DNA-Based Adaptome Profiling for Complete MRD Quantification
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Solution Overview
Problem
Current methods for detecting minimal residual disease (MRD) in hematological malignancies, such as leukemias and lymphomas, are limited by low sensitivity, typically detecting only 1 in 10,000 cells and are restricted to specific DNA markers like chromosomal translocations or RNA-based tests, failing to account for incomplete rearrangements.
Innovation Solution
A method involving multiplex PCR and high-throughput sequencing of T and B cell receptor gene rearrangements, using a library of oligonucleotides to amplify TRα/β/γ/δ, IgH/K/λ, DJ, DD, and kappa-deleting element (KDE) rearrangements, with semi-global alignment and clustering algorithms to correct for PCR and sequencing errors, followed by follow-up monitoring in independent PCR reactions to determine malignant clone concentration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunological based testing for surface proteins is used for MRD testing, then the test can be performed on leukemias with stable cell immunophenotype, but the limit of detection is around 1 in 10,000 cells
Solution Approach 1:
The patent replaces immunological testing (flow cytometry) with DNA-based molecular testing. Specifically, it uses PCR amplification and sequencing of T-cell receptor (TCR) and B-cell receptor (BCR) gene rearrangements to detect malignant cells, achieving detection sensitivity exceeding 1 in 10,000 cells and expanding applicability to leukemias with unstable or absent immunophenotypes
Solution Approach 2:
The patent changes the detection parameter from surface protein expression to genetic rearrangement sequences. By targeting specific V(D)J rearrangements in TCR and BCR genes, the method achieves higher detection sensitivity and can identify malignant cells even when immunophenotypic markers are unstable or unavailable
2Measurement precision
If DNA markers like chromosomal translocations or RNA-based tests are used, then MRD can be detected, but these tests are limited to specific markers and fail to account for incomplete rearrangements
Solution Approach 1:
The patent creates a universal testing platform that simultaneously detects multiple types of rearrangements (complete V(D)J, incomplete V(D), D(D), and kappa-deleting element rearrangements) using a single multiplex PCR assay with multiple primer sets. This multi-functional approach eliminates the need for separate tests for different rearrangement types and improves comprehensive MRD detection
Solution Approach 2:
The patent segments the detection of different rearrangement types into separate primer sets within a unified multiplex PCR system. Each primer set targets specific rearrangement patterns (e.g., V(D)J complete rearrangements, V(D) incomplete rearrangements, D(D) rearrangements, KDE rearrangements), allowing systematic and comprehensive detection of all rearrangement types in a single test
3Adaptability or versatility
If a multiplex PCR with multiple oligonucleotides is used to amplify various rearrangements, then comprehensive MRD detection is achieved, but the complexity of the PCR reaction increases
Solution Approach 1:
The patent merges multiple separate PCR reactions into a single multiplex PCR assay. By combining primer sets for different rearrangement types (V(D)J, V(D), D(D), KDE) into one reaction mixture, the method achieves comprehensive detection of all rearrangement types while reducing operational complexity from multiple separate tests to a single unified test
Solution Approach 2:
The patent uses multiple copies of primer sets targeting different rearrangement patterns within a single multiplex reaction. Each primer set acts as a template copy for its specific rearrangement type, enabling simultaneous amplification of various rearrangements from the same DNA sample without requiring separate reaction systems
4Measurement precision
If follow-up monitoring is performed in multiple independent PCR reactions, then accurate quantification of malignant clone concentration is achieved, but the time and resources required for monitoring increase
Solution Approach 1:
The patent performs follow-up monitoring in a limited number of independent PCR reactions (e.g., 3-5 reactions) rather than exhaustively testing all possible rearrangements. This partial action approach provides sufficient quantification accuracy for clinical decision-making while significantly reducing the time and resources required compared to comprehensive re-testing of all markers
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 by accounting for complete and incomplete rearrangements, enabling precise quantification of malignant clones, thereby improving relapse prediction and therapy outcome evaluation.
Implementation Method 1
detecting rearrangements of T cell receptor genes and B cell receptor genes characteristic for malignant clones in an initial sample from a patient using: a multiplex polymerase chain reaction (PCR) with isolated genomic DNA
Implementation Method 2
high-throughput sequencing of the obtained PCR products
Implementation Method 3
extracting complete and incomplete rearrangements by mapping potential rearrangements to a library comprising V, D, and J gene segments, kappa deletion element (KDE), and IGKC intron sequences, wherein the extraction is performed using a semi-global alignment algorithm to identify flanking sequences
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.