WO2021041765A2 - Kit and methods to detect ntrk gene fusion - Google Patents

Abstract

Provided is a kit for detecting neurotrophin receptor tyrosine kinase (NTRK) gene fusion. The kit includes a set of NTRK fusion-specific primer pairs and a set of NTRK fusion-specific probes. Also provided is a method for detecting NTRK gene fusion, including generating an amplified target cDNA to hybridize with the set of NTRK fusion-specific probes in a single reaction, and detecting the probe-bound product to identify all possible NTRK gene fusions in a biological sample.

Description

KIT AND METHODS TO DETECT NTRK GENE FUSION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of Provisional Application No. 62/893,148, filed on August 28, 2019, the content of which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[0002] The invention relates to a kit and a method for molecular diagnostics and genomics. Particularly, the invention relates to a kit and a method for molecular diagnostics and genomics of cancers.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created August 28, 2020, is named “ACTG-3PCT_SEQList_ST25.txt” and is 55,704 bytes in size.

BACKGROUND OF THE INVENTION

[0004] Neurotrophin receptor tyrosine kinase (NTRK) gene family includes NTRKl, NTRK2, and NTRK3 genes, which codes for three tropomyosin receptor kinases named TrkA, TrkB, and TrkC, respectively. When Trk receptors are activated, they trigger signaling pathways such as PI3K/AKT and MAPK pathways, and regulates cell survival, proliferation, migration, differentiation, and synapse formation and plasticity. Trk receptors are expressed in a widespread of tissues, including neuronal tissue, esophagus, stomach, breast, uterine cervix, thyroid, lung, pancreas, colon, ovary, and skin, to name a few. Genomic alterations, such as gene fusions, involving NTRK can lead to various adult and pediatric tumors. One prevalent type of gene fusion involving NTRK genes is the fusion of the kinase domain of the NTRK gene with a highly expressed 5’ partner, which leads to abnormally high expression and activation of the Trk kinase domain. These alterations may involve the fusion of part of the NTRK gene with many possible fusion partners, leading to a demand to detect many fusion types.

[0005] NTRK gene fusions can be targeted for cancer therapy through TRK inhibitors, such as larotrectinib and entrectinib. These drugs act by inhibiting the kinase activity of the Trk receptors, thereby reducing the downstream signal for cell proliferation. Although TRK inhibitors are efficacious for patients with cancers involving NTRK gene fusions, only a small proportion of cancer patients harbor such genetic aberrations. Therefore, it is essential to detect NTRK gene fusion and identify patients who may benefit from the TRK inhibitor therapy before treatment.

[0006] Several technologies may be adopted for detection of NTRK gene fusion, including next- generation sequencing (NGS), immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). While NGS provides comprehensive information with details, it is not only costly and time consuming, but also requires more samples, thus limiting its clinical application. IHC detects the presence of NTRK proteins, but it is challenging to distinguish the proteins coming from NTRK fusion genes and wild-type NTRK genes. FISH can detect gene fusions, but it requires separate reactions for detecting each fusion type and also requires highly trained specialists to analyze the results. RT-PCR also requires different probes and separate reactions for detecting each fusion type, resulting in the need for more samples as the number of fusion types to be detected increases. Because over a hundred fusion types involving NTRK genes have been discovered, there is a demand for novel detection methods that can detect many fusion types in one reaction.

SUMMARY OF THE INVENTION

[0007] The present disclosure concerns a method for detecting NTRK gene fusion. The method includes the steps of:

(a) obtaining ribonucleic acids (RNA) from a biological sample;

(b) performing reverse transcription of the RNA to obtain complementary deoxyribonucleic acids (cDNA);

(c) amplifying the cDNA with at least two NTRK fusion-specific primer pairs to obtain an amplified product;

(d) mixing the amplified product with at least two probes to obtain a probe-bound product, wherein each of the probes has a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-125 and any complementary sequence thereof; and

(e) detecting the probe-bound product to determine the presence of NTRK gene fusion. [0008] The disclosed method utilizes a set of specifically designed probes, each of which can capture the amplified product including one particular NTRK fusion sequence, to detect all possible NTRK gene fusions. Since the NTRK gene fusion types are numerous, but the exact one or more NTRK fusions in the biological sample are unknown before the detection step, it is important to obtain detectable amounts of the amplified products for all types of NTRK fusions so that any NTRK fusion type can be detected subsequently. Considering that different NTRK fusion-specific primer pairs show different amplification efficiencies, an additional round of DNA amplification may be performed in the aforementioned step (c) using a pair of universal primers so as to ensure sufficient production of the amplified product of any NTRK fusion type. Accordingly, in some preferred embodiments, in step (c) the cDNA is amplified first with the at least two NTRK fusion-specific primer pairs and subsequently with a universal primer pair to obtain the amplified product. In this setting, a NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and a NTRK fusion-specific reverse primer in each of the NTRK fusion- specific primer pairs further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair.

[0009] In another aspect, a kit is also provided for detecting NTRK gene fusion according to the aforementioned method. The kit includes: at least two NTRK fusion-specific primer pairs; and at least two probes each having a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-125 and any complementary sequence thereof.

[0010] In some preferred embodiments, the kit further includes a universal primer pair for an additional round of DNA amplification. In this setting, a NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and a NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair. [0011] In some embodiments, the kit further includes a reverse transcriptase for reverse transcription of the RNA isolated from the biological sample, and also includes a DNA polymerase for amplification of the cDNA generated by the reverse transcription.

[0012] The set of probes used in the method and the kit can target distinct NTRK fusions with high specificities and also show similar hybridization efficiencies, ensuring accurate detection of all possible NTRK gene fusions in one single reaction. Thus, application of the method and the kit in NTRK fusion detection can reduce the need for large sample volume and saves detection time due to no requirement for separate detections of different NTRK fusion types. In addition, the method and the kit are compatible with the platforms designed for multiplex reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiments, with reference to the attached drawings, in which:

[0014] FIG. 1 is a schematic illustration of the method for detecting NTRK gene fusion according to one embodiment of the invention; the detection is based on a single-amplified target- probe-barcoded magnetic bead (BMB) hybridization assay;

[0015] FIG. 2 is a schematic illustration of the method for detecting NTRK gene fusion according to one embodiment of the invention; the detection is based on a double-amplified target- probe hybridization assay; and

[0016] FIG. 3 shows an array of probe spots printed in a well of a plate; the numbers in the first line from the top and the first column from the left are presented as reference coordinates; the squares labeled 1-117 represent the spots of NTRK fusion-specific probes, the squares labeled IC001-IC009 represent the spots of control probes, and the squares labeled R144 represent the spots of an anchor probe.

DETAIFED DESCRIPTION

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Definition

[0018] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0019] The term “gene fusion” refers to a phenomenon where a first gene on a chromosome is fused to a second gene on the same or a different chromosome and a hybrid gene or a fusion gene thus forms. This phenomenon is also commonly referred to as “gene translocation” or “gene rearrangement.” When an NTRK gene is one of the multiple fused genes, such gene fusion is called “NTRK gene fusion” or “NTRK fusion”. Typically, two “partner genes” are fused together. The gene at the 5’ end of the fusion gene is referred to as “5’ partner” or “5’ gene”, and the gene at the 3’ end of the fusion gene is referred to as “3’ partner” or “3’ gene.” The fusion gene has a “fusion junction,” which is the site where the 5’ partner gene fuses to the 3’ partner gene. Fusion junction is located in a fusion region defined by a fusion sequence (also called a fusion junction sequence), which encompasses the sequence from the 5’ gene and the sequence from the 3’ gene. Different combinations of partner genes lead to different “fusion types.” The fusion between two specific genes is further diversified by fusion junction since fusion junction may occur anywhere within the partner genes. For example, the fusion between the first exon of a first gene and the second exon of a second gene is one fusion type, whereas the fusion between the third exon of the first gene and the first exon of the second gene is another fusion type.

[0020] Gene fusions may be detected by identifying a fusion junction in a DNA or in an RNA transcript of that DNA. As used herein, a “fusion type” refers to a unique fusion present in an RNA transcript. In other words, it is considered the same fusion type when the fusions between two specific genes occur at different sites within the same intronic region. For example, a fusion between exon 3 of gene A and exon 5 of gene B may have a DNA fusion region containing a small portion of the intron between exons 3 and 4 of gene A and a large portion of the intron between exons 4 and 5 of gene B. Alternatively, such fusion may have a DNA fusion region containing a large portion of the intron between exons 3 and 4 of gene A and a small portion of the intron between exons 4 and 5 of gene B. These two fusions, though having different DNA fusion junctions, are considered the same “fusion type” because the RNA transcripts generated from the two fusions are the same. [0021] The term “primer” refers to a synthetic single-stranded oligonucleotide that can be used to amplify a target nucleic acid having a specific length. As used herein, the terms “NTRK fusion-specific primer” and “fusion specific primer” are used interchangeably, which refer to a DNA primer that is designed to amplify a target cDNA including a fusion junction originates from a particular NTRK fusion gene. The NTRK fusion-specific primers are used in pairs, including a NTRK fusion-specific forward primer capable of specifically binding to the 5 ’-end of a target cDNA, and a NTRK fusion-specific reverse primer capable of specifically binding to the 3 ’-end of said target cDNA.

[0022] As used herein, the term “universal primer” refers to a DNA primer that is designed to amplify any DNA including the nucleotide sequence of the universal primer. The universal primers are used in pairs, including a universal forward primer and a universal reverse primer.

[0023] Unless defined otherwise, the term “probe” or “NTRK fusion-specific probe” refers to a synthetic single-stranded DNA oligonucleotide that can hybridize to a fusion region originates from a particular NTRK fusion gene.

[0024] As used herein, a “connector” or a “linker” refers to part of a molecule or part of a complex of molecules that connects one molecule to another. The connector or linker can act by covalent bonding, nucleic acid hybridization, or non-covalent interaction between a pair of molecules such as biotin-streptavidin interaction. In this disclosure, a “connector” is used to conjugate a primer with a detectable molecule such as a fluorescent molecule; and a “linker” is used to form linkage between a probe and a detectable molecule or a unique identifier such as a barcoded magnetic bead (BMB).

[0025] In the present disclosure, a method for detecting NTRK gene fusion is provided. The method includes the steps of:

(a) obtaining RNA from a biological sample;

(b) performing reverse transcription of the RNA to obtain cDNA;

(c) amplifying the cDNA with at least two NTRK fusion-specific primer pairs to obtain an amplified product;

(d) mixing the amplified product with at least two probes to obtain a probe-bound product, wherein each of the probes has a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-125 and any complementary sequence thereof; and (e) detecting the probe-bound product to determine the presence of NTRK gene fusion.

[0026] In step (a) of the disclosed method, RNA is prepared from a biological sample. The biological sample may be any sample obtained from an animal and a human subject. Examples of the biological samples include a formalin-fixed paraffin-embedded (FFPE) tissue section, blood, plasma, or cells. In some embodiments, the biological sample originates from a cancer patient. In some embodiments, the biological sample originates from a solid tumor, soft tissue sarcoma, or a hematological cancer. In some embodiments, the biological sample originates from a patient with thyroid cancer, colorectal cancer, melanoma, soft tissue sarcoma, lung cancer, gastrointestinal stromal tumor, appendix cancer, breast cancer, salivary gland cancer, cholangiocarcinoma, pancreas cancer, or infantile fibrosarcoma.

[0027] Preparation of total RNA from the biological sample can be carried out by various methods known in the art. One typical procedure is RNA extraction with organic solvents such as phenol/chloroform and precipitation by centrifugation. There are also commercially available kits for RNA isolation or purification. Once the RNA is obtained, a reverse transcriptase is used along with four kinds of deoxyribonucleoside triphosphates (dNTP, including dATP, dCTP, dTTP, and dGTP) to generate cDNA from the template RNA, a process called reverse transcription. The reverse transcription may be conducted using Superscript cDNA synthesis kit (Cat No: 11754050, Invitrogen).

[0028] In step (c) of the disclosed method, the cDNA is amplified with a DNA polymerase and at least two pairs of NTRK fusion-specific primers to obtain an amplified product for probe detection. The amplification may be conducted using a multiplex PCR kit (Cat No: 206143, Qiagen) which includes a DNA polymerase. The NTRK fusion-specific primers may be provided as a regent before use. In some embodiments, all the NTRK fusion-specific primer pairs are pooled together to form a single pooled reagent. In other embodiments, the NTRK fusion-specific primer pairs are partially pooled to form a plurality of pooled reagents, each of which contains at least one NTRK fusion-specific primer pairs. Thus, the number of the pooled reagent may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some preferred embodiments, the over a hundred NTRK fusion-specific primer pairs are provided in four pooled reagents to be used in four multiplex amplification reactions. DNA amplification in this manner has been demonstrated to show significantly higher performance than a single multiplex amplification reaction carried out with all the NTRK fusion-specific primer pairs, probably due to decreased primer complexity.

[0029] In some preferred embodiments, in step (c) the cDNA is amplified first with the at least two NTRK fusion-specific primer pairs and subsequently with a universal primer pair to obtain the amplified product. When the universal primer pair is utilized in the disclosed method, the NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal forward primer in the pair of universal primers, and the NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal reverse primer in the universal primer pair. The use of the universal primer pair can increase the ultimate yield of any possible amplified product, no matter which NTRK fusion type is to be detected or which NTRK fusion-specific primer pair is used in the first round of amplification. An additional advantage of applying the universal primer pair is that when primers are to be modified to become detectable, only two or even one universal primer needs to be modified, for example, forming a linkage between one universal primer and a connector (such as biotin) so that a detectable molecule can be linked to the universal primer. Otherwise, all NTRK fusion-specific primer pairs have to be modified, which makes the primer modification process more complicated and costly.

[0030] In step (d) of the disclosed method, the amplified product is mixed with at least two probes so that a probe-bound product can form through nucleic acid hybridization. Because the probes are specifically designed based on the fusion sequence of each type of NTRK fusion, the exact NTRK gene fusion type can be determined by detecting a particular probe-bound product. The NTRK gene fusion that can be detected includes, but not limited to, a fusion between an NTRK gene and a partner gene selected from trafficking from TFG (encoding endoplasmic reticulum (ER) to golgi regulator), ETV6 (encoding ETS variant transcription factor 6), QKI (encoding K-homology (KH) domain containing RNA binding protein), AFAPl (encoding actin filament associated protein 1), BCAN (encoding brevican core protein), CD74 (encoding cluster of differentiation 74), CEL (encoding carboxyl ester lipase), CHTOP (encoding chromatin target of PRMT1 protein), EPHB2 (encoding ephrin type B receptor 2), EPS 15 (encoding epidermal growth factor receptor pathway substrate 15), IRF2BP2 (encoding interferon regulatory factor 2 binding protein 2), LMNA (encoding lamin A/C prtein), MIR548F1 (encoding microRNA 548f-l), MPRIP (encoding myosin phosphatase Rho interacting protein), MRPL24 (encoding mitochondrial ribosomal protein L24), NFASC (encoding neurofascin), PEAR1 (encoding platelet endothelial aggregation receptor 1), PLEKHA6 (encoding pleckstrin homology domain containing A6), PPL (encoding periplakin), RNF213 (encoding ring finger protein 213), SQSTM1 (encoding sequestosome 1), SSBP2 (encoding single stranded DNA binding protein 2), TP53 (encoding tumor protein p53), TPM3 (encoding tropomyosin 3), TPM4 (encoding tropomyosin 4), TPR (encoding translocated promoter region, nuclear basket protein), BCR (encoding BCR activator of RhoGEF and GTPase), NACC2 (encoding NACC family member 2), PAN3 (encoding poly(A) specific ribonuclease subunit PAN3), STRN (encoding striatin), STRN3 (encoding striatin 3), TRIM24 (encoding tripartite motif containing 24), TRIM63 (encoding tripartite motif containing 63), VCL (encoding vinculin), AKAP13 (encoding A-kinase anchoring protein 13), EML4 (encoding EMAP like 4), FAT1 (encoding FAT atypical cadherin 1), LYN (encoding a Src family tyrosine kinase), MY05A (encoding myosin VA), RBPMS (encoding RNA binding protein, mRNA processing factor), VPS 18 (encoding VPS 18 core subunit of CORVET and HOPS complexes), DYNC2H1 (encoding dynein cytoplasmic 2 heavy chain 1), LAP3 (encoding leucine aminopeptidase 3), PDE4DIP (encoding phosphodiesterase 4D interacting protein), GON4L (encoding gon-4 like protein) and CTRC (encoding chymotrypsin C). In one preferred embodiment, the detectable NTRK gene fusion types include TFG-NTRK1, ETV6-NTRK3, QKI-NTRK2, TPM3-NTRK1, ETV6-NTRK2, TFG-NTRK3 and NACC2-NTRK2. In another preferred embodiment, the detectable NTRK gene fusion types include PDE4DIP-NTRK1, TRIM63 -NTRK 1 , GON4L-NTRK1 and CTRC-NTRK1.

[0031] Table 1 lists the specifically designed probes for detection of the indicated NTRK gene fusions. Each of these probes has been demonstrated to be specific to the indicated gene fusion type and does not cross-reacts with other fusion types, allowing accurate determination of the particular NTRK gene fusion type. In some embodiments, for detection of one particular NTRK gene fusion, both the probe having any of the sequence of SEQ ID NOs: 1-125 and the probe having a complementary sequence are used to enhance detection efficiency. For example, for detection of the NTRK fusion 001 in Table 1, the probes with the sequences of SEQ ID NO: 1 and SEQ ID NO: 126 may be used together. TABLE 1

[0032] In some embodiments, one NTRK fusion type is detected after one target cDNA is amplified and probed. In other embodiments, multiple NTRK fusion types can be detected simultaneously after two or more target cDNAs with difference sequences are amplified in one reaction (called a multiplex amplification reaction) and/or probed in one reaction (called a multiplex hybridization reaction). When the disclosed method is performed in the multiplex setting, at least two probes for detecting at least two NTRK gene fusion types are applied. In some embodiments, the at least two probes are selected from the group consisting of SEQ ID NOs: 1-125 and any complementary sequence thereof. In some preferred embodiments, the at least two probes are selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 60, 62, 68, 70, 72, 74, 76, 78, 87, 88, 102, 104, 113, 114, 115 and any complementary sequence thereof. More preferably, the at least two probes are selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof. The probes may be provided as a single pooled reagent or as separate reagents.

[0033] Typically, the probe and the amplified product are mixed at a specific temperature to facilitate probe hybridization. The optimal thermo mixing condition for probe hybridization varies depending on probe sequences. Thus, for a multiplex reaction where at least two probes are applied, it is difficult to select a suitable hybridization condition for all the probes. However, by using the probes listed in TABLE 1, a multiplex reaction may be performed at a fixed temperature with agitation at a fixed speed, because these probes are designed to be capable of hybridizing to the respective target at similar hybridization conditions. In some embodiments, the temperature for hybridization is between 35-50°C, 40-50°C, 40-45°C, or 45-50°C. In some embodiments, the hybridization is performed by using a thermomixer at a rotation speed between 700-1000 rpm, 750-1000 rpm, 800-1000 rpm, 900-1000 rpm, 700-750 rpm, 700-800 rpm, 750-800 rpm, or 800-900 rpm.

[0034] Detection of the probe-bound product may be accomplished by detecting the NTRK fusion- specific primers, the universal primers, or the probe in said product. Thus, the primers or probes are usually modified to be detectable. They may be modified to have fluorescence or chemiluminescence activity or become chromogenic or colorimetric by being connected directly or indirectly to a detectable molecule. In some embodiments, one or both primers in the primer pair are connected to biotin or other compounds capable of binding to a streptavi din-conjugated detectable molecule. The detectable molecule may be a dye, a fluorescent molecule such as phycoerythrin (PE) or cyanines, or an enzyme for a chromogenic reaction such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). The enzyme used in a chromogenic reaction catalyzes the production of colored compounds in the presence of a chromogenic substrate.

[0035] In some embodiments, the probe for detecting one particular NTRK fusion type is connected to a unique identifier such that multiple NTRK fusion types can be detected simultaneously and distinguished from one another. The unique identifier may be an oligonucleotide with a unique sequence, or a microbead or a nanoparticle that includes a unique barcode on the surface. The barcode may be a geometric pattern that can be read by an optical scanner with a brightfield imaging system. In some embodiments, the microbead or nanoparticle is a magnetic particle. In some embodiments, the microbead or nanoparticle is made of synthetic polymers.

[0036] The unique identifier may be connected to the probe directly or through a linker. In some embodiments, the unique identifier is connected to the probe by direct chemical coupling and a covalent bond is formed therebetween. In some embodiments, the unique identifier is connected to the probe through a polymer linker. In some embodiments, the unique identifier is connected to the probe by hybridization between complementary nucleotide sequences.

[0037] The disclosed method can be performed on several technology platforms capable of running multiplex reactions, such as a microarray plate, a gene chip, microbeads, nanoparticles, a membrane, or a microfluidic device. In some embodiments, the probes are immobilized on a microarray plate, a gene chip, or a membrane at different positions, for example, in the form of an array of spots, each containing multiple copies of one type of probe. In other embodiments, the probes are coupled with microbeads (such as micro magnetic beads). In still other embodiments, the probes are coated on a substrate plate of a microfluidic device, in which different probes are placed in different regions of the substrate plate.

[0038] When the probes are immobilized on a DNA microarray plate, the microarray plate may further include a set of control spots, each containing multiple copies of a control probe. The control probe binds the cDNA of housekeeping genes such as beta-actin, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), and beta 2-microglobulin. Thus, the control spots can be used as an internal control to validate assay performance. In addition, the microarray plate may further include a set of anchor spots, each containing multiple copies of an anchor probe. The anchor probe is designed to be detected irrespective of the amplified products. Thus, the anchor spots can be used as a position indicator for nearby spots on the microarray plate.

[0039] In the present disclosure, a kit is also provided for detecting NTRK gene fusion according to the disclosed method. The kit includes: at least two NTRK fusion-specific primer pairs; and at least two probes each having a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-125 and any complementary sequence thereof.

[0040] In some preferred embodiments, the kit further includes a universal primer pair. When the NTRK fusion-specific primer pairs are used in combination with the universal primer pair, the NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal forward primer in the universal primer pair, and the NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal reverse primer in the universal primer pair. In addition, the at least two probes each has a different nucleotide sequence preferably selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof.

[0041] In some embodiments, the kit further includes a reverse transcriptase for reverse transcription of the RNA isolated from the biological sample, and also includes a DNA polymerase for amplification of the cDNA generated by the reverse transcription.

[0042] In some embodiments, the kit further includes an internal control. The internal control may be a positive control sample where a NTRK gene fusion is present or may be a negative control sample having no NTRK gene fusion. In some embodiments, the internal control is a FFPE tissue section, blood, plasma, cells, nucleic acids, or oligonucleotides.

[0043] In some embodiments, when one or more NTRK gene fusions are detected in a biological sample from a cancer patient, the patient is expected to respond to a TRK inhibitor, particularly a NTRK inhibitor such as larotrectinib, entrectinib, FOXO-195 or TPX-0005. Examples

Example 1 Detection of NTRK gene fusion by a single-amplified target-probe-BMB hybridization assay

[0044] Single-amplified target-probe-barcoded magnetic bead (BMB) hybridization assay can simultaneously detect multiple possible NTRK fusions in a single reaction. FIG. 1 shows the overall process of this assay, which includes the steps of obtaining RNA from a biological sample, reverse transcription of the RNA to obtain cDNA, PCR amplification of NTRK fusion regions of the cDNA (i.e., the target cDNA) using multiple NTRK fusion- specific primer pairs to obtain an amplified product of the target cDNA, probe hybridization with the amplified target cDNA using BMB-coupled probes and detection of the probe-bound product. Below is an example of this assay.

Prenaration of NTRK-fusion snecific nrobes

[0045] Prior to the assay, five NTRK fusion-specific probes were designed based on the nucleotide sequence of the fusion region in the RNA transcript of each NTRK fusion gene (Table 2). The first 20 and the last 20 base pairs of all the sequences in Table 2 are from 5’ partner genes and 3’ partner genes, respectively. For hybridization at the site underlined in Table 2, each probe was designed to have the sequence listed in Table 3. The probes were synthesized and modified with an amine group at the 5’ end by IDT (Integrated DNA Technologies, Inc, Coralville, IA). Subsequently, the five fusion-specific probes were coupled with five kinds of BMBs having different identification numbers via amine- carboxyl bonding so that different probes were distinguishable from one another when used in a multiplex reaction.

TABLE 2

TABLE 3

PCR amplification using NTRK-fusion specific primer pairs

[0046] To substitute clinical samples harboring NTRK gene fusions, five oligonucleotides having the sequences of SEQ ID NOs:251-255 (termed NTRK fusion oligos) were synthesized by IDT to be used as positive control templates. The five NTRK fusion oligos were amplified by multiplex PCR with five NTRK fusion-specific primer pairs shown in Table 4. Each of these primer pairs, capable of binding to the 5 ’-end and the 3 ’-end of the respective NTRK fusion oligo, was synthesized by IDT. The reverse primer in each of the primer pair was modified at the 5 ’-end with biotin for subsequent interaction with a streptavidin- phycoerythrin (SA-PE) conjugate (Thermo Fisher Scientific). The PCR was performed on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 30 thermal cycles using Platinum Taq DNA polymerase High Fidelity (Thermo Fisher Scientific) according to the manufacturer’s instructions.

TABFE 4

Probe hybridization and signal detection

[0047] The amplified product of each NTRK fusion oligo was mixed with the five kinds of probe- BMBs in the same wells of a 96-well plate for hybridization. Hybridization was performed at 40-45°C for 10-30 minutes with agitation at about 700 rpm. After the hybridization, a fluorescent SA-PE conjugate was added to the wells to bind the biotin of the amplified product, and the probe-BMBs were washed to remove unbound substances. An additional BMB (with an ID number of zero) bearing no probe was also added to the wells as a negative control. Finally, BioCode 2500 analyzer (Applied BioCode Inc., Taipei, Taiwan), equipped with a camera capable of both brightfield and fluorescence imaging, was used to read the barcodes of the BMBs and to detect the fluorescence signals of the BMBs.

[0048] Table 5 shows the fluorescence intensity of BMB59, BMB65, BMB40, BMB52, BMB75 and BMB0. According to Table 5, the fluorescence intensity of one particular BMB, indicating one particular target-probe hybridization (i.e., the presence of one particular NTRK fusion type), was significantly higher than that of the non-specific signals. The results show that the single-amplified target probe-BMB assay can be used to detect and distinguish multiple NTRK gene fusion types.

TABLE 5

Example 2 Detection of NTRK gene fusion by a double-amplified target-probe hybridization assay

[0049] Double-amplified target-probe hybridization assay is another method designed for simultaneous detection of multiple possible NTRK fusions in a single reaction. FIG. 2 shows the overall process of this assay, which includes the steps of obtaining RNA from a biological sample, reverse transcription of the RNA to obtain cDNA, PCR amplification of NTRK fusion regions of the cDNA (i.e., the target cDNA) using multiple NTRK fusion- specific primer pairs to obtain a first amplified product of the target cDNA, PCR amplification of the first amplified product using a universal primer pair to obtain a second amplified product of the target cDNA, probe hybridization with the amplified target cDNA and detection of the probe-bound product. Below is an example of this assay.

RNA extraction and reverse transcription

[0050] Both DNA and RNA were extracted from a FFPE tissue specimen from a cancerous patient by using RecoverAll total nucleic acid isolation kit (Cat No: AM1975, Ambient Technologies) according to the manufacturer’s instructions. Reverse transcription of 100 ng total RNA was carried out at 42°C for 30 to 60 minutes by using Superscript cDNA synthesis kit (Cat No: 11754050, Invitrogen) and random hexanucleotide primers. This step yielded cDNA product in 10 pL.

PCR amplification using NTRK fusion-specific primer pairs [0051 ] Each primer in the NTRK fusion-specific primer pair used in this assay is designed to have two segments. One segment, called a fusion specific segment, is used to bind the 5 ’-end or the 3 ’-end of the fusion sequence of one particular NTRK fusion. The other segment, called a universal segment, encompasses the nucleotide sequence of the universal primer to be used in the second round of PCR. The universal segment is always upstream, or at the 5’ position, relative to the fusion specific segment (FIG. 2). The universal primer may be any of the primers listed in Table 6, where each universal primer can be used as either the universal forward primer or the universal reverse primer. Table 7 shows the fusion specific segments of some fusion specific primer pairs used in this assay.

TABLE 6

TABLE 7

[0052] For fusion specific PCR, 7 pL water was added to 10 pL of the cDNA product, and the resulting mixture (17 pL) was subsequently divided into four equal pools of 4 pL mixture each, leaving 1 pL as dead volume. The number of pools was decided based on primer performance. More specifically, the primer efficiency of each fusion specific primer pair was determined first, and the fusion specific primer pairs with similar efficiencies were mixed to form a single primer pool, resulting in a total of four primer pools (denoted as PI, P2, P3, and P4). Each primer pool, containing 23 to 48 fusion specific primers (Table 8), was added into one pool of cDNA (4 pL). The cDNA in each pool was then amplified on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 25 thermal cycles using multiplex PCR kit (Cat No: 206143, Qiagen) according to the manufacturer’s instructions, yielding a first amplified product in 10 pL. In other words, four multiplex PCR reactions were performed to yield four pools of the first amplified products.

TABLE 8

PCR amplification using a universal primer pair

[0053] Since each fusion specific primer included the nucleotide sequence of a universal primer at the 5’ end, the first amplified products were able to be further amplified by PCR using a universal primer pair, including a universal forward primer with the sequence selected from SEQ ID NOs:266-275 and a universal reverse primer with the sequence selected from SEQ ID NOs: 266-275. The universal reverse primer was biotinylated. For the second round of PCR, each of the four pools of the first amplified products was diluted 100 folds in the final reaction mix and amplified on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 25 thermal cycles using Platinum SuperFi II PCR Master Mix (Cat No: 12368010, Invitrogen) according to the manufacturer’s instructions, yielding a second amplified product in 10 pL. In other words, four PCR reactions were performed to yield four pools of the second amplified products.

Probe hybridization and signal detection

[0054] The four pools of the second amplified products (a total of 40 pL) were combined, and 18 pL of the resulting pool was mixed with 3 pL water to yield a mixture. The mixture was placed in a 96-well PCR plate (Cat No: R46-4Ή- 1000/C, 4titude). The second amplified products were denatured at 96°C for 5 minutes and transferred to pre-blocked wells, each of which was previously printed with an array of probe spots, including 117 spots of NTRK fusion-specific probes, 9 spots of control probes, and 10 spots of an anchor probe. The NTRK fusion-specific probes included the probes with the sequences of SEQ ID NOs:2, 4, 6, 8, 10, 60, 62, 68, 70, 72, 74, 76, 78, 87, 88, 102, 104, 113, 114, 115. FIG. 3 shows the distribution of different probes in one well. Target-probe hybridization was performed at 50°C for 15 minutes with vibration. After the hybridization, the wells were cooled and washed twice. A buffer containing a streptavi din-alkaline phosphatase conjugate was subsequently added to the wells to allow biotin-streptavidin interaction, and a substrate for the alkaline phosphatase was then added so that color products formed at the position where a probe-target hybrid was present. By photographing the wells with a camera and identifying the position of color spots in the wells, the particular hybridization indicating the presence of a particular NTRK fusion was determined. The position of color spots can be analyzed by a computer.

Claims

WHAT IS CLAIMED IS:

1. A kit for detecting neurotrophin receptor tyrosine kinase (NTRK) gene fusion, comprising: at least two NTRK fusion-specific primer pairs; and at least two probes each having a different nucleotide sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof.

2. The kit of claim 1, further comprising a universal primer pair, wherein a NTRK fusion- specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and a NTRK fusion-specific reverse primer in each of the NTRK fusion- specific primer pairs further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair.

3. The kit of claim 1 , further comprising an additional probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11-61, 63-69, 71-86, 88-103, 105-112, 114-125 and any complementary sequence thereof.

4. The kit of claim 1 , wherein a NTRK fusion-specific forward primer or a NTRK fusion- specific reverse primer in each of the NTRK fusion-specific primer pairs is connected to a detectable molecule.

5. The kit of claim 4, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

6. The kit of claim 2, wherein the universal forward primer or the universal reverse primer is connected to a detectable molecule.

7. The kit of claim 6, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

8. The kit of claim 1, wherein the at least two probes are immobilized on a microarray plate at different positions.

9. The kit of claim 8, wherein the microarray plate comprises a position indicator.

10. The kit of claim 1, further comprising a control probe for detecting a housekeeping gene.

11. The kit of claim 1 , further comprising a reverse transcriptase and a DNA polymerase.

12. A method for detecting neurotrophin receptor tyrosine kinase (NTRK) gene fusion, comprising the steps of:

(a) obtaining ribonucleic acids (RNA) from a biological sample;

(b) performing reverse transcription of the RNA to obtain complementary deoxyribonucleic acids (cDNA);

(c) amplifying the cDNA with at least two NTRK fusion-specific primer pairs to obtain an amplified product;

(d) mixing the amplified product with at least two probes to obtain a probe-bound product, wherein each of the probes has a different nucleotide sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof; and

(e) detecting the probe-bound product to determine the presence of NTRK gene fusion.

13. The method of claim 12, wherein in step (c) the cDNA is amplified first with the at least two NTRK fusion-specific primer pairs and subsequently with a universal primer pair to obtain the amplified product, wherein a NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and a NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair.

14. The method of claim 12, wherein in step (d) the amplified product is mixed further with an additional probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11-61, 63-69, 71-86, 88-103, 105-112, 114-125 and any complementary sequence thereof.

15. The method of claim 12, wherein a NTRK fusion-specific forward primer or a NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs is connected to a detectable molecule.

16. The method of claim 15, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

17. The method of claim 13, wherein the universal forward primer or the universal reverse primer is connected to a detectable molecule.

18. The method of claim 17, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

19. The method of claim 12, wherein in step (d) the at least two probes and the amplified product are mixed at a temperature between 35-50°C.

20. The method of claim 12, wherein in step (d) the at least two probes and the amplified product are mixed at a rotation speed between 700-1000 rpm.

21. The method of claim 12, wherein the at least two probes are immobilized on a microarray plate at different positions.