MRD Detection via Patient-Specific Sequencing and PCR
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Solution Overview
Problem
Current methods for detecting minimal residual disease (MRD) in treated proliferative diseases lack sensitivity, reproducibility, and accuracy, and require access to external databases and specific molecular markers, making them inefficient and lab-dependent.
Innovation Solution
A method involving PCR amplification and sequencing of genomic DNA before and after treatment, using locus-specific primers to determine the degree of similarity and calculate MRD levels based on point mutations and indels, allowing for automated and standardized detection of MRD without external database reliance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry with multiple markers is used to detect MRD, then detection capability is improved, but device complexity and operational difficulty increase
Solution Approach 1:
The patent extracts the essential detection function from complex multi-marker flow cytometry by focusing on a single genetic marker (mutation-specific DNA sequence). This simplifies the detection system while maintaining sensitivity by targeting the unique molecular fingerprint of the patient's disease cells through PCR amplification and sequencing of the specific mutation.
Solution Approach 2:
The patent replaces the mechanical/optical flow cytometry system with a molecular biology-based PCR and sequencing system. This substitution enables highly sensitive detection of MRD by amplifying and sequencing specific genetic markers, achieving comparable or superior sensitivity without requiring multiple physical markers or complex optical equipment.
2Measurement precision
If allele specific oligonucleotide PCR is used to detect MRD, then detection specificity is improved, but applicability decreases to only 40% of cases
Solution Approach 1:
The patent creates a universal detection method that works for any patient with a detectable mutation by using next-generation sequencing technology. Unlike ASO-PCR which requires pre-designed patient-specific primers, this method can identify and quantify MRD for any mutation type (point mutations, indels, rearrangements) through a single universal sequencing platform, making it applicable to 100% of cases rather than just 40%.
Solution Approach 2:
The patent changes the detection parameter from requiring pre-specified known mutations (ASO-PCR) to detecting any mutation present in the sample through comprehensive sequencing. This parameter change enables the method to adapt to any patient's specific mutation profile, greatly expanding versatility while maintaining high specificity through mutation-specific sequence analysis.
3Ease of operation
If manual MRD monitoring methods are used, then flexibility is maintained, but reproducibility and standardization decrease
Solution Approach 1:
The patent implements an automated computational system that performs sequence alignment, mutation identification, and MRD quantification without manual intervention. The system automatically processes sequencing data, compares it to reference sequences, and calculates MRD levels, ensuring consistent and reproducible results across different laboratories while maintaining operational simplicity through automated workflows.
Solution Approach 2:
The patent incorporates quality control feedback mechanisms where the system monitors sequencing data quality, validates mutation detections against control samples, and adjusts analysis parameters automatically. This feedback loop ensures high reproducibility by identifying and correcting potential errors, while maintaining ease of operation through automated quality assurance processes.
4Loss of information
If methods requiring external databases are used, then comprehensive analysis is achieved, but access requirements and complexity increase
Solution Approach 1:
The patent segments the reference data into patient-specific pre-treatment sequences that are obtained directly from each patient's own sample. This eliminates the need for external population databases by using the patient's own baseline genetic information as the reference, thereby achieving complete disease characterization without external database dependencies or associated complexity.
Solution Approach 2:
The patent uses the patient's own pre-treatment DNA sequence as an intermediary reference standard. This personal reference sequence serves as the mediator between the sequencing data and disease detection, enabling comprehensive mutation identification without requiring external databases, thus simplifying the system while maintaining complete disease characterization capability.
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
This approach provides a sensitive, specific, and reproducible method for detecting MRD, enabling personalized treatment by accurately identifying residual disease and minimizing unnecessary therapy.
Implementation Method 1
amplifying by polymerase chain reaction using a pair of primers comprising a locus-specific forward primer and a locus-specific reverse primer, at least one nucleotide sequence comprised in genomic DNA
Implementation Method 2
sequencing each amplified nucleotide sequence, whereby a first list of characters reading from left to right is obtained from each nucleotide sequence thus sequenced
Data Source
Figure 1A
Figure 1B
Figure 2
AI summary
The present invention is focused on a method, kit and system for determining the presence or absence of minimal residual disease in a subject who has been treated for a proliferative disease wherein said method, kit and system comprise: (A) amplifying and sequencing at least one nucleotide sequence comprised in genomic DNA from a biological sample obtained from said subject prior to treatment for said disease, to obtain a first list of characters reading from left to right; (B) amplifying and sequencing at least one nucleotide sequence comprised in genomic DNA from a biological sample obtained from said subject after treatment for said disease, to obtain a second list of characters reading from left to right, wherein when a nucleotide sequence is mutated it is a genetic marker for said proliferative disease; (C) determining, for each second list of characters obtained in step (B), the degree of similarity, DS, with each first list of characters obtained in step (A); (D) selecting, for each second list of characters obtained in step (B), the DS of highest value, DSHV; (E) adding up the number of second lists of characters which have a DSHV that is greater than a threshold value, T, to obtain Lc, (F) adding up the total number of second lists of characters, Lt; (G) calculating the level of minimal residual disease, MRD, according to any of the following formulae: MRD = (Lc x k) / (Lt x D) or MRD = Lc / Lt or MRD = g x Lc x (D / k) / Lt 2; (H) determining (i) the minimum variant read frequency, minVRF, of said genetic marker, (ii) the limit ofdetection, D-limit, of said genetic marker (iii) the average mutation noise, avMut and (iv) the average position noise, avPos; (I) determining the experimental sensitivity, ES, from the greater of the minVRF, D-limit, avMut and avPos or from the greater of minVRF and D-limit; (J) determining the presence or absence of minimal residual disease in said subject by comparing the value of the level of minimal residual disease with the value of ES, the values of minVRF, D-limit, avMut and avPos, or the values of minVRF and D-limit; wherein when said level of minimal residual disease is equal to or greater than said ES, minVRF, D-limit, avMut or avPos values, minimal residual disease is present in said subject.