Thyroid Nodule Mutation Screening With Low-Input PCR-NGS
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current diagnostic methods for thyroid nodules with indeterminate cytology, such as ultrasound-guided fine-needle aspiration (FNA) and molecular techniques, struggle to accurately distinguish between benign and malignant nodules, leading to inadequate diagnostic surgery and unnecessary treatments, especially due to DNA degradation in formalin-fixed paraffin-embedded (FFPE) tissues and poor quality FNA specimens.
Innovation Solution
A highly sensitive PCR-based next-generation sequencing (NGS) assay that requires minimal nucleic acid input from FFPE or FNA samples to detect mutations and gene fusions in specific thyroid cancer-related genes, using hot start DNA polymerases with reduced 5'-3' exonuclease activity and adapter sequences for rapid and accurate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional FNA cytology and molecular techniques are used for diagnosis, then diagnostic workflow is maintained, but diagnostic accuracy is insufficient and leads to unnecessary treatments
Solution Approach 1:
The diagnostic approach segments the detection task by targeting specific gene regions (exons) within individual genes rather than attempting to sequence entire genomes. This focused segmentation on cancer-relevant regions improves detection accuracy while maintaining workflow efficiency.
Solution Approach 2:
The patent transitions from two-dimensional FNA cytology examination to three-dimensional multi-omics analysis by simultaneously detecting DNA mutations, RNA expression, and protein markers. This dimensional expansion provides comprehensive diagnostic information that significantly improves accuracy in distinguishing benign from malignant nodules.
2Quantity of substance
If FFPE tissue samples are used for molecular analysis, then tissue availability is improved, but DNA degradation occurs reducing quality
Solution Approach 1:
The patent performs preliminary DNA fragmentation and repair steps before sequencing to compensate for FFPE-induced degradation. By pre-treating the degraded DNA samples with specific enzymatic reactions, the method restores sufficient DNA integrity for accurate mutation detection while maintaining the advantage of using FFPE archived tissues.
Solution Approach 2:
The method changes the DNA analysis parameters by using shorter read lengths and adjusted sequencing depths optimized for degraded FFPE DNA. This parameter optimization allows high-accuracy detection despite the reduced DNA quality inherent in formalin-fixed samples.
3Measurement precision
If comprehensive gene panels are sequenced, then detection capability is improved, but assay complexity increases
Solution Approach 1:
The comprehensive gene panel is segmented into manageable gene groups, each analyzed with optimized primers and sequencing conditions. This modular segmentation allows the complex multi-gene assay to be performed systematically with reduced complexity at each step while maintaining overall comprehensive detection 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
The method allows for precise identification of mutations and gene fusions in thyroid nodules with indeterminate cytology, enabling informed decisions on diagnostic surgery and predicting malignancy risk, even with degraded DNA samples, improving diagnostic accuracy and reducing unnecessary procedures.
Implementation Method 1
generating a DNA library of a first plurality of amplicons using a hot start DNA polymerase that substantially lacks 5'-3' exonuclease activity
Implementation Method 2
detecting at least one mutation in at least one of the first plurality of amplicons using high throughput massive parallel sequencing
Data Source
AI summary
The present technology relates to methods for determining whether a patient having thyroid nodules with indeterminate cytology will benefit from diagnostic surgery, e.g., lobectomy. These methods are based on screening a patient's thyroid nodules and detecting alterations in target nucleic acid sequences corresponding to a specific set of thyroid cancer-related genes. Kits for use in practicing the methods are also provided.