of malignant clones at lower levels, thereby improving relapse prediction and therapy outcome evaluation.
Implementation Method 1
multiplex polymerase chain reaction (PCR) with isolated genomic DNA wherein the PCR reaction comprises a library of oligonucleotides amplifying one or more of the following: TRα/β/γ/δ, IgH/K/λ, DJ, DD, VD, and kappa-deleting element (KDE) rearrangements
Implementation Method 2
high-throughput sequencing of the obtained PCR products
Data Source
AI summary
The present disclosure relates to target sequencing of T and B cell receptor gene rearrangements at the DNA level and using this technology to detect and quantify lymphoid malignant T or B cells (minimal residual disease, MRD) during and after therapy.