Orthogonal Tumor Assay Validation for Low-ctDNA MRD Detection
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Solution Overview
Problem
Current methods for detecting minimal residual disease (MRD) in cancer patients are prone to false negatives and false positives due to biases in PCR and difficulties with next-generation sequencing (NGS), particularly in low amounts of circulating tumor DNA (ctDNA) and homopolymer runs.
Innovation Solution
A method involving sequencing tumor nucleic acid to identify tumor-specific variants, followed by orthogonal validation using techniques like digital PCR, Sanger sequencing, or atomic force microscopy to confirm the presence of these variants, thereby reducing errors associated with primary detection methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next-generation sequencing (NGS) is used to detect tumor variants in low amounts of ctDNA, then detection sensitivity is improved, but false positives and false negatives increase due to biases in PCR and difficulties with homopolymer runs and short read assembly
Solution Approach 1:
The detection process is segmented into two independent stages: primary detection using NGS to identify candidate tumor variants, and orthogonal validation using alternative methods (such as digital PCR or long-read sequencing) to confirm findings. This segmentation allows each method to play to its strengths while mitigating individual weaknesses, thereby reducing false positives and false negatives.
Solution Approach 2:
An orthogonal validation step serves as an intermediary between the primary NGS detection and the final diagnostic conclusion. This intermediate validation layer uses methods with different technical characteristics (e.g., different PCR approaches, different sequencing technologies) to independently verify variant calls, acting as a buffer against errors from either method alone.
2Quantity of substance
If PCR amplification is used to increase the amount of ctDNA for detection, then detection capability is improved, but biases are introduced that reduce measurement accuracy
Solution Approach 1:
The patent employs parameter changes by switching between different amplification methods with distinct characteristics. Digital PCR uses partition-based amplification that reduces competition effects and provides absolute quantification, while long-read sequencing uses different polymerase and buffer conditions. These parameter changes allow detection of low-abundance ctDNA while minimizing amplification biases that plague traditional PCR.
Data Source
AI summary
The invention provides methods for analyzing tumor nucleic acid from a tumor from a subject to discover one or more variants that are specific to the tumor and confirming by orthogonal testing that nucleic acid of the tumor harbors the variants and that the variants are specific to the tumor and thus useful as a tumor biomarker in an independent assay for the presence of the tumor in the subject.