Histochemical and cytochemical methods for detecting NTRK fusion proteins

Abstract

The present disclosure provides materials and methods for detecting NTRK rearrangements via affinity staining. A sample is stained with a biomarker-specific reagent (e.g., an antibody) that binds to the 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 the sample has at least a threshold number of cells with a threshold staining intensity. In some cases, the same assessment method is applied regardless of the staining localization pattern. In other cases, cytoplasmic localization and / or membrane localization are assessed by a first method, while nuclear localization is assessed by a second method. The methods disclosed herein can be applied to all non-endocrine solid tumor types.

Description

[0001] Cross - reference to related patent applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 731,032, filed on September 13, 2018, entitled HISTOCHEMICAL AND CYTOCHEMICAL METHODS FOR DETECTING NTRK FUSION PROTEINS, the content of which is incorporated herein by reference in its entirety.

[0003] Incorporation of sequence listing by reference

[0004] This application hereby incorporates by reference the sequence listing submitted herewith in computer - readable format, which has the file name "P35023 - WO PCT FLING SEQUENCE LISTING", was created on September 9, 2019, and is 21,260 bytes in size. Technical field

[0005] Among other things, the present disclosure relates to methods for histochemical and cytochemical detection of fusion proteins involving NTRK gene products; materials, kits, and systems useful in such methods; and products resulting from the performance of such methods. Background art

[0006] Neurotrophic tyrosine receptor kinases (NTRK1, NTRK2, and NTRK3) are a family of genes encoding receptor tyrosine kinase proteins (TRKA, TRKB, and TRKC), which play roles in the development and maturation of the central and peripheral nervous systems (Barbacid I, Barbacid II, Lemmon & Schlessinger, and Klein). In cancer, the intact kinase domain of one of the three NTRK genes can fuse with various upstream partners (such as ETV6, LMNA, TPM3), which replace the ligand - binding domain of the TRK protein using a structural motif that promotes dimerization (Eide, Luberg, & Vaishnavi). This results in constitutive activation of TRK signaling and unchecked proliferation, thereby triggering the expression of fusion proteins.

[0007] NTRK gene fusions have specific signatures in certain rare tumors, such as infantile fibrosarcoma, congenital mesoblastic nephroma, secretory breast carcinoma, and mammary analogue secretory carcinoma (MASC) (Argani, Bishop, Bourgeois, Rubin, & Tognon). Conversely, NTRK gene fusions rarely occur in a variety of adult and pediatric solid tumors, which include but are not limited to: appendiceal cancer, breast cancer, cholangiocarcinoma, colorectal cancer (CRC), GIST, lung cancer, melanoma, pancreatic cancer, thyroid cancer, and various sarcomas (DeBraud, Brzezianska, Fernandez-Cuesta, Leeman-Neill, & Ross). Identifying NTRK gene fusions has important clinical implications because small molecule inhibitors are currently being developed for the treatment of solid tumors with NTRK gene fusions (Vaishnavi & De Braud).

[0008] To date, there is no gold standard for the detection of NTRK fusions in tumors due to the inconsistent specificity and sensitivity of different methods. Current detection methods include fluorescence in situ hybridization (FISH), in situ hybridization (ISH), next-generation sequencing (NGS), and immunohistochemistry (IHC). Optimization and enumeration / interpretation of FISH and ISH assays can be difficult. NGS is highly specific but lacks sensitivity. In contrast, IHC can be highly sensitive, but it detects protein expression rather than the presence of the actual fusion. Additionally, due to the endogenous presence of wild-type TRK protein in some tumors (i.e., neuroendocrine tumors and GIST), optimizing IHC assays for fusion products and subsequently developing specific scoring algorithms to select for this fusion product can be challenging. SUMMARY OF THE INVENTION

[0009] The present disclosure relates to methods for identifying non-neuroendocrine tumors driven by TRK fusion proteins.

[0010] In one embodiment, a method for detecting TrkA, TrkB, or TrkC fusion proteins in a non-neuroendocrine tumor sample is provided, the method comprising: performing affinity histochemical staining of the sample with a biomarker-specific reagent that specifically binds to one or more of the following: an amino acid sequence comprising residues 363-760 of SEQ ID NO:1, consisting essentially of or consisting of the same; an amino acid sequence comprising residues 646-838 of SEQ ID NO:2, consisting essentially of or consisting of the same; or an amino acid sequence comprising residues 718-839 of SEQ ID NO:3, consisting essentially of or consisting of the same; detecting the staining pattern in the sample; and assessing the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if any of the following occurs: the sample has a cytoplasmic staining pattern and / or a membrane staining pattern and has a first threshold percentage of tumor cells with staining above a predetermined specific staining intensity; or the sample has a nuclear staining pattern and is greater than or equal to a second threshold percentage of cells specifically stained at any intensity within a threshold tumor cell area.

[0011] In one embodiment, a method for detecting TrkA, TrkB, or TrkC fusion proteins in a non-neuroendocrine tumor sample is provided, the method comprising: performing affinity histochemical staining of the sample with a biomarker-specific reagent that specifically binds to: an amino acid sequence comprising residues 363-760 of SEQ ID NO:1, consisting essentially of or consisting of the same; an amino acid sequence comprising residues 646-838 of SEQ ID NO:2, consisting essentially of or consisting of the same; and an amino acid sequence comprising residues 718-839 of SEQ ID NO:3, consisting essentially of or consisting of the same; detecting the staining pattern in the sample; and assessing the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if any of the following occurs: the sample has a cytoplasmic staining pattern and / or a membrane staining pattern and has a first threshold percentage of tumor cells with staining above a predetermined specific staining intensity; or the sample has a nuclear staining pattern and is greater than or equal to a second threshold percentage of cells specifically stained at any intensity within a threshold tumor cell area.

[0012] In one embodiment, a method for detecting TrkA, TrkB, or TrkC fusion proteins in a non-neuroendocrine tumor sample is provided, the method comprising: performing affinity histochemical staining of the sample with a biomarker-specific reagent that specifically binds to the amino acid sequence consisting of residues 816-838 of SEQ ID NO:2; detecting the staining pattern in the sample; and assessing the sample as positive for a fusion protein involving TrkA, TrkB, or TrkC if: the sample has a cytoplasmic staining pattern and / or a membrane staining pattern and has a first threshold percentage of tumor cells with staining above a predetermined specific staining intensity; or the sample has a nuclear staining pattern and is greater than or equal to a second threshold percentage of cells specifically stained at any intensity within the threshold tumor cell area.

[0013] In one embodiment, a method for detecting NTRK rearrangements in a non-neuroendocrine tumor sample is provided, the method comprising detecting the presence of Trk fusion proteins in the sample according to the methods described herein, and if the sample is assessed as positive for a fusion protein involving TrkA, TrkB, or TrkC, screening the sample by sequencing, reverse transcriptase polymerase chain reaction, or in situ hybridization to confirm the presence of NTRK rearrangements.

[0014] In one embodiment, a method for selecting a patient to receive Trk-directed therapy is provided, the method comprising detecting the presence of Trk fusion proteins or NTRK rearrangements in a non-neuroendocrine tumor sample according to the methods described herein, and if the sample is assessed as positive for a fusion protein involving TrkA, TrkB, or TrkC or an NTRK rearrangement is detected, selecting the patient to receive therapy.

[0015] In one embodiment, a method for staining a sample for the presence or absence of TrkA, TrkB, or TrkC and fusion proteins involving their kinase domains is also provided, the method comprising: (a) performing a heat-induced epitope retrieval process on the sample; and (b) contacting the sample with a biomarker-specific reagent and a set of detection reagents to deposit a bright-field dye in the vicinity of any biomarker-specific reagent bound to the sample, wherein the biomarker-specific reagent specifically binds to one or more of the following: the amino acid sequence comprising residues 363-760 of SEQ ID NO:1, consisting essentially of or consisting of the same; the amino acid sequence comprising residues 646-838 of SEQ ID NO:2, consisting essentially of or consisting of the same; or the amino acid sequence comprising residues 718-839 of SEQ ID NO:3, consisting essentially of or consisting of the same.

[0016] In one embodiment, there is also provided a method for staining a sample for the presence or absence of TrkA, TrkB, or TrkC and fusion proteins involving their kinase domains, the method comprising: (a) subjecting the sample to a heat-induced epitope retrieval process; and (b) contacting the sample with a biomarker-specific reagent and a set of detection reagents to deposit a brightfield dye in the vicinity of any biomarker-specific reagent bound to the sample, wherein the biomarker-specific reagent is a primary antibody that specifically binds to one or more of the following: an amino acid sequence comprising residues 363-760 of SEQ ID NO:1, consisting essentially of or consisting of the same; an amino acid sequence comprising residues 646-838 of SEQ ID NO:2, consisting essentially of or consisting of the same; or an amino acid sequence comprising residues 718-839 of SEQ ID NO:3, consisting essentially of or consisting of the same; and wherein the set of detection reagents includes: a secondary antibody that is immunoreactive with the primary antibody; a tertiary antibody that is immunoreactive with the secondary antibody, the secondary antibody being conjugated to an enzyme; and a set of reagents that are reactive with the enzyme to effect deposition of the brightfield dye on the sample.

[0017] In one embodiment, there is also provided a method for staining a sample for the presence or absence of TrkA, TrkB, or TrkC and fusion proteins involving their kinase domains, the method comprising: (a) subjecting the sample to a heat-induced epitope retrieval process; and (b) contacting the sample with a biomarker-specific reagent and a set of detection reagents to deposit a brightfield dye in the vicinity of any biomarker-specific reagent bound to the sample, wherein the biomarker-specific reagent is a primary antibody that specifically binds to each of the following: an amino acid sequence comprising residues 363-760 of SEQ ID NO:1, consisting essentially of or consisting of the same; an amino acid sequence comprising residues 646-838 of SEQ ID NO:2, consisting essentially of or consisting of the same; or an amino acid sequence comprising residues 718-839 of SEQ ID NO:3, consisting essentially of or consisting of the same; and wherein the set of detection reagents includes: a secondary antibody that is immunoreactive with the primary antibody; a tertiary antibody that is immunoreactive with the secondary antibody, the secondary antibody being conjugated to an enzyme; and a set of reagents that are reactive with the enzyme to effect deposition of the brightfield dye on the sample.

[0018] Other embodiments will be apparent from the following disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Sequence alignment between exemplary conserved portions of TrkA (amino acid residues 363 - 760 of SEQ ID NO:1), TrkB (amino acid residues 646 - 838 of SEQ ID NO:2), and TrkC (amino acid residues 718 - 839 of SEQ ID NO:3). The Kalign multiple sequence alignment tool from EMBL-EBI (https: / / www.ebi.ac.uk / Tools / msa / kalign / ) was used to generate the sequence alignment. Default settings were used, which include ClustalW output format, gap open penalty of 11, gap extension penalty of 0.85, end gap penalty of 0.45, and reward score of 0.

[0020] Figure 2A IHC results for Trk fusions in multiple tumor types. Orthogonal testing was used to evaluate the presence of Trk fusions.

[0021] Figure 2B IHC results for Trk fusions in multiple tumor types. Orthogonal testing was used to evaluate the absence of Trk fusions.

[0022] Figure 3A Staining distribution of tumor types with ISH-confirmed fusion-positive cases in the dataset by percentage of tumor cell staining (0 - 100%) and staining intensity (0 - 3+). Circles represent fusion-negative cases, while triangles represent fusion-positive cases. Diamonds indicate cases where fusions were detected by ISH, but a fixed gradient was observed. Patterns represent various tumor histologies. Figure 5 a and Figure 5 b show the staining distribution of neuroendocrine tumors in which no fusion-positive cases were detected.

[0023] Figure 3B Staining distribution of tumor types with ISH-confirmed fusion-negative cases in the dataset by percentage of tumor cell staining (0 - 100%) and staining intensity (0 - 3+).

[0024] Figure 4 Subclassification of solid tumors based on IHC and ISH tests.

[0025] Figure 5Examples of differences in the localization of staining patterns obtained with the staining methods described herein. (A) Cytoplasmic staining pattern / membrane staining pattern in a colorectal tumor determined by break-apart in situ hybridization to have an NTRK1 rearrangement and confirmed by next-generation sequencing to be an NTRK1-TPM3 fusion. (B) Cytoplasmic staining pattern in a colorectal tumor determined by break-apart in situ hybridization to have an NTRK1 rearrangement and confirmed by next-generation sequencing to be an NTRK1-EML4 fusion. (C) Nuclear staining pattern / cytoplasmic staining pattern in a mammary analogue secretory carcinoma (MASC) determined by break-apart in situ hybridization to have an NTRK3 rearrangement and confirmed by next-generation sequencing to be an NTRK3-ETV6 fusion.

[0026] Figure 6 Examples of fixed gradients in tissues and showing that nuclear localization appears to be more affected by the gradient than cytoplasmic / membrane localization. The bottom row shows low-magnification image segments, and the top row shows high-magnification of the image segments immediately below them. Detailed Description

[0027] I. Definitions

[0028] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). The terms "a" or "an" are intended to mean "one or more / one or more kinds." Before listing steps or elements, the terms "comprising," "including," and "containing" are intended to mean that the addition of further steps or elements is optional and not exclusive.

[0029] Antibody: The term "antibody" as used herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0030] Antibody fragment: "Antibody fragment" refers to a molecule other than a whole antibody that contains a part of the whole antibody and binds to the antigen to which the whole antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments.

[0031] Biomarker: As used herein, the term "biomarker" shall refer to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or the subject from whom the biological sample was obtained. For example, a biomarker can be a molecule or a group of molecules whose presence, absence, or relative abundance is:

[0032] · Characteristic of a specific cell or tissue type or state;

[0033] · Characteristic of a specific pathological condition or state; or

[0034] · Indicating the severity of a pathological condition, the likelihood of progression or regression of a pathological condition, and / or the likelihood of response of a pathological condition to a specific treatment.

[0035] As another example, a biomarker can be a cell type or microorganism (such as bacteria, mycobacteria, fungi, viruses, etc.), or a substituent molecule or a group of its molecules.

[0036] Biomarker-specific reagent: A specific detection reagent capable of specifically binding to one or more biomarkers in a cell sample, such as a primary antibody.

[0037] Cell sample: As used herein, the term "cell sample" refers to any sample containing intact cells, such as a cell culture, a body fluid sample, or a surgical sample, which is collected for pathological, histological, or cytological interpretation.

[0038] Cytochemical detection: A process involving labeling a biomarker or other structure in a cytological sample with a biomarker-specific reagent and a detection reagent, which is carried out in such a way as to allow microscopic detection of the biomarker or other structure in the context of intact cells.

[0039] Cytological sample: As used herein, the term "cytological sample" shall refer to a cell sample that has no cross-sectional spatial relationship in vivo (such as a cell sample derived from a blood sample, a urine sample, a sputum, etc.), or a cell sample in which the cross-sectional spatial relationship has been at least partially disrupted (such as a tissue smear, a liquid-based cytological sample, a fine needle aspirate, etc.).

[0040] Detection Reagent: "Detection reagent" refers to any reagent used to deposit a stain near a biomarker-specific reagent in a cell sample. Non-limiting examples include biomarker-specific reagents (such as primary antibodies), secondary detection reagents (such as secondary antibodies capable of binding to primary antibodies), tertiary detection reagents (such as tertiary antibodies capable of binding to secondary antibodies), enzymes directly or indirectly associated with biomarker-specific reagents, chemicals that react with such enzymes to affect the deposition of fluorescent or chromogenic stains, washing reagents used between staining steps, etc.

[0041] Detectable Moiety: A molecule or material that can generate a detectable signal (such as visually, electronically, or otherwise), where the detectable signal indicates the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of the detectable moiety deposited on the sample. The detectable signal can be generated by any known or yet-to-be-discovered mechanism, which includes the absorption, emission, and / or scattering of photons (including radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency photons). The term "detectable moiety" includes chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another to provide a detectable difference (such as by converting a colorless substance into a colored substance, or vice versa, or by producing a precipitate or increasing the turbidity of the sample). In some examples, the detectable moiety is a fluorophore, which belongs to several common chemical classes, including coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorufin, luminophore, and cyanine. Additional examples of fluorescent molecules can be found in Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11th Edition. In other embodiments, the detectable moiety is a molecule detectable via brightfield microscopy, such as dyes including 3,3'-Diaminobenzidine (DAB), 4-(Dimethylamino)azobenzene-4'-sulfonamide (DABSYL), Tetramethylrhodamine (DISCOVERY Purple), N,N'-Bis(carboxypentyl)-5,5'-disulfonate-Indodicarbocyanine (Cy5), and Rhodamine 110.

[0042] Histochemical Detection: A process involving labeling a biomarker or other structure in a tissue sample with a biomarker-specific reagent and a detection reagent, which is carried out in such a way as to permit microscopic detection of the biomarker or other structure in the context of the cross-sectional relationships between the structures of the tissue sample.

[0043] Monoclonal antibody: An antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody of the population is identical and / or binds the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody preparation, such variants typically being present in trace amounts). In contrast to polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus or combinations thereof.

[0044] "Retention portion" shall mean any portion of the wild-type counterpart of the oncogenic fusion protein that is retained in the oncogenic fusion protein.

[0045] Specific binding: As used herein, the terms "specifically bind", "binds specifically", "is specific for" or other similar iterations refer to a measurable and reproducible interaction between a target and a specific detection reagent that identifies the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target is one that binds that target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the degree of binding of the specific detection reagent to an irrelevant target is less than about 10% of the measured binding of the antibody to the target (e.g., by radioimmunoassay (RIA)). In certain embodiments, the dissociation constant (Kd) of a biomarker-specific reagent that specifically binds to a target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In another embodiment, specific binding can include but does not require exclusive binding.

[0046] Specific detection reagent: Any substance composition capable of specifically binding to the chemical structure of a target in the context of a cell sample.

[0047] Stain: When used as a noun, the term "stain" shall refer to any substance that can be used to visualize a particular molecule or structure in a cell sample for microscopic analysis, including bright-field microscopy, fluorescence microscopy, electron microscopy, etc. When used as a verb, the term "stain" shall refer to any process that results in the deposition of a stain on a cell sample.

[0048] Subjects: As used herein, the terms "subject" or "individual" are mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0049] Test sample: A tumor sample obtained from a subject, the tumor sample having an unknown outcome at the time of sample acquisition.

[0050] Tissue sample: As used herein, the term "tissue sample" shall refer to a sample of cells that maintains the cross-sectional spatial relationships between the cells as they exist in the subject from which the sample was obtained.

[0051] Tumor sample: A tissue sample obtained from a tumor.

[0052] II. Staining Methods

[0053] The present disclosure is based on affinity histochemical or cytochemical staining of samples for non-neuroendocrine NTRK fusion proteins using a first biomarker-specific reagent that targets a retained portion of one or more of TrkA, TrkB, and TrkC. The biomarker-specific reagent is selected by identifying the breakpoint of the wild-type protein that gives rise to the fusion protein and targeting a portion of the wild-type protein on one side of the breakpoint that is retained in the fusion protein. Many resources are available for identifying fusion protein breakpoints by genomic location and by the first exon expressed in the resulting fusion protein, including, for example, the COSMIC database that contains notations of breakpoints. Exemplary wild-type protein sequences are listed in Table 1:

[0054] Table 1

[0055] Wild-type protein Exemplary amino acid sequence Background references TrkA Uniprot P04629-3 (SEQ ID NO:1) Amatu et al.; Stransky et al. TrkB Uniprot Q16620-4 (SEQ ID NO:2) Amatu et al.; Stransky et al. TrkC Uniprot Q16288-1 (SEQ ID NO:3) Amatu et al.; Stransky et al.

[0056] Exemplary breakpoints and shared retained portions for fusion proteins that retain the C-terminus of TrkA, TrkB, or TrkC are shown in Table 2:

[0057] Table 2

[0058]

[0059] In one embodiment, the biomarker-specific reagent specifically binds to a retained portion of one or more of TrkA, TrkB, and TrkC. In one embodiment, the biomarker-specific reagent specifically binds to the retained portion of each of TrkA, TrkB, and TrkC. Figure 1Shows an alignment between exemplary conserved portions of TrkA, TrkB, and TrkC. In another specific embodiment, the biomarker-specific reagent specifically binds to one or more of the following: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3. In another specific embodiment, the biomarker-specific reagent specifically binds to: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; and (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3.

[0060] A biomarker-specific reagent can be any type of entity that can be used to detect in situ a protein expressed by a cell sample (such as by histological or cytological staining methods). Exemplary biomarker-specific reagents include antibodies and their antigen-binding fragments and engineered specific binding compositions such as ADNECTIN (a scaffold based on the 10FN3 fibronectin domain; Bristol-Myers-Squibb Co.), AFFIBODY (a scaffold based on the Z domain of protein A from Staphylococcus aureus; Affibody AB, Solna, Sweden), AVIMER (a scaffold based on the domain A / LDL receptor; Amgen, Thousand Oaks, CA), dAb (a scaffold based on the VH or VL antibody domain; GlaxoSmithKline PLC, Cambridge, UK), DARPin (a scaffold based on ankyrin repeat proteins; Molecular Partners AG, Zürich, CH), ANTICALIN (a scaffold based on lipocalins; Pieris AG, Freising, DE), NANOBODY (a scaffold based on VHH (camel Ig); Ablynx N / V, Ghent, BE), TRANS-BODY (a scaffold based on transferrin; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (a scaffold based on the C-type lectin domain (CTLD), tetranectin; Borean Pharma A / S, Aarhus, DK). The description of such engineered specific binding structures is reviewed by Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the content of which is incorporated by reference.

[0061] In a particular embodiment, the biomarker-specific reagent is an antibody. In another particular embodiment, the antibody is a monoclonal antibody (such as a mouse monoclonal or a rabbit monoclonal antibody). In another particular embodiment, the antibody specifically binds to a conserved portion of one or more of TrkA, TrkB, or TrkC. In another particular embodiment, the antibody specifically binds to an epitope contained in one or more of the following: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; or (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3. In another particular embodiment, the antibody specifically binds to the following: (a) an amino acid sequence comprising, consisting essentially of, or consisting of residues 363-760 of SEQ ID NO:1; (b) an amino acid sequence comprising, consisting essentially of, or consisting of residues 646-838 of SEQ ID NO:2; and (c) an amino acid sequence comprising, consisting essentially of, or consisting of residues 718-839 of SEQ ID NO:3. In one embodiment, the antibody is a commercially available antibody according to Table 3:

[0062] Table 3

[0063]

[0064] In a particular embodiment, the antibody is EPR17341.

[0065] The biomarker-specific reagent is used for affinity histochemistry or affinity cytochemistry staining of a sample suspected of carrying an NTRK fusion protein. Affinity histochemistry and cytochemistry staining techniques generally involve contacting the sample deposited on a slide or other solid support with the biomarker-specific reagent under conditions sufficient to allow specific binding between the biomarker-specific reagent and the target biomarker. The binding of the biomarker-specific reagent to the biomarker helps deposit a detectable moiety on the sample near the location containing the biomarker. The detectable moiety can be used to localize and / or quantify the biomarker involved by the biomarker-specific reagent. Thus, the presence and / or relative amount of the target in the sample can be detected by detecting the signal generated by the detectable moiety.

[0066] The staining process can be manual, automated, or a combination of manual and automated steps. In one embodiment, the staining process can be performed on an automated advanced staining platform. An automated advanced staining platform typically includes at least: reservoirs for the various reagents in the staining protocol, a reagent dispensing unit in fluid communication with the reservoirs for dispensing the reagents onto the slides, a waste removal system for removing the used reagents and other waste from the slides, and a control system for coordinating the actions of the reagent dispensing unit and the waste removal system. In addition to performing the staining steps, many automated slide stainers can also perform steps that assist with the staining (or are compatible with a separate system that performs such assisting steps), which include: slide baking (for adhering the sample to the slide), dewaxing (also known as deparaffinization), epitope retrieval, counterstaining, dehydration and cleaning, and coverslipping. Prichard describes several specific examples of automated IHC / ISH slide stainers and their various features, which include the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS, and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer (Thermo Scientific) automated slide stainers. Additionally, Ventana Medical Systems, Inc. is the assignee of a number of U.S. patents that disclose systems and methods for performing automated analysis, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety.Commercially available staining units typically operate according to one of the following principles: (1) open single-slide staining, where the slide is positioned horizontally and the reagent is dispensed as a puddle on the surface of the slide containing the tissue sample (such as implemented on the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and intelliPATH (Biocare Medical) stainers); (2) liquid-overlay techniques, where the reagent is covered by or dispensed through an inert fluid layer deposited over the sample (such as implemented on the VENTANA BenchMark and DISCOVERY stainers); (3) capillary-gap staining, where the slide surface is placed near another surface (which can be another slide or a cover slip) to create a narrow gap, and capillary forces cause the liquid reagent to contact and hold the sample through this gap (such as the staining principle used by the DAKO TECHMATE, Leica BOND, and DAKO OMNIS stainers). Some iterations of capillary-gap staining do not mix the fluid in the gap (such as on the DAKO TECHMATE and Leica BOND). In a variant of capillary-gap staining called dynamic-gap staining, capillary forces are used to apply the sample to the slide, and then during incubation, the parallel surfaces are translated relative to one another to agitate the reagent to achieve reagent mixing (such as the staining principle implemented on the DAKO OMNIS slide stainer (Agilent)). In translational-gap staining, a translatable head is positioned above the slide. The lower surface of the head is spaced from the slide by a first gap small enough to allow a liquid meniscus to form from the liquid on the slide during translation of the slide. A mixing extension having a lateral dimension smaller than the width of the slide extends from the lower surface of the translatable head to define a second gap smaller than the first gap between the mixing extension and the slide. During translation of the head, the lateral dimension of the mixing extension is sufficient to generate a lateral movement in the liquid on the slide in a direction generally extending from the second gap to the first gap. See WO 2011-139978 A1. Recently, it has been proposed to use inkjet technology to deposit reagents on slides. See WO 2016-170008 A1. This list of staining techniques is not intended to be comprehensive, and any fully automated or semi-automated system for performing biomarker staining can be used.

[0067] A staining method is practiced on a cell sample of a suspected tissue, the cell sample including a tissue sample and a cytology sample. In some embodiments, the cell sample is obtained from a subject having or suspected of having a tumor. In some embodiments, the sample is obtained directly from the tumor. In some embodiments, the tumor is a solid tumor, such as a carcinoma, lymphoma, or sarcoma. In one embodiment, the tumor is a non-neuroendocrine tumor. In one embodiment, the non-neuroendocrine tumor is a solid tumor of the salivary gland, thyroid, skin, breast, head and / or neck, lung, upper digestive tract (including esophagus and stomach), female reproductive system (including uterine, fallopian tube, and ovarian tumors), lower digestive tract (including colorectal, rectal, and anal tumors), urogenital tract, exocrine, endocrine, kidney, or a solid tumor of lymphocyte origin. In one embodiment, the subject has melanoma, salivary gland carcinoma, thyroid carcinoma, breast carcinoma, ovarian carcinoma, pancreatic carcinoma, head and neck carcinoma, lung carcinoma, esophageal carcinoma, gastric carcinoma (excluding gastrointestinal stromal tumor (GIST)), colorectal carcinoma (including carcinomas of the colon, rectum, and anus), prostate carcinoma, urothelial carcinoma, or lymphoma.

[0068] In the case of using a tissue sample, the tissue sample is processed in a manner compatible with histochemical staining, the manner including, for example, fixation, embedding in a wax matrix (such as paraffin), and sectioning (such as with a microtome). The present disclosure does not require a specific processing step, as long as the obtained sample is compatible with histochemical staining of the sample with the set of biomarker-specific reagents. In a particular embodiment, ultrathin sections of formalin-fixed, paraffin-embedded (FFPE) samples are used during the staining process. In the case of using a cytology sample, the sample is fixed in formalin.

[0069] Depending on the biomarker-specific reagent used and the sample used, an epitope retrieval process (also known as antigen retrieval) may be performed on the sample prior to application of the biomarker-specific reagent. Exemplary epitope retrieval processes include: heat-induced epitope retrieval (HIER), which involves heating the sample in various buffers at different pH levels; protease-based epitope retrieval (PBER), in which the sample is digested by a proteolytic enzyme prior to staining; and combinations of HIER and PBER. Various specific epitope retrieval processes have been reviewed by Shi et al., D'Amico et al., Yamashita et al., Vinod et al., and Warford et al., although this is not exhaustive. Whether to perform the epitope retrieval to be used and the specific form of epitope retrieval depend on the particular biomarker-specific reagent selected and may require empirical determination for each biomarker-specific reagent used.

[0070] Depending on the reagents and samples used, it may also be necessary to block the activity of endogenous proteins before adding biomarker-specific reagents and / or detection reagents. For example, in cases where the detection reagent depends on biotin and biotin-binding proteins, it may be necessary to use, for example, free, unlabeled biotin-binding proteins to block endogenous biotin. Similarly, many detection protocols rely on the activity of enzymes, including phosphatases and peroxidases, which requires neutralizing endogenous enzymes with similar activity. Commercially available kits can be used for such blocking processes, such as, for example, the Endogenous Biotin Blocking Kit (Catalog No. E21390, ThermoFisher Scientific), the Endogenous Avidin / Biotin Blocking Kit (Catalog No. ab64212, Abcam, plc.), the Endogenous Biotin Blocking Kit Catalog No. 760-050, Ventana Medical Systems, Inc.), the Hydrogen Peroxide Blocking Reagent (Catalog No. ab64218, Abcam plc.), the Peroxidase and Alkaline Phosphatase Blocking Reagent (Code S2003, Agilent Technologies), etc.

[0071] It may also be useful to block sites on the sample to which the biomarker-specific reagent can bind nonspecifically before applying the biomarker-specific reagent to the sample. Common blocking agents include buffered solutions of normal serum, skim milk powder, BSA (bovine serum albumin), and gelatin, as well as commercially available blocking agents such as eBioscience TM IHC / ICC Blocking Buffer - High Protein (Catalog No. 00-4952-54, ThermoFisher Scientific), eBioscience TM IHC / ICC Blocking Buffer - Low Protein (Catalog No. 00-4953-54, ThermoFisher Scientific), DISCOVERY Antibody Blocking Agent (Catalog No. 760-4204, Ventana Medical Systems, Inc.), etc.

[0072] After each of these pretreatment steps, the washing step can be performed by washing one or more times with a washing buffer. The washing buffer is typically a neutral buffered physiological saline solution, which may also contain a small amount of detergent. Exemplary washing buffers include, for example, phosphate buffered saline (PBS), PBS-Tween20, Tris buffered saline (TBS), TBS-Tween20 (polysorbate 20), Tris-HCl, Tris-HC-Tween20, phosphate buffer (PB), AP buffer, etc.

[0073] Once the sample is ready for staining, a biomarker-specific reagent is applied to the sample and incubated for a sufficient period of time and under conditions to promote specific binding between the biomarker and the biomarker-specific reagent. After incubating the sample with the biomarker-specific reagent, the washing step can be performed by applying a wash buffer one or more times. This removes unbound or non-specifically bound biomarker-specific reagent from the sample to reduce off-target and / or background staining.

[0074] Detection of the biomarker in the sample is achieved by depositing a detectable moiety in close proximity to the biomarker-specific reagent bound to the sample. In some embodiments, the detectable moiety is directly conjugated to the biomarker-specific reagent and thus deposited on the sample when the biomarker-specific reagent binds to its target (commonly referred to as the direct labeling method). In other embodiments, deposition of the detectable moiety is achieved by applying a set of detection reagents to the sample after application of the biomarker-specific reagent, where the detection reagents bind to or otherwise react with the biomarker-specific reagent in a manner that affects the deposition of the detectable moiety (commonly referred to as the indirect labeling method).

[0075] In some embodiments using the indirect method, the detectable moiety is deposited via an enzymatic reaction localized to the biomarker-specific reagent. Suitable enzymes for such reactions are well known and include, but are not limited to, oxidoreductases, hydrolases, and peroxidases. Specific enzymes that are expressly included are horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, and β-lactamase. The enzyme can be directly conjugated to the biomarker-specific reagent or can be indirectly associated with the biomarker-specific reagent via a labeling conjugate. As used herein, "labeling conjugate" includes:

[0076] (a) a specific detection reagent; and

[0077] (b) an enzyme conjugated to the specific detection reagent, where the enzyme reacts with a chromogenic substrate, a signal transduction conjugate, and / or an enzyme-reactive dye under appropriate reaction conditions to effect in situ generation of a dye and / or deposition of the dye on the tissue sample.

[0078] In non-limiting examples, the specific detection reagent for the labeled conjugate can be a secondary detection reagent (such as a class-specific secondary antibody that binds to a primary antibody, an anti-hapten antibody that binds to a hapten-conjugated primary antibody, or a streptavidin that binds to a biotinylated primary antibody), a tertiary detection reagent (such as a class-specific tertiary antibody that binds to a secondary antibody, an anti-hapten antibody that binds to a hapten-conjugated secondary antibody, or a streptavidin that binds to a biotinylated secondary antibody), or other such arrangements. Thus, the enzyme localized to the biomarker-specific reagent bound to the sample can then be used in a plurality of protocols to deposit a detectable moiety.

[0079] In some cases, the enzyme reacts with a chromogenic compound / substrate. Specific non-limiting examples of chromogenic compounds / substrates include 4-nitrophenyl phosphate (pNPP), Fast Red, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, Fast Red, AP Orange, AP Blue, 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), o-nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium chloride (INT), tetrazolium blue, or tetrazolium violet.

[0080] In some embodiments, the enzyme can be used in a metallographic detection protocol. Metallographic detection methods include using an enzyme (such as alkaline phosphatase) in combination with a water-soluble metal ion and a redox-inactivating substrate of the enzyme. In some embodiments, the substrate is converted by the enzyme into a redox-active agent, and the redox-active agent reduces the metal ion to form a detectable precipitate. (See, for example, U.S. Patent Application No. 11 / 015,646, filed December 20, 2004, PCT Publication No. 2005 / 003777, and U.S. Patent Application Publication No. 2004 / 0265922; each of the patents is incorporated herein by reference in its entirety). Metallographic detection methods include using a redox enzyme (such as horseradish peroxidase) in combination with a water-soluble metal ion, an oxidizing agent, and a reducing agent, again for forming a detectable precipitate. (See, for example, U.S. Patent No. 6,670,113, which is incorporated herein by reference in its entirety).

[0081] In some embodiments, the enzymatic action occurs between the enzyme and the dye itself, where the reaction converts the dye from a non-conjugated class to a class that deposits on the sample. For example, the reaction of DAB with a peroxidase (such as horseradish peroxidase) oxidizes DAB and causes it to precipitate.

[0082] In other embodiments, the detectable moiety is deposited via a signaling conjugate that includes a latent reactive moiety configured to react with an enzyme to form a reactive species that can bind to the sample or to other detection components. These reactive species are capable of reacting with the sample proximal to their site of generation, i.e., near the enzyme, but are rapidly converted to non-reactive species such that the signaling conjugate does not deposit at sites distant from the enzyme deposition site. Examples of latent reactive moieties include: quinomethide (QM) analogs, such as those described in WO2015124703A1; and tyramide conjugates, such as those described in WO2012003476A2, each of which patents is hereby incorporated by reference in its entirety. In some instances, the latent reactive moiety is directly conjugated to a dye (such as N,N'-bis-carboxypentyl-5,5'-disulfonate-indodicarbocyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCO Purple), and rhodamine 110). In other instances, the latent reactive moiety is conjugated to one member of a specific binding pair, and the dye is linked to the other member of the specific binding pair. In other instances, the latent reactive moiety is linked to one member of a specific binding pair, and the enzyme is linked to the other member of the specific binding pair, where the enzyme (a) reacts with a chromogenic substrate to effect the generation of the dye, or (b) reacts with the dye to effect the deposition of the dye (such as DAB). Examples of specific binding pairs include:

[0083] (1) Biotin or a biotin derivative (such as desthiobiotin) linked to the latent reactive moiety, and a biotin-binding entity (such as avidin, streptavidin, deglycosylated avidin (such as NEUTRAVIDIN), or a biotin-binding protein having nitrated tyrosine at its biotin-binding site (such as CAPTAVIDIN)) linked to the dye or to the enzyme, where the enzyme reacts with a chromogenic substrate or with the dye (e.g., when the dye is DAB, peroxidase linked to the biotin-binding protein); and

[0084] (2) A hapten linked to the latent reactive moiety, and an anti-hapten antibody linked to the dye or to the enzyme, where the enzyme reacts with a chromogenic substrate or with the dye (e.g., when the dye is DAB, peroxidase linked to the biotin-binding protein).

[0085] Specifically includes non-limiting examples of combinations of biomarker-specific reagents and detection reagents listed in Table 4.

[0086] Table 4

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] In a particular embodiment, the biomarker-specific reagent and the specific detection reagent listed in Table 4 are antibodies. As will be recognized by one of ordinary skill in the art, the detection protocols for each of the biomarker-specific reagents may be the same or may be different.

[0098] Non-limiting examples of commercially available detection reagents or kits containing detection reagents suitable for the present method include: VENTANA ultraView detection system (secondary antibodies conjugated with enzymes, the enzymes including HRP and AP); VENTANA iVIEW detection system (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes); VENTANA OptiView detection system (OptiView) (anti-species secondary antibodies conjugated with haptens and anti-hapten tertiary antibodies conjugated with enzyme polymers); VENTANA amplification kit (unconjugated secondary antibodies, which can be used with any of the aforementioned VENTANA detection systems to amplify the amount of enzyme deposited at the primary antibody binding sites); VENTANA OptiView amplification system (anti-species secondary antibodies conjugated with haptens, anti-hapten tertiary antibodies conjugated with enzyme polymers, and tyramide conjugated with the same hapten. In use, the secondary antibody is contacted with the sample to achieve binding to the primary antibody. Then the sample is incubated with anti-hapten antibodies to achieve association of the enzyme with the secondary antibody. Then the sample is incubated with tyramide to achieve deposition of additional hapten molecules. Then the sample is incubated with anti-hapten antibodies again to achieve deposition of additional enzyme molecules. Then the sample is incubated with a detectable moiety to achieve dye deposition); VENTANA DISCOVERY, DISCOVERY OmniMap, DISCOVERY UltraMap anti-hapten antibodies, secondary antibodies, chromophores, fluorophores, and dye kits, each of the above products being available from Ventana Medical Systems, Inc. (Tucson, Arizona); PowerVision and PowerVision+ IHC detection systems (secondary antibodies directly polymerized with HRP or AP into compact polymers carrying a high ratio of enzyme to antibody); and DAKO EnVision TM + system (enzyme-labeled polymers conjugated with secondary antibodies).

[0099] If desired, the slide stained with the biomarker can be counterstained to assist in identifying morphologically relevant regions. Examples of counterstains include chromogenic nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains nuclei dark blue and polysaccharides pink to red), nuclear fast red (also known as Kernechtrot dye, stains red), and methyl green (stains green); non-nuclear chromogenic stains such as eosin (stains pink); fluorescent nuclear stains including 4′,6-diamidino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst stains (stains blue), Nuclear Green DCS1 (stains green), Nuclear Yellow (Hoechst S769121, stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), DRAQ7 (stains red); fluorescent non-nuclear stains such as fluorophore-conjugated phalloidin, (stains filamentous actin, color depends on conjugated fluorophore).

[0100] III. Staining Assessment

[0101] In one embodiment, a set of stained samples generated by the presently disclosed method is used to determine the presence or absence of a fusion protein in a patient sample. Typically, as described above, a sample is obtained from a patient and prepared for analysis. A portion of the sample is prepared (e.g., the first tissue section of a biopsy of a tumor resection sample, or the first slide prepared from a cytological sample of tumor cells such as a cytospin (such as a cervical smear), a fine needle aspirate, isolated circulating tumor cells, etc.) and stained with a biomarker-specific reagent. The stained sample is then evaluated for the percentage of cells stained and / or the percentage of cells with a staining percentage equal to or higher than a predetermined threshold level. Preferably, the sample is fixed in a formalin solution. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity ranges from 25% to 75%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 75%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity ranges from 25% to 75%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 75%.

[0102] In some cases, the stained samples are stratified based on the expression pattern and then evaluated using an evaluation method for the staining pattern. For example, in the case of observing a fixed gradient, a first evaluation method is applied to samples with a cytoplasmic expression pattern and / or a membrane expression pattern, while a second evaluation method is applied to samples with a nuclear cell localization. For example, samples with a cytoplasmic staining pattern and / or a membrane staining pattern can be evaluated by determining whether the sample meets or exceeds a threshold level of tumor cells (the tumor cells being equal to or higher than the threshold staining intensity), which is considered positive for the TRK fusion protein. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity ranges from 25% to 75%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 1.5+, and the threshold level of tumor cells with the threshold staining intensity is 75%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity ranges from 25% to 75%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 50%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 60%. In one embodiment, the threshold staining intensity is at least 2+, and the threshold level of tumor cells with the threshold staining intensity is 75%.

[0103] As another example, a sample having a nuclear staining pattern can be evaluated by determining whether the smallest contiguous tumor cell region of the sample has a tumor cell concentration that is stained at an expression level equal to or higher than a threshold. In one embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is in the range of 25% to 75% or 25% to 80%. In another embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is in the range of 25% to 75% or 25% to 80%. In another embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold contiguous tumor cell region is at least 20 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is in the range of 25% to 75% or 25% to 80%. In another embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity is any specific staining above background, and the threshold percentage of cells is at least 80%. In one embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is in the range of 25% to 75% or 25% to 80%. In another embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is at least 75%. In another embodiment, the threshold contiguous tumor cell region is at least 50 cells, the threshold staining intensity ≥ 0.5+, and the threshold percentage of cells is at least 80%.

[0104] VI. Clinical Applications

[0105] In one embodiment, the assays described herein are used to characterize tumor samples from a patient. For example, a biopsy section or an excised sample is obtained, fixed, embedded in paraffin, sectioned, and stained. As described above, the stained sections are evaluated. In some embodiments, a tumor with an evaluation indicating the presence of a fusion protein is characterized as "fusion positive", while a tumor with an evaluation not indicating the presence of a fusion protein is characterized as "fusion negative". In other embodiments, intermediate category cells between "fusion positive" and "fusion negative" are characterized as "of unknown nature". For example, where the evaluation method is based on a threshold percentage of cells with staining equal to or above a threshold level, a percentage range below the threshold percentage can be defined as "of unknown nature", and all other samples with a percentage of cells below the range of unknown nature are considered "fusion negative". As another example, where the evaluation method is based on a threshold percentage of cells with staining equal to or above a threshold level within a threshold tumor cell area, a percentage range below the threshold tumor cell area can be defined as "of unknown nature", and all other samples with a percentage of cells below the range of unknown nature are considered "fusion negative".

[0106] In some embodiments, the assay is used as a screening test to identify patients eligible for nucleic acid-based assays to confirm the presence of a fusion protein. For example, the assay can be used to screen samples for the presence or absence of a fusion protein, and only those samples characterized as fusion positive are subjected to sequencing- or PCR-based assays to confirm the presence and / or identity of the fusion detected in the assay. In other embodiments, the assay is a reflex test to confirm the presence and expression of a fusion protein identified by a nucleic acid-based assay. For example, sequencing- or PCR-based assays can be used to screen samples for the presence or absence of an NTRK gene rearrangement, and only those samples characterized as rearrangement positive by the sequencing- or PCR-based assay are screened by the assays described herein to confirm the presence and / or expression of the fusion detected by the nucleic acid assay. In other embodiments, the presence or absence of a fusion protein is characterized only based on the assay. In other embodiments, samples characterized as "fusion negative" or "of unknown nature" can be screened for the presence of an NTRK gene rearrangement by sequencing- or PCR-based assays, while cells characterized as "fusion positive" are not.

[0107] In some embodiments, the assay is used to select a therapy for a patient. For example, a patient with a tumor or sample characterized as "fusion positive" receives a targeted therapy against the wild-type counterpart, optionally in combination with a standard course of treatment for the tumor. Exemplary targeted therapies include those listed in Table 5:

[0108] Table 5

[0109]

[0110] Patients with a tumor or sample characterized as fusion-negative receive standard therapy, excluding targeted therapy against the wild-type counterpart.

[0111] VIII. Examples

[0112] To evaluate whether an assessment algorithm could be developed to predict the presence of Trk fusions in different tumor types, over 3,000 tissues were stained and analyzed by IHC in multiple indicators.

[0113] Commercially available formalin-fixed, paraffin-embedded (FFPE) normal and tumor tissues. Tissues were screened using a prototype IHC assay with the pan-TRK antibody clone EPR17341, which included heat-induced epitope retrieval, followed by antibody incubation and chromogenic development using OptiView DAB Detection (RTD, Tucson, Arizona) on a BenchMark ULTRA automated slide stainer (RTD, Tucson, Arizona).

[0114] A subset of cases with mostly specific staining by the pan-TRK (EPR17341) assay was further evaluated by in situ hybridization (ISH), which used break-apart probes for NTRK1, NTRK2, and NTRK3. The NTRK 1, 2, and 3 break-apart oligo probes each consisted of two pools of oligonucleotides (oligos) targeting genomic regions spanning the 5' and 3' ends of the NTRK1, NTRK2, and NTRK3 genes. During synthesis, the DNP and fluorescein haptens were attached to the oligo in a substantially intercalated configuration using DNP-TEG phosphoramidite (Link Technologies Ltd, Bellshill, Lanarkshire) and 6-Fluorescein phosphoramidite (Link Technologies Ltd, Bellshill, Lanarkshire). The synthesized oligos were purified using a reverse-phase chromatography column and mass spectrometry was performed to verify the removal of truncated oligos.

[0115] Criteria for defining the break status of the ISH assay were determined based on an assessment of the average break rate in normal tissues and tumors without pan-TRK IHC expression. Tumors with a break rate greater than 5 standard deviations above the mean were considered to represent true NTRK gene fusions.

[0116] Figures 2A-2BRepresentative images of pan-TRK staining in different tumor types with various genotyping are shown. When defined as tumor cell staining >0%, 9% (n = 324) of tumors exhibited specific pan-TRK IHC staining (correspondingly, ranging from 0% - 54% in gastric cancer and salivary adenocarcinoma). When considering only specimens with IHC-specific staining in ≥10% and ≥25% of tumor cells respectively, the overall percentage of cases with specific staining decreased to 5% (n = 191) and 4% (n = 133).

[0117] 164 cases showed that any staining performed by IHC was available for ISH testing. Among them, 12 cases were found to carry NTRK fusions (5 cases of CRC, 2 cases of melanoma, 2 cases of papillary thyroid cancer, 2 cases of salivary adenocarcinoma, and 1 case of pancreatic cancer). The pan-TRK positive case rates in different types of tumors at various tumor cell staining percentages (including the results of confirmation tests performed by ISH) are shown in Table 6:

[0118] Table 6

[0119]

[0120] The staining intensity and the percentage of tumor cell staining varied with different tissue types, but cases carrying fusions tended to have higher staining intensity and larger percentages of tumor cell staining (see Figure 3A &3B). Figure 3A and 3B show the distribution of ISH-confirmed samples by percentage of tumor cell staining (0 - 100%) and staining intensity (0 - 3+). Circles represent fusion-negative cases, while triangles represent fusion-positive cases. Colors represent various tumor histologies. Figure 3A shows the staining distribution of tumor types with ISH-confirmed fusion-positive cases in the dataset, and Figure 3B shows the staining distribution of neuroendocrine tumors in which no fusion-positive cases were detected.

[0121] The results of the percentage of tumor cell staining performed by IHC in all tumors in the dataset are shown in Table 7:

[0122] Table 7

[0123]

[0124] In 12 / 164 (7%) cases, where IHC specific staining was greater than 0% of tumor cells, this demonstrated the presence of an NTRK fusion detected by ISH. In 10 / 88 (11%) cases, where IHC specific staining was greater than 25% of tumor cells, this demonstrated the presence of a fusion detected by ISH (Table 6). Tumors showing a fusion detected by ISH tended to be stained by pan-TRK IHC at higher intensity and higher tumor percentage compared to non-fused tumors (upper right quadrant; Figure 3A ), except for neuroendocrine tumors ( Figure 3B ). Using a higher definition of IHC specific positive staining (>25%) would have excluded two fusion-positive melanomas from the ISH test (Table 6). These two tissue samples were not adequately stained by IHC, which demonstrated evidence of fixation gradient. Importantly, these tissues did show high-intensity nuclear staining in a high percentage (>90%) of adjacent cells in areas not affected by fixation-related artifacts.

[0125] Figure 4 Subclassification of solid tumors based on IHC and ISH tests is shown.

[0126] Figure 5 and Figure 6 show examples of the staining patterns observed. As Figure 5 shown, the staining patterns can include aspects of membrane staining (A), cytoplasmic staining (A - C), and nuclear staining (C). Figure 7 shows that some samples experienced a fixation gradient, which may affect the staining levels of some cells. It should be noted that samples with nuclear localization are more sensitive to the fixation gradient than samples with cytoplasmic / membrane localization.

[0127] In summary, pan-TRK IHC staining varied in intensity and percentage of tumor cell staining across different tumor types. Compared to tumors with wild-type TRK protein expression, ISH-based solid tumors harboring fusions showed higher intensity and IHC staining in a greater percentage of tumor cells, except for neuroendocrine and spindle cell tumors. In tumor types with low wild-type TRK protein expression rates (such as CRC and papillary thyroid cancer), pan-TRK IHC staining can be used to help identify tumors harboring fusions. In contrast, in tumors with high wild-type TRK protein expression rates (such as neuroendocrine tumors, GIST), the distribution of IHC staining percentage and intensity did not support a clear distinction between wild-type expression and fusion. According to Table 6, as the percentage of tumor cell staining using pan-TRK IHC increased, the number of specimens undergoing ISH (out of a total of 164 cases) gradually decreased; however, this decrease did not affect the identification of tumors with NTRK gene fusions by ISH in well-preserved tissue. Combining the increase in IHC staining intensity, this metric seemed to further highlight those tumors with NTRK gene fusions by ISH testing.

[0128] Two melanomas and one pancreatic ductal adenocarcinoma did not follow the above observations. The lower percentage of tumor cell staining observed may be related to fixation artifacts. Interestingly, however, at high intensity, small areas of adjacent viable tumor did show a high percentage of nuclear staining.

[0129] VI. References

[0130] The following references are hereby incorporated by reference in their entirety:

[0131] Amatu et al., NTRK gene fusions as novel targets of cancer therapy across multiple tumour types, ESMO Open, Vol. 1, No. 2, e000023 doi:10.1136 / esmoopen-2015-000023 (2016).

[0132] Bailey et al., Tropomyosin receptor kinase inhibitors: an updated patent review for 2010 - 2016–Part II, Expert Opinion on Therapetuic Patents, Vol. 27, No. 7, pp. 831–49 (Mar. 2017).

[0133] D’Amico et al., State of the art in antigen retrieval for immunohistochemistry, J Immunol Methods. Vol. 341(1-2), pp. 1-18 (Feb. 28, 2009).

[0134] Hechtman et al., Pan-Trk Immunohistochemistry Is an Efficient and Reliable Screen for the Detection of NTRK Fusions, Am. J. Surg. Path., Vol. 41, No. 11, pp. 1547–51 (Nov. 2017).

[0135] Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578–1582 (2014)

[0136] Shi et al., Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades, J Histochem Cytochem, Vol. 59, No. 1, pp. 13-32 (Jan. 2011).

[0137] Stransky et al., The landscape of kinase fusions in cancer, Nature Communications, Vol. 5, Article No. 4846 (2014) doi:10.1038 / ncomms5846.

[0138] Vinod et al., A simple and effective heat induced antigen retrieval method, MethodsX, Vol. 3, pp. 315–19 (published online Apr. 8, 2016).

[0139] Warford et al., Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: A review and practical evaluation of tyramide and rolling circle amplification systems, Methods, Vol. 70, No. 1, pp. 28–33 (Nov. 2014).

[0140] Wellcome Sanger Institute, COSMIC – the Catalogue of Somatic Mutations in Cancer (COSMIC database), available at http: / / cancer.sanger.ac.uk / cosmic / fusion (last accessed on 13 - SEP - 2018).

[0141] Yamashita et al., Mechanisms of Heat - induced Antigen Retrieval: Analyses In Vitro Employing SDS - PAGE and Immunohistochemistry, J. Histochemistry and Cytochemistry, Vol. 53, No. 1, pp. 13–21 (2005).

[0142] Barbacid M. et al. Biochim. Biophys. Acta Rev. Cancer 1991.

[0143] Barbacid M. Annals New York Academy of Sciences. 1995:442 - 458.

[0144] Lemmon MA and Schlessinger J. Cell 2010; 141:1117 - 1134.

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[0153] Rubin BP et al. Am J Pathol. 1998;153(5):1451-1458.

[0154] Tognon C et al. Cancer Cell. 2002;2:367-376.

[0155] Brzezianska E et al. Mutat Res. 2006;599(1-2):26-35.

[0156] Fernandez-Cuesta L et al. 105th Annual Meeting of the American Association for Cancer Research, 2014, San Diego, California, AACR.

[0157] Leeman-Neill RJ et al. Cancer. 2014;120(6):799-807.

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[0159] Farago et al. JCO Precision Oncology. Published online on July 23, 2018.

[0160] Gatalica Z et al. Mod Pathol. Published online on 23 August 2018. Sequence Listing <110> Ventana Medical Systems, Inc. F. 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5 10 15 Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys 20 25 30 Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro 35 40 45 Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp 50 55 60 Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu 65 70 75 80 Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu 85 90 95 Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu 100 105 110 Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr 115 120 125 Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile 130 135 140 Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys 145 150 155 160 Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys 165 170 175 Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro 180 185 190 Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val 195 200 205 Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro 210 215 220 Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met 225 230 235 240 Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser 245 250 255 Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val 260 265 270 Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr 275 280 285 Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro 290 295 300 Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn 305 310 315 320 Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val 325 330 335 Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr 340 345 350 His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly 355 360 365 Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile 370 375 380 Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr 385 390 395 400 Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu 405 410 415 Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser 420 425 430 Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu 435 440 445 Val Met Leu Phe Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met 450 455 460 Lys Asp Phe Ser Trp Phe Gly Phe Gly Lys Val Lys Ser Arg Gln Gly 465 470 475 480 Val Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro 485 490 495 Leu His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly 500 505 510 Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu 515 520 525 Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr 530 535 540 Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu 545 550 555 560 Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu 565 570 575 Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp 580 585 590 Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu 595 600 605 Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val 610 615 620 Glu Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp 625 630 635 640 Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala 645 650 655 Glu Gly Asn Pro Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile 660 665 670 Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe 675 680 685 Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu 690 695 700 Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr 705 710 715 720 Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met 725 730 735 Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val 740 745 750 Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln 755 760 765 Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln 770 775 780 Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu 785 790 795 800 Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Met Arg Lys Asn Ile 805 810 815 Lys Gly Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val 820 825 830 Tyr Leu Asp Ile Leu Gly 835 <210> 3 <211> 839 <212> PRT <213> Homo sapiens <400> 3 Met Asp Val Ser Leu Cys Pro Ala Lys Cys Ser Phe Trp Arg Ile Phe 1 5 10 15 Leu Leu Gly Ser Val Trp Leu Asp Tyr Val Gly Ser Val Leu Ala Cys 20 25 30 Pro Ala Asn Cys Val Cys Ser Lys Thr Glu Ile Asn Cys Arg Arg Pro 35 40 45 Asp Asp Gly Asn Leu Phe Pro Leu Leu Glu Gly Gln Asp Ser Gly Asn 50 55 60 Ser Asn Gly Asn Ala Ser Ile Asn Ile Thr Asp Ile Ser Arg Asn Ile 65 70 75 80 Thr Ser Ile His Ile Glu Asn Trp Arg Ser Leu His Thr Leu Asn Ala 85 90 95 Val Asp Met Glu Leu Tyr Thr Gly Leu Gln Lys Leu Thr Ile Lys Asn 100 105 110 Ser Gly Leu Arg Ser Ile Gln Pro Arg Ala Phe Ala Lys Asn Pro His 115 120 125 Leu Arg Tyr Ile Asn Leu Ser Ser Asn Arg Leu Thr Thr Leu Ser Trp 130 135 140 Gln Leu Phe Gln Thr Leu Ser Leu Arg Glu Leu Gln Leu Glu Gln Asn 145 150 155 160 Phe Phe Asn Cys Ser Cys Asp Ile Arg Trp Met Gln Leu Trp Gln Glu 165 170 175 Gln Gly Glu Ala Lys Leu Asn Ser Gln Asn Leu Tyr Cys Ile Asn Ala 180 185 190 Asp Gly Ser Gln Leu Pro Leu Phe Arg Met Asn Ile Ser Gln Cys Asp 195 200 205 Leu Pro Glu Ile Ser Val Ser His Val Asn Leu Thr Val Arg Glu Gly 210 215 220 Asp Asn Ala Val Ile Thr Cys Asn Gly Ser Gly Ser Pro Leu Pro Asp 225 230 235 240 Val Asp Trp Ile Val Thr Gly Leu Gln Ser Ile Asn Thr His Gln Thr 245 250 255 Asn Leu Asn Trp Thr Asn Val His Ala Ile Asn Leu Thr Leu Val Asn 260 265 270 Val Thr Ser Glu Asp Asn Gly Phe Thr Leu Thr Cys Ile Ala Glu Asn 275 280 285 Val Val Gly Met Ser Asn Ala Ser Val Ala Leu Thr Val Tyr Tyr Pro 290 295 300 Pro Arg Val Val Ser Leu Glu Glu Pro Glu Leu Arg Leu Glu His Cys 305 310 315 320 Ile Glu Phe Val Val Arg Gly Asn Pro Pro Pro Thr Leu His Trp Leu 325 330 335 His Asn Gly Gln Pro Leu Arg Glu Ser Lys Ile Ile His Val Glu Tyr 340 345 350 Tyr Gln Glu Gly Glu Ile Ser Glu Gly Cys Leu Leu Phe Asn Lys Pro 355 360 365 Thr His Tyr Asn Asn Gly Asn Tyr Thr Leu Ile Ala Lys Asn Pro Leu 370 375 380 Gly Thr Ala Asn Gln Thr Ile Asn Gly His Phe Leu Lys Glu Pro Phe 385 390 395 400 Pro Glu Ser Thr Asp Asn Phe Ile Leu Phe Asp Glu Val Ser Pro Thr 405 410 415 Pro Pro Ile Thr Val Thr His Lys Pro Glu Glu Asp Thr Phe Gly Val 420 425 430 Ser Ile Ala Val Gly Leu Ala Ala Phe Ala Cys Val Leu Leu Val Val 435 440 445 Leu Phe Val Met Ile Asn Lys Tyr Gly Arg Arg Ser Lys Phe Gly Met 450 455 460 Lys Gly Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro 465 470 475 480 Leu His His Ile Asn His Gly Ile Thr Thr Pro Ser Ser Leu Asp Ala 485 490 495 Gly Pro Asp Thr Val Val Ile Gly Met Thr Arg Ile Pro Val Ile Glu 500 505 510 Asn Pro Gln Tyr Phe Arg Gln Gly His Asn Cys His Lys Pro Asp Thr 515 520 525 Tyr Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu Leu 530 535 540 Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu 545 550 555 560 Ser Pro Thr Lys Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Asp 565 570 575 Pro Thr Leu Ala Ala Arg Lys Asp Phe Gln Arg Glu Ala Glu Leu Leu 580 585 590 Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Gly 595 600 605 Asp Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp 610 615 620 Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Met Ile Leu Val 625 630 635 640 Asp Gly Gln Pro Arg Gln Ala Lys Gly Glu Leu Gly Leu Ser Gln Met 645 650 655 Leu His Ile Ala Ser Gln Ile Ala Ser Gly Met Val Tyr Leu Ala Ser 660 665 670 Gln His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly 675 680 685 Ala Asn Leu Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val 690 695 700 Tyr Ser Thr Asp Tyr Tyr Arg Leu Phe Asn Pro Ser Gly Asn Asp Phe 705 710 715 720 Cys Ile Trp Cys Glu Val Gly Gly His Thr Met Leu Pro Ile Arg Trp 725 730 735 Met Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp 740 745 750 Val Trp Ser Phe Gly Val Ile Leu Trp Glu Ile Phe Thr Tyr Gly Lys 755 760 765 Gln Pro Trp Phe Gln Leu Ser Asn Thr Glu Val Ile Glu Cys Ile Thr 770 775 780 Gln Gly Arg Val Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr 785 790 795 800 Asp Val Met Leu Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Asn 805 810 815 Ile Lys Glu Ile Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr Pro 820 825 830 Ile Tyr Leu Asp Ile Leu Gly 835

Claims

1. Use of a biomarker - specific reagent for preparing a kit for use in a method for identifying TrkA, TrkB, or TrkC fusion proteins in a non - neuroendocrine tumor sample, the method comprising: Performing affinity immunohistochemical staining on the sample with a biomarker - specific reagent that specifically binds to: an amino acid sequence comprising residues 363 - 760 of SEQ ID NO:1 or consisting thereof; an amino acid sequence comprising residues 646 - 838 of SEQ ID NO:2 or consisting thereof; and an amino acid sequence comprising residues 718 - 839 of SEQ ID NO:3 or consisting thereof; Detecting the staining pattern in the sample; And If the sample has a staining pattern in which greater than or equal to a threshold percentage of tumor cells are stained above a threshold specific staining intensity, then the sample is rated positive for a fusion protein involving TrkA, TrkB, or TrkC, wherein The staining pattern is a membrane staining pattern and / or a cytoplasmic staining pattern, with greater than or equal to a first threshold percentage of tumor cells stained above a first threshold specific staining intensity, and wherein the first threshold percentage of tumor cells is in the range of 25% to 75% and the first threshold specific staining intensity is at least 1.5 or more; or The staining pattern is a nuclear staining pattern, with greater than or equal to a second threshold percentage of cells specifically stained at a second staining intensity within a threshold tumor cell region, and wherein the threshold tumor cell region is at least 20 adjacent cells, the second staining intensity is any specific staining above background, and the second threshold percentage of cells is in the range of 25% to 80%.

2. The use according to claim 1, wherein the first threshold percentage of tumor cells is in the range of 50% to 75% and the first threshold specific staining intensity is at least 1.

5.

3. The use according to claim 1, wherein the threshold tumor cell region is at least 50 adjacent cells, the second staining intensity is at least 0.5 or more, and the second threshold percentage of cells is in the range of 25% to 75%.

4. The use according to claim 1, wherein the biomarker - specific reagent is an antibody.

5. The use according to claim 4, wherein the antibody is immunospecific for an epitope arranged in the amino acid sequence consisting of amino acids 816 - 838 of SEQ ID NO:

2.

6. The use according to claim 5, wherein the antibody is monoclonal antibody clone EPR17341.

7. The use according to any one of claims 1 to 6, wherein the affinity immunohistochemical staining comprises: (a) Performing a heat - induced epitope retrieval process on the sample; And (b) Contacting the sample with the biomarker - specific reagent and a set of detection reagents to deposit a bright - field dye in the vicinity of any biomarker - specific reagent bound to the sample.

8. Use of a biomarker-specific reagent for preparing a kit for use in a method for identifying NTRK rearrangements in a non-neuroendocrine tumor sample, the method comprising detecting the presence of Trk fusion protein in the sample according to the method in any one of claims 1 to 7, and if the sample is assessed as positive for a fusion protein involving TrkA, TrkB or TrkC, screening the sample for the NTRK rearrangement situation by in situ hybridization, using cleavage oligonucleotide probes for NTRK1, NTRK2 and NTRK3, wherein each of the cleavage oligonucleotide probes targets regions spanning the 5' and 3' ends of the NTRK1, NTRK2 and NTRK3 genes, and wherein each of the cleavage oligonucleotide probes is conjugated to one or more haptens.

9. Use of a biomarker-specific reagent for preparing a kit for use in a method for identifying NTRK rearrangements in non-neuroendocrine tumor samples, said method comprising: Detecting the presence of Trk fusion protein in the sample according to the method in any one of claims 1 to 7; Identifying the sample as Trk fusion positive or Trk fusion negative; and screening the Trk fusion negative samples for the NTRK rearrangement situation by in situ hybridization, using cleavage oligonucleotide probes for NTRK1, NTRK2 and NTRK3, wherein each of the cleavage oligonucleotide probes targets regions spanning the 5' and 3' ends of the NTRK1, NTRK2 and NTRK3 genes, and wherein each of the cleavage oligonucleotide probes is conjugated to one or more haptens.

10. Use of a biomarker-specific reagent for preparing a kit for use in a method of identifying NTRK rearrangements in non-neuroendocrine tumor samples, said method comprising: Detecting the presence of Trk fusion protein in the sample according to the method in any one of claims 1 to 7; Identifying the sample as Trk fusion positive, of undetermined Trk fusion nature or Trk fusion negative; and screening the samples of undetermined Trk fusion nature for the NTRK rearrangement situation by in situ hybridization, using cleavage oligonucleotide probes for NTRK1, NTRK2 and NTRK3, wherein each of the cleavage oligonucleotide probes targets regions spanning the 5' and 3' ends of the NTRK1, NTRK2 and NTRK3 genes, and wherein each of the cleavage oligonucleotide probes is conjugated to one or more haptens.

11. Use of a biomarker-specific reagent for preparing a kit for use in a method for selecting a patient to receive Trk-directed therapy, the method comprising detecting the presence of Trk fusion protein in a non-neuroendocrine tumor sample according to the method in any one of claims 1 to 8, and if the sample is assessed as positive for a fusion protein involving TrkA, TrkB or TrkC or the NTRK rearrangement is detected, selecting the patient to receive the therapy.

12. Use of a biomarker-specific reagent for preparing a kit for use in a method of selecting a patient for receiving a Trk-directed therapy, said method comprising detecting the presence or NTRK rearrangement of a Trk fusion protein in a non-neuroendocrine tumor sample according to the method in any one of uses according to claims 9 to 10, and selecting said patient for receiving said therapy if said sample is evaluated as positive for a fusion protein involving TrkA, TrkB or TrkC or the NTRK rearrangement is detected.

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