Multiplex TCR CDR3 Sequencing for Lymphoid MRD Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for diagnosing lymphoid hematological malignancies and detecting minimal residual disease (MRD) are limited by poor sensitivity and specificity, particularly due to the heterogeneity of adaptive immune receptors and the need for laborious, patient-specific probes in molecular assays.
Innovation Solution
A method using multiplex PCR and sequencing of TCR V- and J-segment primers to amplify and quantify TCR CDR3-encoding regions, allowing for the detection of unique rearranged DNA sequences and their relative frequencies in biological samples, enabling diagnosis of clonal lymphoid malignancies and MRD.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If molecular assays using patient-specific probes are used to detect minimal residual disease, then sensitivity can be improved, but the device complexity and ease of manufacture deteriorate due to the need for laborious, patient-specific probe development
Solution Approach 1:
The patent applies universality by developing a standardized panel of primers that can detect multiple TCR gene rearrangements across different patients simultaneously. Instead of creating patient-specific probes, the universal primer panel binds to conserved regions of TCR genes, enabling broad detection capability with a single standardized assay platform that works across diverse patient populations.
Solution Approach 2:
The patent segments the TCR gene detection into targeted amplicons covering specific V and J gene segments. By dividing the complex TCR genome into manageable segments that can be amplified and detected individually, the assay achieves high sensitivity for minimal residual disease detection while maintaining standardized procedures that reduce overall complexity.
2Measurement precision
If patient-specific probes are developed for each patient, then measurement precision improves, but productivity deteriorates due to the laborious and time-consuming nature of probe development
Solution Approach 1:
The universal primer panel enables a single assay to detect multiple TCR rearrangements simultaneously, eliminating the need for separate patient-specific probe development. This increases throughput by allowing parallel detection of numerous clonotypes in one experiment while maintaining the detection accuracy needed for minimal residual disease monitoring.
Solution Approach 2:
The patent performs preliminary action by pre-designing and validating a comprehensive panel of primers that cover multiple TCR V and J gene segments before patient testing. This upfront preparation creates a ready-to-use standardized assay that can be immediately applied to multiple patients without requiring individual probe development for each case, thereby significantly improving productivity.
3Productivity
If standardized multiplex PCR with sequencing is used, then ease of manufacture and productivity improve, but measurement precision may deteriorate due to the heterogeneity of adaptive immune receptors
Solution Approach 1:
The patent applies local quality by designing primers that target specific conserved regions within the heterogeneous TCR gene family. Rather than attempting to detect all possible variations uniformly, the assay focuses on locally conserved sequences in V and J segments that are sufficient to identify clonal rearrangements with high accuracy, thereby maintaining measurement precision while enabling standardized high-throughput processing.
Solution Approach 2:
The sequencing-based detection creates digital copies of the amplified TCR rearrangements, allowing for precise quantification and identification of clonotypes. This copying approach enables the standardized assay to accurately distinguish between different TCR rearrangements despite heterogeneity, maintaining detection accuracy while achieving high throughput through automated sequencing analysis.
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 sensitivity and specificity in diagnosing lymphoid malignancies and detecting MRD by accurately identifying unique rearranged DNA sequences, providing a standardized and efficient approach for clinical decision-making.
Implementation Method 1
amplifying DNA extracted from the biological sample in a multiplex polymerase chain reaction (PCR) that comprises: (i) a plurality of V-segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a human T cell receptor (TCR) V-region polypeptide
Implementation Method 2
each V-segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR Vγ-encoding or TCR Vβ-encoding gene segment and wherein the plurality of V-segment primers specifically hybridize to substantially all functional TCR Vγ-encoding or TCR Vβ-encoding gene segments
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
Methods are described for diagnosis of a lymphoid hematological malignancy in a subject prior to treatment, and for detecting minimal residual disease (MRD) in the subject after treatment for the malignancy, by high throughput quantitative sequencing (HTS) of multiple unique adaptive immune receptor (TCR or Ig) encoding DNA molecules that have been amplified from DNA isolated from blood samples or other lymphoid cell-containing samples. Amplification employs oligonucleotide primer sets designed to amplify CDR3-encoding sequences within substantially all possible human VDJ or VJ combinations. Disease-characteristic adaptive immune receptor clonotypes occur, prior to treatment, at a relative frequency of at least 15-30% of rearranged receptor CDR3-encoding gene regions. Following treatment, persistence of at least one such clonotype at a detectable frequency of at least 10-6 or at least 10-5 receptor CDR3-encoding regions indicates MRD. Improved quantitative embodiments are provided by inclusion of a template composition for amplification factor determination and related methods.