FISH Probe Set for Tumor Suppressor Gene Deletion Detection
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Solution Overview
Problem
Current fluorescence in situ hybridization (FISH)-based assays for detecting tumor suppressor gene deletions, such as PTEN, face challenges due to nuclear truncation artifacts, leading to false positives and reduced sensitivity, especially in formalin-fixed paraffin embedded (FFPE) samples, which can result in inconclusive results and missed deletions in low-frequency or heterogeneous samples.
Innovation Solution
The use of probe sets comprising first and second flanking probes that hybridize centromeric and telomeric to the tumor suppressor gene, respectively, along with a target probe, to enumerate FISH signals and determine artifactual and apparent deletion frequencies, allowing for the distinction between actual and artifactual deletions and improving the detection of small and large deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If FISH-based assays are used to detect tumor suppressor gene deletions in FFPE samples, then diagnostic information can be obtained, but nuclear truncation artifacts cause false positives and reduced sensitivity
Solution Approach 1:
The patent divides the detection system into multiple probe types (target probe, centromeric control probe, telomeric control probe) that can independently assess different regions. By segmenting the detection function across multiple probes, the assay can distinguish between true deletions and artifacts affecting specific regions.
Solution Approach 2:
The patent introduces control probes (centromeric and telomeric) as intermediary elements that mediate the detection process. These control probes serve as reference points to determine whether target probe signal loss is due to actual deletions or nuclear truncation artifacts, enabling differentiation between true positives and false positives.
2Reliability
If minimum thresholds are set to guard against false positives, then specificity improves, but sensitivity decreases making it difficult to detect low-frequency deletions
Solution Approach 1:
The patent changes the parameters used for deletion calling from simple binary thresholds to a multi-parameter system based on signal ratios and control probe performance. By using ratio-based metrics and control probe validation, the system can adjust sensitivity and specificity dynamically rather than relying on fixed thresholds.
Solution Approach 2:
The patent implements feedback mechanisms where control probe signals are used to validate and adjust the interpretation of target probe signals. The control probes provide feedback about nuclear integrity, allowing the system to adjust deletion calling decisions based on the observed control signal patterns rather than applying uniform thresholds.
3Reliability
If control probes are added to distinguish artifactual from real deletions, then false positives are reduced, but assay complexity increases
Solution Approach 1:
The patent merges multiple detection functions into a single integrated FISH assay. The target probe, centromeric control probe, and telomeric control probe are combined in one hybridization reaction, allowing simultaneous assessment of deletion status and nuclear integrity without requiring separate assays.
Solution Approach 2:
The control probes serve multiple functions: they verify nuclear integrity, provide reference points for signal normalization, and help distinguish artifacts from true deletions. This multi-functionality reduces the need for separate validation assays, offsetting the added complexity with consolidated utility.
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 reduces the incidence of nuclear truncation artifacts, enhances the specificity and sensitivity of FISH-based assays, enabling more accurate detection of tumor suppressor gene deletions, particularly in FFPE samples, and provides a method to distinguish between small and large deletions, which is clinically relevant for prognosis and treatment decisions.
Implementation Method 1
performing fluorescence in situ hybridization (FISH) with a probe set on a cellular sample comprising a plurality of cells, wherein the probe set comprises at least one first flanking probe that hybridizes to a position centromeric to the tumor suppressor gene, at least one second flanking probe that hybridizes to a position telomeric to the tumor suppressor gene
Data Source
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AI summary
Methods, probe sets, kits, and compositions for gene deletion assays are disclosed. In some embodiments, the methods relate to preparing probes for a deletion assay, performing a deletion assay, or optimizing a deletion assay. In some embodiments, the methods and probe sets can provide reduced artifactual deletion frequency, for example, when analyzing samples subject to truncation artifacts. In some embodiments, the methods and probe sets can distinguish between small and large deletions.