Histochemical and cytochemical methods for detecting NTRK fusion proteins
Inventors
Feng, Janine • Hansen, Frances • Kivi, Linda • Nuzzo, Patricia • Palting, John • Pate, John
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12631634-B2
Publication Date
2026-05-19
Expiration Date
Abstract
Materials and methods for detecting NTRK rearrangements via affinity staining. Samples are stained with a biomarker-specific reagent (such as an antibody) that binds to a retained portion of TrkA, TrkB, and/or TrkC. The staining pattern is evaluated, and the presence of a Trk fusion is determined by detecting whether or not the sample has at least a threshold number of cells having a threshold staining intensity. In some cases, the same scoring methodology is applied regardless of the staining localization pattern. In other cases, a cytoplasmic and/or membranous localization is scored by a first methodology, whereas a nuclear localization is scored by a second methodology. The methods disclosed herein may be applied across non-endocrine solid tumor types.
Core Innovation
The invention provides a method of detecting a fusion protein of TrkA, TrkB, or TrkC in a non-neuroendocrine tumor sample by affinity histochemically staining the sample with an antibody that specifically binds to defined amino acid sequences comprising residues 363-760 of SEQ ID NO: 1, residues 646-838 of SEQ ID NO: 2, and residues 718-839 of SEQ ID NO: 3. The antibody specifically binds retained Trk portions associated with NTRK fusion proteins.
The method includes detecting nuclear staining in the sample and scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC when the detected nuclear staining shows a staining pattern meeting threshold criteria. Positivity is determined based on a staining pattern that is greater than or equal to a threshold percentage of tumor cells within a threshold contiguous tumor cell area that are specifically stained above a threshold staining intensity.
In additional refinements described in dependent claims, the affinity histochemical staining includes heat-induced epitope retrieval followed by contacting the sample with the antibody and a set of detection reagents to deposit a brightfield dye near any antibody bound to the sample. The detection reagents include a secondary antibody, a tertiary enzyme-conjugated antibody reactive with the secondary antibody, and enzyme-reactive reagents for depositing a brightfield dye, with 3,3′-diaminobenzidine (DAB) identified as an example brightfield dye.
Claims Coverage
The independent claim is directed to a detection method for TrkA/TrkB/TrkC fusion proteins in non-neuroendocrine tumor samples. Across the claim set, five core inventive features recur: affinity histochemical staining with antibodies bound to specified retained Trk residue segments, nuclear-staining-based positivity scoring using contiguous tumor cell area, tumor-cell percentage, and staining intensity thresholds, heat-induced epitope retrieval, multi-tier detection reagents for brightfield dye deposition, and use of antibody EPR17341.
Affinity histochemical staining with antibodies binding retained Trk residue segments
An affinity histochemical staining step using an antibody that specifically binds to defined amino acid sequences comprising residues 363-760 of SEQ ID NO: 1, residues 646-838 of SEQ ID NO: 2, and residues 718-839 of SEQ ID NO: 3 for detecting a fusion protein of TrkA, TrkB, or TrkC in a non-neuroendocrine tumor sample.
Nuclear staining detection and contiguous-area threshold scoring
Detecting nuclear staining and scoring the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if the detected nuclear staining has a staining pattern greater than or equal to a threshold percentage of tumor cells within a threshold contiguous tumor cell area that are specifically stained above a threshold staining intensity, where the threshold contiguous tumor cell area is at least 20 contiguous cells and the threshold percentage of tumor cells ranges from 25% to 75% or 25% to 80%.
Heat-induced epitope retrieval and brightfield dye deposition
Affinity histochemical staining includes heat-induced epitope retrieval followed by contacting the sample with the antibody and a set of detection reagents to deposit a brightfield dye near any antibody bound to the sample.
Multi-tier detection reagents for brightfield dye deposition
The set of detection reagents includes a secondary antibody, a tertiary enzyme-conjugated antibody reactive with the secondary antibody, and enzyme-reactive reagents that deposit a brightfield dye on the sample.
Use of antibody EPR17341
The method is performed using the antibody EPR17341.
The claim set covers detection of TrkA/TrkB/TrkC fusion proteins in non-neuroendocrine tumor samples via affinity histochemical staining with antibodies directed to defined retained Trk residue segments, followed by detection of nuclear staining and positivity scoring based on threshold contiguous tumor cell area, tumor-cell percentage, and staining intensity.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Detection and scoring of TrkA/TrkB/TrkC (NTRK) fusion proteins in non-neuroendocrine tumor samples using affinity histochemical staining and nuclear-staining threshold criteria.
Pan-TRK IHC evaluation supported with in situ hybridization (ISH) confirmation across large tissue sets, as described in the partial content.
Interested in licensing this patent?