Multiplex PCR Detection of ALK, RET, and ROS1 Fusions
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting gene fusions associated with cancers, such as ALK, RET, and ROS1, are invasive, require large biopsy samples, and have high false positive rates, limiting their effectiveness and accessibility for cancer patients, especially in later stages.
Innovation Solution
Development of multiplex assay compositions using specific primer sets and labeled probes for amplifying and detecting ALK, RET, and ROS1 fusion genes, along with an internal control, allowing for sensitive detection of multiple fusion genes from small biological samples, including circulating free RNA from plasma.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FISH or IHC methods are used for gene fusion detection, then detection can be performed on tissue sections, but the false positive rate increases and background noise increases due to shearing during sectioning
Solution Approach 1:
The patent extracts the detection process from formalin-fixed paraffin-embedded tissue sections to circulating free RNA in plasma. By taking out the detection from the problematic fixed tissue matrix and performing it on circulating RNA instead, the method eliminates shearing artifacts and associated false positives while maintaining detection capability
Solution Approach 2:
The patent uses circulating free RNA as an intermediary medium to detect gene fusions. Instead of directly analyzing fixed tissue sections, the method detects fusion transcripts in circulating RNA, which serves as a mediator that preserves genetic information without the artifacts of tissue fixation and sectioning
2Measurement precision
If multiple tissue sections are observed by skilled cytologists, then detection accuracy improves, but the biopsy size increases and patient burden increases
Solution Approach 1:
The patent develops a universal detection method using circulating free RNA that can detect multiple gene fusions (ALK, RET, ROS1) simultaneously in a single plasma sample. This multi-functional approach eliminates the need for multiple tissue sections and expert cytological review while maintaining comprehensive detection capability
Solution Approach 2:
The patent uses circulating free RNA as a copy or surrogate of the tumor's genetic information. Instead of requiring large amounts of original tissue for multiple sections, the method detects fusion transcripts that are copied and circulating in the plasma, allowing detection from a minimal or no tissue biopsy
3Measurement precision
If RT-PCR is used for gene fusion detection, then detection sensitivity improves, but the amount and quality of genetic material required from tumor tissue increases
Solution Approach 1:
The patent uses circulating free RNA as an intermediary that provides sufficient genetic material for sensitive RT-PCR detection without requiring large amounts of tumor tissue. The circulating RNA serves as a readily available source of fusion transcripts that can be amplified with high sensitivity
Solution Approach 2:
The patent changes the source parameter from fixed tissue to circulating RNA, which fundamentally alters the availability and quality of genetic material. This parameter change enables sensitive RT-PCR detection because circulating RNA is more accessible and requires smaller sample amounts while maintaining detection sensitivity
4Loss of information
If biopsy is performed for gene fusion detection, then diagnostic information is obtained, but patient invasiveness increases and feasibility decreases for later stage patients
Solution Approach 1:
The patent extracts the diagnostic information need from invasive tissue biopsy and satisfies it through analysis of circulating free RNA in plasma. By taking out the requirement for tissue extraction, the method eliminates patient invasiveness while maintaining the ability to obtain critical diagnostic information about gene fusions
Solution Approach 2:
The patent uses circulating free RNA as a copy of tumor genetic information that can be obtained without invasive biopsy. This copying approach allows diagnostic information to be retrieved from easily accessible plasma samples, making the test feasible for patients who would be too weakened to undergo repeated biopsies
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
Enables sensitive detection of gene fusions down to 25 copies, reducing the need for invasive biopsies and improving the accuracy of cancer diagnosis and treatment by identifying patients responsive to kinase inhibitor therapies.
Implementation Method 1
multiplex assay compositions comprising: (A) at least one primer set and labeled probe that specifically amplify and detect at least one ALK fusion gene; (B) at least one primer set and labeled probe that specifically amplify and detect at least one RET fusion gene
Data Source
AI summary
Provided herein are methods and compositions for multiplex detection of a large number of actionable gene fusions with very high sensitivity and specificity. The present methods and compositions can detect ALK, RET, and ROS1 gene fusions, optionally in combination with other mutations and fusions.