[028] As used herein, "determining the presence of" an analyte means performing a test on a sample that leads to a result indicating that the analyte is present. The presence of an analyte may be determined, e.g., as an initial or confirmatory step— with or without prior knowledge, belief, suspicion, or expectation as to the presence of the analyte. In contrast, "determining the presence or absence of" an analyte means performing a test on a sample that leads to a result indicating either that the analyte is present or that the analyte is absent (wherein "absent" includes the existence of an undetectably low amount or concentration under the circumstances of the test).

[029] "Premalignant" means that a cell or tissue, while not cancerous, is at an increased risk for becoming malignant. Dysplasias, hyperplasias, and adenomas that are non-cancerous are examples of premalignancies.

[030] As used herein, "RET/PTC" refers to any of the more than 10 rearrangements, including RET/PTC1 , RET/PTC2, and RET/PTC3, in which the RET gene (which encodes a tyrosine kinase not normally expressed in thyroid cells) is fused to a 5' fusion partner such that the promoter of the 5' fusion partner, drives expression of the fusion gene, which are described in, e.g., Patel et al., Cancer Control, 13, 1 11 -1 18 (2006); Santoro et al., Br. J. Cancer, 82, 315-322 (2000); and Vecchio et al., Clin. Chem. Lab. Med. 38, 1 13-1 16 (2000). It has been described that the 5' fusion partner in a RET/PTC rearrangement is ubiquitously expressed. See, e.g., Patel et al., Cancer Control, 13, 1 1 1 -1 18 (2006). Thus, for purposes of this specification, reference to RET/PTC is to be understood as equivalent to referring to a fusion nucleic acid comprising a 5' fusion partner and a sequence of the RET gene, the fusion encoding a polypeptide with tyrosine kinase activity, and/or the encoded fusion polypeptide (as appropriate for the context), wherein the fusion may be RET/PTC 1 , RET/PTC2, RET/PTC3, or another RET/PTC fusion, such as those described in the three articles cited earlier in this paragraph.

[031 ] In general, attachment to a solid phase, such as of a reagent, can be covalent or non-covalent. Examples of non-covalent attachment include attachment through a binding interaction, e.g., avidin-biotin. One of the components involved in the binding interaction can itself be attached to the solid phase.

[032] Reference will now be made in detail to various embodiments of the invention.

[033] NTRK2-TERT fusion determination

[034] In some embodiments, the presence of an NTRK2-TERT fusion is determined in a thyroid-related cell or sample. In other embodiments, the presence or absence of an NTRK2-TERT fusion is determined in a thyroid-related cell or sample, such as a thyroid sample chosen from a fine needle aspirate of thyroid tissue, a formalin-fixed paraffin-embedded thyroid tissue sample, a thyroid biopsy, isolated thyroid cells, metastatic cells descended from a thyroid cancer, or biomolecules isolated from thyroid cells; or a biological sample obtained from a subject having thyroid cancer, a subject who previously had thyroid cancer, or a subject in need of diagnosis of a thyroid condition, which may be chosen from, e.g., a buccal swab sample or a biological fluid sample, such as a blood sample, a serum sample, a plasma sample, a urine sample, or a saliva sample. Unless otherwise indicated, discussion of a determination step "regarding" an analyte refers to either one of determining the presence of the analyte or to determining the presence or absence of the analyte. Thus, both determining the presence of an NTRK2-TERT fusion or and determining the presence or absence of an NTRK2-TERT fusion qualify individually as a determination step regarding an NTRK2- TEf?7fusion.

[035] In some embodiments, a determination step regarding an NTRK2- TERTfusion comprises an assay for an NTRK2-TERT fusion polypeptide, an NTRK2-TERT fusion RNA, or an NTRK2-TERT fusion DNA. In some embodiments, the assay is a binding assay, examples of which are discussed below. In other embodiments, the fusion is detected by a binding-independent assay, examples of which are also discussed below.

[036] Nucleic acid determination - hybridization and/or primer extension

[037] A determination step regarding an NTRK2-TERT fusion can be performed at the nucleic acid level (DNA or RNA), e.g., in a binding assay using one or more probes or primers that hybridize to the fusion to form a hybridization complex.

[038] In some embodiments, the probe, primer or primers used in such reactions comprise or consist of the exemplary primers discussed below in the section entitled "NTRK2-TERT fusion nucleic acids, polypeptides, primers, and probes." Those skilled in the art will understand that primers and/or probes with suitable hybridization characteristics can be designed based on known NTRK2 and TERT sequence data in. light of the information disclosed herein regarding fusion structure and junction locations. Probes or primers are non-naturally occurring nucleic acid molecules that form a hybridization complex.

[039] In some embodiments, the hybridization complex is detected using a label attached to a primer or probe. In other embodiments, the hybridization complex is detected using a label attached to analyte nucleic acid, e.g., when the primer or probe is associated with a solid phase such as a slide, surface, or bead. The label may be any form of detectable label, e.g., a fluorescent label (e.g., FITC, FAM, HEX, etc.), radioactive label (e.g.,³²P,³H,¹⁴C, etc.), enzymatic label (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, etc.), or affinity label (e.g., biotin, digoxygenin, etc.).

[040] Any form of nucleic acid hybridization assay suitable for specific detection of a nucleic acid can be used. Types of hybridization assays include blots including slot blots, Sourthern blots, or Northern blots and fluorescence hybridization assays including FISH and hybridization of isolated nucleic acids, e.g., to a microarray or other source of solid-phase-immobilized probe.

[041 ] In some embodiments, a determination step regarding an NTRK2- TEftTfusion comprises a primer extension assay. For example, a primer comprising an NTRK2 subsequence and a TERT subsequence can be contacted with a sample, wherein the primer anneals specifically to an NTRK2- TERT ius\on nucleic acid. Extension of such an annealed primer by a polymerase can then be determined, and such extension is indicative of the presence of the NTRK2-TERT fusion nucleic acid. Alternatively, a primer can be provided which anneals upstream of the fusion junction and extended; product of this reaction can be analyzed in a further step to determine the presence of the fusion if the analysis shows that sequence from both NTRK2 and TERT is present in the extension product. The further analysis can comprise, e.g., hybridization, restriction analysis, sequencing, etc.

[042] In some embodiments, the primer extension assay is a comparative primer extension assay comprising contacting a sample (or separate aliquots of a sample) with a first primer that anneals to wild-type TERT but not the NTRK2- TERT fusion and a second primer that anneals to both wild-type TERT and the NTRK2- TERTiusion. Extension of the second primer by a polymerase to a greater degree than the first primer (accounting for experimental/measurement error) is indicative of the presence of the NTRK2- TERT ius\on nucleic acid. The sample may comprise gDNA, primary transcript RNA, or mRNA and/or or cDNA, and further details regarding suitable samples are provided below. In some embodiments, the first primer anneals to a position upstream of TERT exon 3, e.g., in TERT exon 1 or 2 or upstream of TERT exon 1 , such as in the 5'-UTR of wild-type TERT mRNA, or, in gDNA, more than 12 kb upstream of TERT exon 1 . It is understood that "upstream" in the context of TERT and generally unless otherwise indicated means in the 5' direction relative to the TERT coding strand, while "downstream" in the context of TERT and generally unless otherwise indicated means in the 3' direction relative to the TEflTcoding strand. In some embodiments, the first primer anneals to TERT exon 1 or 2 or the TERT 5'-UTR; such a first primer can be used with mRNA or cDNA samples. In some embodiments, the second primer anneals to TERTexon 3, 4, 5, 6, 7, 8, 9, 10, 1 , 12, 13, 14, 15, or 16. In some embodiments, the second primer anneals to TERT at a location downstream of the beginning of exon 3. It is understood that primers with intronic annealing sites are suitable for use with gDNA or primary transcript RNA.

[043] Nucleic acid determination - amplification

[044] In some embodiments, a determination step regarding an NTRK2- TERT fusion comprises a nucleic acid amplification assay. Generally speaking, an amplification assay is performed on a sample and involves a reaction that amplifies a nucleic acid if the nucleic acid is present in the sample. Examples of amplification reactions include, without limitation, PCR, NASBA (nucleic acid sequence based amplification), SDA (strand displacement amplification), LAMP (loop-mediated isothermal amplification), and RCA (rolling circle amplification). See, e.g., U.S. Patent 4,683,202 (PCR); U.S. Patent 6,326,173 and Journal of Virological Methods 151 :283-293 (2008) (NASBA); U.S. Patent 5,648,21 1 (SDA); U.S. Patent 6,410,278 (LAMP); and U.S. Patent 6,287,824 (RCA). These reactions involve DNA synthesis, and as such involve the use of DNA Polymerases, nucleotides, and divalent cations (supplied as a salt), particularly magnesium, in a solution conducive to DNA polymerization and in which the template is present. The methods vary in terms of providing additional catalytic activities, the use of thermocycling or isothermal incubation, and the use and structure of primers. A buffer at a suitable pH such as between 7 and 8, between 6.5 and 8.5, between 6 and 9, or about 7.4 or 7.5 is also typically provided.

[045] In some embodiments, the primer or primers used in such reactions comprise or consist of the exemplary primers discussed below in the section entitled "NTRK2-TERT fusion nucleic acids, polypeptides, primers, and probes." Those skilled in the art will understand that primers with suitable hybridization

characteristics can be designed based on known NTRK2 and TEftT sequence data in light of the information disclosed herein regarding fusion structure and junction locations.

[046] In PCR, a pair of primers are provided that bind at each end of a target region, on opposite strands such that they each prime synthesis toward the other primer. The reaction is thermocycied so as to drive denaturation of the substrate in a high temperature step, annealing of the primers at a lower temperature step, and extension at a temperature which may be but is not necessarily higher than that of the annealing step. Amplification occurs because the products of one cycle can serve as template in the next cycle.

[047] In NASBA, an RNA polymerase (RNAP) is provided in addition to the DNA polymerase, which may also be a reverse transcriptase (RT) (e.g., an enzyme that can catalyze DNA synthesis using either an RNA or DNA template). Primers are provided that are similar to those used in PCR except that at least one primer additionally comprises a promoter sequence that is recognized by the RNAP. Thus, the product of the RT serves as template for the RNAP, which synthesizes RNA that serves as template for the RT, leading to amplification. In some forms of NASBA, RNase H is provided to produce single-stranded DNA after synthesis of an RNA- DNA hybrid by RT. Amplification occurs via the combined action of the RT and RNAP, in the absence of repeated thermal denaturation.

[048] SDA is a technique in which DNA is amplified in an isothermal and asynchronous manner, meaning that cyclic thermal denaturation is not used to separate the strands; instead, strand displacement occurs through DNA synthesis itself, wherein extension of a 3' OH causes displacement of the downstream strand. The 3' OH is provided initially by an exterior primer and subsequently by a nicking reaction. Two pairs of primers are provided. One 'interior' pair binds surrounding the amplicon and additionally comprises 5' flaps containing a restriction site. The other, 'exterior' pair is positioned distally, i.e., further from the target region. An interior primer may bind the template, be extended, and then be displaced by synthesis from the corresponding exterior primer.. Subsequently, the displaced DNA is made double-stranded, e.g., by second strand synthesis. The next step is to nick one strand of the double stranded molecule, which may be done by using modified nucleotides and a restriction site wherein the cleavage site is inactivated on one strand (but not the other) by the modified nucleotide. The restriction enzyme corresponding to this site is provided in the reaction and generates the nick. The 3' OH at the resulting nick is then extended by the DNA polymerase, displacing one strand (which may again serve as a template) and the regenerated double strand molecule is again a substrate for nicking followed by extension and displacement, leading to amplification. Note that some displaced strands will not initially be full- length but will lack the complement of the distal portion of the interior primer flap, as a consequence of the nicking. This does not impair primer binding (the non-flap portion of the primer has sufficient length to anneal stably) and, upon primer binding, a 5' overhang is generated that the polymerase is able to fill in. Repeated thermal denaturation is not necessary.

[049] LAMP is an amplification procedure designed to be highly specific, that is, it can discriminate between templates differing by only a single nucleotide polymorphism (SNP), in that one allele is a substrate for amplification and the other is not. It is also isothermal. As in SDA, two pairs of primers, interior and exterior, are provided; the interior primers also have a 5' flap. However, in LAMP, the 5' flap of each interior primer contains a sequence matching a sequence within the template strand to which it binds, interior to the site where the 3' portion of the primer binds. For example, if the primer anneals to the (+) strand of a template molecule, which contains the downstream sequence A, then the primer flap may also contain sequence A. Notably, the SNP locus which is to be discriminated by this reaction is located at the edge of the region bound by the flap, corresponding to the last base at the 5' end of the flap. The last base at the 5' end of the reverse interior primer flap also corresponds to the SNP locus. As in SDA, the interior primer is extended and then displaced by extension of the exterior primer. When this occurs, the 5' flap forms a loop by binding its complement (which is now part of the same molecule; continuing the above example, the displaced strand contains the reverse complement of sequence A, designated sequence T, and the sequence A in the flap binds intramolecularly to sequence T). The reverse interior primer anneals to the looped displaced strand, interior to its 3' end (which corresponds to the reverse exterior primer) and primes synthesis, which displaces the loop and forms a partially double-stranded, partially single stranded DNA. Then, a reverse exterior primer anneals to the single stranded portion and primes synthesis, causing strand displacement. The displaced strand can now form a loop wherein its 3' end is paired to an internal portion of the molecule. Only if the SNP locus matches the 3' end (which is derived from an interior primer flap that was exogenously supplied) does extension occur. Further primer annealing, looping, and extension/displacement events, described in the reference cited above, result in selective amplification of templates with the SNP allele matching the primer flap.

[050] In RCA, a circular DNA template is used. A primer anneals to the circle and is extended continuously, with the polymerase displacing the DNA synthesized during the previous revolution as it proceeds. This reaction proceeds with linear kinetics and produces long, concatemerized products. In double-primed RCA, a second primer is provided that anneals to the concatemerized product of the above reaction. This version of the reaction allows use of product as template, and therefore results in exponential kinetics. As in other isothermal reactions, product is made suitable for annealing to primer in double-primed RCA through strand displacement due to extension of upstream primers; in this case the primers are bound to other concatemers further upstream in the template strand.

[051] Nucleic acid determination - sequencing

[052] In some embodiments, a determination step regarding an NTRK2- TERTfusion comprises sequencing, for example, obtaining sequence data comprising an NTRK2 subsequence fused to a TEfiT subsequence. In some embodiments, in sequence data comprising an NTRK2 subsequence fused to a TEflTsubsequence, the NTRK2 subsequence comprises at least 10, 15, 20, 50, 100 or more bases, and/or the TERT subsequence comprises at least 1 0, 15, 20, 50, 100 or more bases.

[053] Examples of nucleic acid sequencing techniques that can be used in a determination step regarding an NTRK2-TERT fusion include, but are not limited to, chain terminator (Sanger) sequencing, dye terminator sequencing, Maxam-Gilbert sequencing, pyrosequencing, 454 sequencing, reversible terminator sequencing, ligation sequencing, nanopore sequencing, and third-generation sequencing.

Because RNA is generally less stable and more susceptible to nuclease attack, RNA may be reverse transcribed to DNA before sequencing in some embodiments.

[054] In some embodiments, sequencing comprises using one or more primers in a sequencing reaction. The primer or primers may comprise or consist of the exemplary primers discussed below in the section entitled "NTRK2-TERT fusion nucleic acids, polypeptides, primers, and probes." Those skilled in the art will understand that primers with suitable hybridization characteristics can be designed and made based on known NTRK2 and TEf?Tsequence data in light of the information disclosed herein regarding fusion structure and junction locations.

[055] Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, or other labeled, oligonucleotide primer complementary to the template at that region. The

oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di-deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di-deoxynucleotide. The products are size-separated by electrophoresis, e.g., in a slab gel or capillary. Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di-deoxynucleotide chain- terminators with a separate fluorescent dye, which fluoresces at a different wavelength. [056] Maxam-Gilbert sequencing, which involves performing multiple partial chemical degradation reactions on fractions of the nucleic acid sample followed by detection and analysis of the fragments to infer the sequence, is also well known in the art; see, e.g., Maxam et al., Proc Natl Acad Sci USA 74, 560-564 (1977).

[057] In sequencing by hybridization, the sequence of a sample is deduced based on its hybridization properties to a plurality of sequences, e.g., on a microarray or gene chip; see, e.g., Drmanac, et al., Nat Biotechnol 16, 54-58 (1998).

[058] Pyrosequencing, reversible terminator sequencing, and ligation sequencing are considered to be second-generation sequencing methods.

Generally, these methods use amplification products generated from a single molecule, which are spatially segregated from amplification products generated from other molecules. The spatial segregation can be implemented by using an emulsion, a picoliter well, or by attachment to a glass slide. Sequence information is obtained via fluorescence upon incorporation of a nucleotide; after acquiring data, the fluorescence of the newly incorporated nucleotide is eliminated and the process is repeated for the next nucleotide.

[059] In pyrosequencing, the pyrophosphate ion released by the

polymerization reaction is reacted with adenosine 5' phosphosulfate by ATP sulfurylase to produce ATP; the ATP then drives the conversion of luciferin to oxyluciferin plus light by luciferase. As the fluorescence is transient, no separate step to eliminate fluorescence is necessary in this method. One type of

deoxyribonucleotide triphosphate (dNTP) is added at a time, and sequence information is discerned according to which dNTP generates significant signal at a reaction site. The commercially available Roche GS FLX instrument acquires sequence using this method. This technique and applications thereof are discussed in detail, for example, in Ronaghi et a\.,Anal Biochem 242, 84-89 (1996) and Margulies et al., Nature 437, 376-380 (2005) (corrigendum at Nature 441 , 120 (2006)).

[060] In reversible terminator sequencing, a fluorescent dye-labeled nucleotide analog that is a reversible chain terminator due to the presence of a blocking group is incorporated in a single-base extension reaction. The identity of the base is determined according to the fluorophore; in other words, each base is paired with a different fluorophore. After fluorescence/sequence data is acquired, the fluorophore and the blocking group are chemically removed, and the cycle is repeated to acquire the next base of sequence information. The lllumina GA instrument operates by this method. This technique and applications thereof are discussed in detail, for example, in Ruparel et al., Proc Natl Acad Sci USA 102, 5932-5937 (2005), and Harris et al., Science 320, 106-109 (2008).

[061 ] In ligation sequencing, a ligase enzyme is used to join a partially double-stranded oligonucleotide with an overhang to the nucleic acid being sequenced, which has an overhang; in order for ligation to occur, the overhangs must be complementary. The bases in the overhang of the partially double-stranded oligonucleotide can be identified according to a fluorophore conjugated to the partially double-stranded oligonucleotide and/or to a secondary oligonucleotide that hybridizes to another part of the partially double-stranded oligonucleotide. After acquisition of fluorescence data, the ligated complex is cleaved upstream of the ligation site, such as by a type lis restriction enzyme, for example, Bbvl, which cuts at a site a fixed distance from its recognition site (which was included in the partially double stranded oligonucleotide). This cleavage reaction exposes a new overhang just upstream of the previous overhang, and the process is repeated. This technique and applications thereof are discussed in detail, for example, in Brenner et al., Nat Biotechnol 18, 630-634 (2000). In some embodiments, ligation sequencing is adapted to the methods of the invention by obtaining a rolling circle amplification product of a circular nucleic acid molecule, and using the rolling circle amplification product as the template for ligation sequencing.

[062] In nanopore sequencing, a single stranded nucleic acid molecule is threaded through a pore, e.g., using an eiectrophoretic driving force, and sequence is deduced by analyzing data obtained as the single stranded nucleic acid molecule passes through the pore. The data can be ion current data, wherein each base alters the current, e.g., by partially blocking the current passing through the pore to a different, distinguishable degree. See, e.g., US Patent App. Pub. No.

US2013/0244340 A1 and PCT Patent App. Pub. No. WO2013/185137 A1.

[063] In third-generation sequencing, a slide with an aluminum coating with many small (^"50 nm) holes is used as a zero mode waveguide (see, e.g., Levene et al., Science 299, 682-686 (2003)). The aluminum surface is protected from attachment of DNA polymerase by polyphosphonate chemistry, e.g.,

polyvinylphosphonate chemistry (see, e.g., Korlach et al., Proc Natl Acad Sci USA 105, 1 176-1181 (2008)). This results in preferential attachment of the DNA polymerase molecules to the exposed silica in the holes of the aluminum coating. This setup allows evanescent wave phenomena to be used to reduce fluorescence background, allowing the use of higher concentrations of fluorescently labeled dNTPs. The fluorophore is attached to the terminal phosphate of the dNTPs, such that fluorescence is released upon incorporation of the dNTP, but the fluorophore does not remain attached to the newly incorporated nucleotide, meaning that the complex is immediately ready for another round of incorporation. By this method, incorporation of dNTPs into an individual primer-template complexes present in the holes of the aluminum coating can be detected. See, e.g., Eid et al., Science 323, 133-138 (2009).

[064] In some embodiments, a determination step regarding an NTRK2- TERTfusion comprises a ligation assay. A ligation assay can be performed, for example, with oligonucleotides that anneal to immediately adjacent segments of the sequence to be determined. One of the selected oligonucleotide probes has an end region wherein one of the end region nucleotides is complementary to either the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence. A ligase is provided which covalently connects the two probes when they are correctly base paired and are located immediately adjacent to each other. The presence or amount of the linked probes is an indication of the presence of the target sequence, e.g., fusion. See, e.g., U.S. Pat. No. 4,988,617. The linked probes can be detected by any appropriate step, e.g., by size (such as through electrophoresis) or fluorescence (such as fluorescence resonance, energy transfer involving a donor fluor on one probe and an acceptor fluor on the other probe, or association of fluorescence with a solid phase, e.g., bead or surface, where one fluor is labeled and the other is attached to the solid phase). Thus, a pair of probes specific for an ΝΤΠΚ2 subsequence and a TERT subsequence can be used to determine the presence of a fusion comprising the NTRK2 subsequence fused to the TERT subsequence. In some embodiments, a ligation assay is performed in a multiplexed format, wherein a plurality of probes comprising NTRK2 subsequence and/or a plurality of probes comprising TERT subsequence is provided. In this way, the determination can provide for the detection of any of a number of possible fusions. Additional forms of ligation assay are described in, e.g., WO 2007/100243 A1 and documents cited therein.

[065] In some embodiments, a determination step regarding an NTRK2- TEflTfusion comprises a NanoString assay. In a NanoString assay, a reporter probe and capture probe can be used to assay for individual copies of a nucleic acid. See Geiss et al., Nat Biotech 26, 317-325 (2008). a multiplexed probe library is made with two sequence-specific probes for each gene of interest. The capture probe contains a 35- to 50-base sequence complementary to a particular target plus a short common sequence coupled to an affinity tag such as biotin. The second probe, the reporter probe, contains a second 35- to 50-base sequence

complementary to the target, which is coupled to a color-coded tag that provides the detection signal. The tag is a single-stranded DNA molecule, the backbone, annealed to a series of complementary in vitro transcribed RNA segments each labeled with a specific fluorophore. The linear order of these differently colored RNA segments creates a unique code for each target, thus allowing for multiplexed use of many probes per assay. After hybridization and removal of unhybridized probes, ternary target-capture probe-reporter probe complexes are associated with a surface through a capture reagent on the surface, e.g., streptavidin if the capture probe affinity tag is biotin. Then, the complexes are oriented and extended using an electric field, and the complexes are immobilized and imaged. Target molecules are identified based on the ordered fluorescent segments of the reporter probe.

[066] Polypeptide assays

[067] In some embodiments, a determination step regarding an NTRK2- TERT fusion comprises a polypeptide assay. For example, a determination step regarding an NTRK2-TERT fusion polypeptide can comprise an affinity assay, such as an antibody binding assay, an RNA aptamer assay, or a telomerase RNA affinity assay. Appropriate procedures for isolation of an antibody or aptamer specific for a given polypeptide are known in the art. In some embodiments, an antibody or aptamer specific for the fusion is used, which antibody lacks or has significantly lower affinity for NTRK2 and TERT individually. Alternatively, two or more antibodies or aptamers with specificities to different parts of the fusion can be used, e.g., in a sandwich assay. For example, a first antibody or aptamer against an NTRK2 epitope and a second antibody or aptamer against a TERT epitope can be employed in various ways to detect the presence of the fusion. In some embodiments, the first antibody or aptamer is associated with a solid phase, such as a surface or bead, and association of the second antibody or aptamer with the solid phase indicates the formation of a ternary complex or sandwich involving the two antibodies or aptamers and the fusion. In general, an antibody or aptamer can be detected by any suitable method, such as with a label, e.g., fluorescence of a fluorophore, radioactive emission by a radioisotope, chemiluminescence, or the action of an enzyme such as alkaline phosphatase. In some embodiments, a label such as one of the labels mentioned above is attached covalently to the antibody or aptamer. In some embodiments, where an antibody is used, a label such as one of the labels mentioned above is attached to a detection reagent that binds the aptamer, such as a secondary antibody, protein A, or protein G.

[068] Telomerase RNA affinity assays can be used for a determination step regarding an NTRK2-TERT fusion polypeptide wherein the NTRK2-TERT fusion polypeptide comprises a functional RNA-binding telomerase ribonucleoprotein complex domain, which can be a fragment of the wild-type domain, e.g., a fragment comprising amino acids 326-613 of wild-type hTERT, which was reported to bind telomerase RNA in Lai et al., Mol. Cell. Biol. 21 , 990-1000 (2001 ) (doi:

10.1 128/MCB.21 .4.990-1000.2001 ) (see, e.g., Fig. 5 therein).

[069] A sequential binding assay such as immunoprecipitation-Western blot (I P-WB) can also be used to determine the presence of an NTRK2-TERT fusion. In a first example of an IP-WB assay, a first antibody specific for NTRK2 would be used to prepare an immunoprecipitate from a sample, and the immunoprecipitate would then be analyzed via Western blot with a primary antibody specific for TERT. In a second example of an I P-WB assay, a first antibody specific for TERT would be used to prepare an immunoprecipitate from a sample, and the immunoprecipitate would then be analyzed via Western blot with a primary antibody specific for NTRK2.

[070] In some embodiments, a determination regarding an NTRK2-TERT fusion comprises a proteomic assay. Proteomic assays generally can detect a plurality of polypeptides. Examples of proteomic assays include protein mass spectrometry, 2D protein electrophoresis, and protein microarrays.

[071 ] In protein mass spectrometry, polypeptides are ionized and characterized according to their mass to charge ratio (m/z). For example, in some mass spectrometers, ionized polypeptides are accelerated and deflected

electromagnetically before being detected; details about the detection such as the location of contact and/or time of flight provide the basis for calculating m/z. Various forms of mass spectrometry are discussed, e.g. , in U.S. Pat. App. Pub. No.

2009/0189069 A1 . In tandem mass spectrometry, m/z can be determined for an ionized polypeptide and then for fragmented, ionized pieces of the polypeptide. In this way a "fingerprint" of fragments can be generated for individual polypeptide ions. Alternatively, polypeptides can be fragmented in a predictable manner, e.g., using a protease such as trypsin or chymotrypsin that cleaves substrate polypeptides in an amino-acid sequence-dependent manner, and the resulting cleavage products can be analyzed mass spectrometrically. Mass spectrometry can also be performed following a separation step, e.g., a chromatographic or electrophoretic separation step. A determination regarding a NTRK2-TERT fusion polypeptide can be performed mass spectrometrically, e.g., by detecting one or more polypeptide ions or polypeptide fragment ions with an m/z value characteristic of an NTRK2-TERT fusion, such as an m/z equal to the molecular weight of an NTRK2-TERT fusion times 1 , 1/2, 1/3, 1/4, etc. (with it being understood that the ionization process may slightly alter the molecular weight through the gain or loss of certain chemical moieties, as is known in the art, such that the molecular weight of the NTRK2-TERT fusion is adjusted to account for such alteration). Alternatively, where a separation step or tandem mass spectrometry is employed, a determination regarding a NTRK2-TERT fusion polypeptide can be performed by detecting one or more first polypeptide fragment ions with an m/z value characteristic of an NTRK2 fragment ion and one or more second polypeptide fragment ions with an m/z value characteristic of a TEftTfragment, wherein the one or more first polypeptide fragment ions and one or more second polypeptide fragment ions originated from the same polypeptide species.

[072] In 2D electrophoresis, a sample can be analyzed by separating polypeptides electrophoretically in two dimensions, with the electrophoretic separation in the first dimension being under conditions different from the conditions of the electrophoretic separation in the second dimension. For example, one form of 2D electrophoresis involves separation based on acidity/alkalinity (e.g., pi) in one dimension (e.g., isoelectric focusing) and separation based on molecular weight in the other dimension (e.g., electrophoresis of denatured, SDS-associated polypeptide). In a determination regarding an NTRK2-TERT fusion polypeptide by 2D electrophoresis, the fusion can be detected based on observation of a polypeptide signal (e.g., a spot after staining of the gel) in a location consistent with the properties of the fusion, such as its molecular weight and pi. Procedures for estimating the molecular weight and pi of a polypeptide are well known in the art, such that locations in a 2D gel at which an NTRK2-TERT fusion is expected to appear can be easily determined given knowledge of the NTRK2-TERT fusion as disclosed herein. In some embodiments, the identity of the NTRK2-TERT fusion in a gel can be confirmed, e.g., based on antibody binding to one or both of an NTRK2 antibody or a TERT antibody, or based on antibody binding to an antibody specific for the NTRK2-TERT fusion, using a procedure such as a Western blot.

[073] Protein. microarrays are discussed generally in Poetz et al., Mech. Ageing Devel. 126, 161 -170 (2005). For example, an array surface can be provided, which comprises spotted affinity reagents such as antibodies. A labeled sample can be applied to the array, and binding (measured by signal from the label) at a particular spot indicates the presence of the analyte for which the reagent at that spot has affinity. Thus, in an embodiment, a protein array comprising a spotted affinity reagent specific for an NTRK2-TERT fusion, such as an antibody specific for the fusion as discussed above, is provided.

[074] In some embodiments, a determination regarding an NTRK2-TERT fusion is made to characterize the genotype of a cell, which can be a mammalian cell, such as a human cell. In some embodiments, the cell is malignant or premalignant. In some embodiments, the cell is a thyroid cell, such as a thyroid cell obtained from a thyroid biopsy. In some embodiments, the cell is a metastatic cancer cell, such as a metastatic cancer cell descended from a primary thyroid cancer. In some embodiments, the cell is isolated from a subject. Alternatively, the cell itself need not be isolated; for example, its genotype can be characterized through analysis of a lysate, released nucleic acid or polypeptide, or a tissue sample, wherein the cell is not isolated from its surrounding tissue. Immunohistochemistry, immunofluorescence, and fluorescence in situ hybridization are examples of techniques suitable for analysis of a cell that has not been isolated from its surrounding tissue.

[075] In some embodiments, the methods further comprise characterizing the genotype of the cell with respect to the status, presence, or absence of additional genes, polypeptides, and/or mutations. For example, the methods may further comprise determinations regarding one or more of a RAS mutation (e.g., an HRAS, KRAS, or NRAS mutation), a BRAF mutation, a RET/PTC fusion, a PAX8- PPARgamma fusion, a PIK3CA mutation. Some embodiments comprise a determination regarding a fusion involving NTRK1 , NTRK3, ALK, PPARg, or BRAF. Some embodiments comprise a determination regarding a fusion involving a point mutation in EIF1 AX, TP53, or PTEN. Some embodiments comprise a determination regarding a BRAF V600E mutation.

[076] In some embodiments, the presence of an NTRK2-TERT^'fusion is determined to characterize a biological sample. In some embodiments, the sample comprises tissue, at least one isolated cell, isolated nucleic acid, isolated DNA, isolated RNA (e.g., DNA and/or RNA), or isolated polypeptide.

[077] NTRK2-TERT Fusion characteristics

[078] In some embodiments, in a determination regarding an NTRK2-TERT fusion, the NTRK2-TERT iuslon is an in-frame fusion. Thus, codons from TERT are in the same reading frame as codons from NTRK2 in an NTRK2-TERT fusion mRNA. In some embodiments, amino acid subsequences from NTRK2 and TERT are present in an NTRK2-TERT ius on polypeptide.

[079] In some embodiments, in a determination regarding an NTRK2-TERT fusion, the NTRK2-TERT fusion genomic DNA comprises a junction point between NTRK2 and ΤΕΉΤ intronic or untranslated region subsequences. For example, upon transcription and mRNA splicing, the 3'-terminal nucleotide of an exon of NTRK2^'\s joined to the 5'-terminal nucleotide of an exon of TERT. As a further example, NTRK2-TERT fusion genomic DNA comprises a junction point between a subsequence of the NTRK2 intron following NTRK2 exon 8 and a subsequence of the region 10-15 kb upstream of TERT exon 1 in gDNA, e.g., 1 1.5-13 kb upstream, 11 -13 kb upstream, or 1 1.5-12.5 kb upstream, which can be upstream of the TERT transcription start site. As a further example, the NTRK2-TERT fusion mRNA comprises a junction point between the 3'-terminal nucleotide of NTRK2 exon 8 and the 5'-terminal nucleotide of TERT exon 3. It is possible that sequences that function as exons in an unfused gene become intronic in a fusion. Thus, for example, it has been observed that an NTRK2- TERT fusion genomic DNA with a junction point between a subsequence of the intron following NTRK2 exon 8 and a subsequence of the TERT 5' UTR can be spliced into an mRNA with a junction point between the 3'- terminal nucleotide of NTRK2 exon 8 and the 5'-terminal nucleotide of TERT exon 3. Thus, as a result of the splicing of the fusion primary transcript, TERTexons 1 and 2 may be omitted from the fusion mRNA.

[080] For exon designations for human TERT, see, e.g., the exon information under entry NX_014746 in the neXtprot™ database or RefSeq/GenBank accession nos. N _198253.2 or NM_001 193376.1 , each of which is incorporated herein by reference. For exon designations for NTRK2, see, e.g., the exon information under entry NX_Q16620 in the neXtprot database or RefSeq/GenBank accession nos. NM_001018066.2, NM_001018065.2, NM_001018064.1 ,

N J306180.3, or NM_001007097.1 , each of which is incorporated herein by reference. Exon designations are similarly available for TERT and NTRK2 in other species, including other mammalian species. Where multiple possible transcripts exist, as for TERT, a reference to a specific exon encompasses that exon of each variant unless otherwise indicated.

[081 ] In some embodiments, in a determination regarding an NTRK2-TERT fusion, an NTRK2- TERT fusion mRNA comprises exons 1 -8 of NTRK2. In some embodiments, in a determination regarding an NTRK2-TERT fusion, an NTRK2- TERT fusion mRNA comprises exons 3-16 of TERT. In some embodiments, in a determination regarding an NTRK2-TERT fusion, an NTRK2-TERT fusion polypeptide comprises an amino acid sequence translated from exons 1 -8 of NTRK2. In some embodiments, in a determination regarding an NTRK2-TERT fusion, an NTRK2- TERT fusion polypeptide comprises an amino acid sequence translated from exons 3-16 of TERT.

[082] In some embodiments, in a determination regarding an NTRK2-TERT fusion, an NTRK2-TERT fusion mRNA comprises a sequence spanning the NTRK2- TEflTjunction point chosen from CCCAATTGTGGGGTTGGC,

CCAATTGTGGGGTTGG, C AATTGTG G G GTTG , A ATTGTG G G GTT,

ATTGTGGGGT, TTGTGGGG, TGTGGG, CCCAAUUGUGGGGUUGGC,

CCAAUUGUGGGGUUGG, CAAUUGUGGGGUUG, AAUUGUGGGGUU,

AUUGUGGGGU, UUGUGGGG, or UGUGGG.

[083] In some embodiments, the NTRK2-TERT fusion is a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide shows telomere elongation activity in a telomere elongation assay. Telomere elongation assays are known and can be performed as described in, e.g., Cohen et al., Nature Methods 5, 355-60 (2008). In this assay, bead-associated DNA substrate comprising 3'-terminal TTAGGG repeats is contacted with a preparation that may contain telomerase. After incubation under conditions that permit extension, the DNA substrate is released from the beads and a determination regarding extension of the DNA substrate is performed.

[084] Sample types

[085] In some embodiments, the determining step is performed on a thyroid sample chosen from a fine needle aspirate of thyroid tissue, a formalin-fixed paraffin- embedded thyroid tissue sample, a thyroid biopsy, isolated thyroid cells, or biomolecules isolated from thyroid cells. In some embodiments, the determining step is performed on a biological sample such as a buccal swab sample or a biological fluid sample, such as a blood sample, a serum sample, a plasma sample, a urine sample, or a saliva sample; the biological sample, including any of the foregoing examples of a biological sample, may be obtained from a subject having thyroid cancer, a subject who previously had thyroid cancer, or a subject in need of diagnosis of a thyroid condition. Generally, a determining step is considered to be performed on a sample of a given type if the material actually used in the

determining step is isolated from that type of sample, e.g., nucleic acid or polypeptide isolated from a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a buccal swab sample, or a thyroid sample such as those listed above. Thus, in some embodiments, the determining step is performed on a biological sample comprising isolated DNA, isolated RNA, or isolated polypeptide. In some embodiments, the determining step is performed on cDNA prepared from RNA.

[086] Distinguishing non-malignant thyroid cells from malignant thyroid cells; likelihood of cancer being present

[087] This disclosure provides methods of distinguishing non-malignant thyroid cells from malignant thyroid cells comprising a determination regarding an NTRK2-TERT fusion. A determination that an NTRK2-TERT fusion is present in a thyroid cell can indicate that the cell is cancerous. A determination that an NTRK2- TERTIus on is present in a thyroid tissue sample or subject can indicate that the thyroid tissue or subject comprises cancerous thyroid cells. Alternatively, a determination that an NTRK2-TERT fusion is present in a thyroid cell can indicate an increased likelihood that the cell is cancerous, relative to the likelihood in the absence of the fusion. A determination that an NTRK2-TERT^'fusion is present in a thyroid tissue sample or subject can indicate an increased likelihood that the thyroid tissue sample or subject comprises cancerous thyroid cells, relative to the likelihood in the absence of the fusion. In these methods, determinations regarding an NTRK2-TERT fusion can be made, e.g., using any of the approaches discussed above.

[088] In some embodiments, the methods of the preceding paragraph further comprise one or more additional characterizations of thyroid cells, e.g., a

microscopic characterization of cell morphology, karyotyping, comparative genomic hybridization, a determination regarding one or more gene fusions other than NTRK2-TERT, determination of the expression level of one or more genes other than NTRK2, TERT, or an NTRK2-TERT 1us\on, or sequencing of one or more genetic loci or RNAs other than NTRK2, TERT, or an NTRK2- TEf?7f usion, such as high-throughput sequencing of genomic DNA or targeted sequencing of one or more individual genetic loci or RNAs. In some embodiments, the genes or genetic loci comprise one or more of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RET/PTC, PAX8-PPARgamma, and PIK3CA. In some embodiments, the methods comprise a determination regarding a RAF mutation. In some embodiments, in the presence of a RAF mutation and/or wherein a determination of the presence of a BRAF mutation is made, determining the presence of an NTRK2-TERT fusion indicates the presence of cancer, e.g., that thyroid cells are malignant, while determining the absence of an NTRK2-TERT fusion may indicate the absence of cancer, e.g., that thyroid cells are pre-malignant. A determination or determinations that indicate the presence of thyroid cancer can be followed with appropriate thyroid cancer monitoring and/or treatments, as discussed in the section entitled Cancer Monitoring and Treatment.

[089] Stratifying a thyroid cancer

[090] The disclosure provides methods of stratifying a thyroid cancer comprising a determination regarding an NTRK2-TERT fusion. In general, stratifying a thyroid cancer can refer to assigning a subtype to a thyroid cancer, with the understanding that the subtype may have implications regarding the outcome or progression of the thyroid cancer and/or the likelihood that one or more possible treatments will be effective in bringing about a desired result, e.g., slowing or halting the progression of the thyroid cancer, reducing the number of thyroid cancer cells or thyroid tumor size, producing remission of the thyroid cancer, rendering thyroid cancer undetectable in the patient, or curing the thyroid cancer.

[091 ] In some embodiments, a method of stratifying a thyroid cancer comprises determining the presence of an NTRK2-TERT fusion, wherein the thyroid cancer is stratified as an NTRK2- TERT positive thyroid cancer. Such methods may further comprise determinations regarding one or more of wild-type or mutated forms of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RAF, RET/PTC, PAX8- PPARgamma, or PIK3CA. In some embodiments, the one or more determinations regarding wild-type or mutated forms of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RAF, RET/PTC, PAX8-PPARgamma, or PIK3CA are determinations of the presence of the wild-type form of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RAF, RET/PTC, PAX8-PPARgamma, or PIK3CA. In some embodiments, the one or more determinations regarding wild-type or mutated forms of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RAF, RET/PTC, PAX8-PPARgamma, or PIK3CA are determinations of the absence of a mutated form of RAS (e.g., KRAS, NRAS, or HRAS), BRAF, RAF, RET/PTC, PAX8-PPARgamma, or PIK3CA.

[092] Methods comprising determining the presence of an NTRK2-TERT fusion, wherein the thyroid cancer is stratified as an NTRK2-TERT positive thyroid cancer^' may also comprise, alternatively or in addition, a determination regarding the cytology and/or histology of a sample comprising thyroid cells. In some

embodiments, the determination regarding the cytology and/or histology of a sample comprising thyroid cells is negative, i.e., does not provide an indication that the sample comprises cancerous thyroid cells.

[093] In some embodiments, stratification of a thyroid cancer as an NTRK2- TERT positive cancer indicates that telomerase inhibition and/or a tyrosine kinase inhibition is more likely to be effective in bringing about a desired result, e.g., slowing or halting the progression of the thyroid cancer, reducing the number of thyroid cancer cells or thyroid tumor size, producing remission of the cancer, rendering thyroid cancer undetectable in the patient, or curing the thyroid cancer. Exemplary forms of telomerase inhibition are discussed below. [094] Thyroid Cancer Phenotype; Follicular Thyroid Cancer

[095] In some embodiments, a thyroid cancer that is the subject of a determination as discussed above, or of monitoring and/or treatment as discussed below, comprises cancer cells having a phenotype of high telomerase expression or activity. In some embodiments, a thyroid cancer that is the subject of a

determination as discussed above, or of monitoring and/or treatment as discussed below, comprises cancer cells determined to have a phenotype of high telomerase expression or activity. The high telomerase expression or activity can be, for example, at least 2, 3, 4, 5, 10, 20, 30, 40, 50, or 100 times higher than a normal level. The normal level can be, e.g., the average level in healthy thyroid cells of the individual with the cancer, or of a reference sample of healthy thyroid cells, such as cells located in normal adjacent tissue. Telomerase expression can refer to either RNA or polypeptide levels.

[096] Where telomerase expression is or has been determined, the determination can comprise a determination at the RNA level, e.g., by measuring the amount of mRNA comprising a subsequence of the TERT reverse transcriptase catalytic domain. Alternatively, telomerase expression can be determined at the polypeptide level, e.g., using an antibody or aptamer to TERT, such as an antibody or aptamer to the TERT reverse transcriptase domain. Where telomerase activity is or has been determined, the determination can comprise an assay for DNA synthesis, e.g., reverse transcription. Procedures for telomerase activity assays are known to those skilled in the art.

[097] In some embodiments, a thyroid cancer that is the subject of a determination as discussed above, or of monitoring and/or treatment as discussed below, is a follicular thyroid cancer. [098] Thyroid Cancer Monitoring and Treatment

[099] Disclosed herein are methods of cancer monitoring and/or treatment of subjects with a thyroid cancer determined to have an NTRK2-TERT fusion. Also disclosed herein are methods of thyroid cancer monitoring and/or treatment of subjects with a thyroid cancer determined not to have an NTRK2-TERT fusion. In some embodiments, the methods comprise a determination regarding the NTRK2-^' TERTfusion, which may be, e.g., a determination chosen from those discussed above.

[0100] Thus, an embodiment is monitoring a thyroid cancer in a patient, wherein the thyroid cancer has been determined not to have an NTRK2-TERT fusion. Monitoring means performing one or more follow-up tests that provide information about the status of the cancer. The one or more tests may comprise two or more tests separated by a period of time, e.g., at least 1 or 2 weeks or 1 , 2, 3, 4, 5, 6, 9, or 12 months. In some embodiments, the monitoring is performed without administering any anti-cancer agents. In some embodiments, the monitoring is performed without any surgical resection. In some embodiments, the thyroid cancer has been determined to have a RAS mutation (e.g., an HRAS, NRAS, or KRAS mutation). The absence of an NTRK2-TERT fusion in a thyroid cancer with a RAS mutation can indicate that the cancer has not progressed to an aggressive form, such that treatment may not be necessary. In further embodiments, the thyroid cancer may also lack and/or have been determined not to have a mutation chosen from one or more of a TERT promoter mutation, a RET/PTC fusion, a PAX8- PPARgamma fusion, or a PIK3CA mutation.

[0101 ] Another embodiment is treating a thyroid cancer in a patient, wherein the thyroid cancer was determined not to have an NTRK2-TERT fusion. The treatment can comprise, e.g., hemithyroidectomy and/or administering radioactive iodine. In some embodiments, the thyroid cancer was determined to have one or more mutations comprising a PAX8-PPARgamma fusion or a PIK3CA mutation.

[0102] Another embodiment is treating a thyroid cancer in a patient, wherein the thyroid cancer was determined to have an NTRK2-TERT fusion. The treatment can comprise inhibiting telomerase, for example, by administering at least one reverse transcriptase inhibitor and/or telomerase inhibitor. For example, the at least one reverse transcriptase inhibitor and/or telomerase inhibitor can be chosen from one or more of GRN163L, lamivudine, abacavir, zidovudine, emtricitabine, tenofovir, GV1001 , BIBR1532, Vx-001 , telomestatin, BRACO-19, RHPS4, and their equivalents. GRN163L is a telomerase inhibitor. See, e.g., Burchett et al., "Telomerase Inhibitor Imetelstat (GRN163L) Limits the Lifespan of Human

Pancreatic Cancer Cells," PLoS ONE 9(1 ): e85155 (2014),

doi:10.1371/journal. pone.0085155, which is incorporated herein by reference.

Lamivudine, abacavir, zidovudine, emtricitabine, tenofovir, and their equivalents are reverse transcriptase inhibitors; reverse transcriptase inhibitors have also been shown to inhibit telomerase. See Leeansyah et al., "Inhibition of telomerase activity by human immunodeficiency virus (HIV) nucleoside reverse transcriptase inhibitors: a potential factor contributing to HIV-associated accelerated aging," J Infect Dis. 2013 Apr;207(7):1 157-65, doi: 10.1093/infdis/jit006, which is incorporated herein by reference. Additionally or alternatively, inhibiting telomerase can comprise administering GRNVAC1 or its equivalents. GRNVAC1 and its equivalents are a vaccine-based therapy against telomerase. GRNVAC1 and additional telomerase inhibitors including GV1001 , BIBR1532, Vx-001 , telomestatin, BRACO-19, and RHPS4 are discussed in Puri, et al., "Novel Therapeutics Targeting Telomerase and Telomeres," J Cancer Sci Ther 5 (2013): e127, doi:10.4172/1948-5956.1000e127, which is incorporated herein by reference.

[0103] Alternatively or in addition to inhibiting telomerase as discussed above, treating a thyroid cancer in a patient, wherein the thyroid cancer was determined to have an NTRK2-TERT fusion, can comprise administration of at least one anticancer agent and/or thyroidectomy. In some embodiments, the at least one anticancer agent is other than a telomerase or reverse transcriptase inhibitor. In some embodiments, the at least one anti-cancer agent comprises radioiodine and/or at least one tyrosine kinase inhibitor. In some embodiments, the at least one tyrosine kinase inhibitor comprises at least one EGFR and/or at least one VEGFR inhibitor. Exemplary inhibitors including tyrosine kinase, EGFR, and VEGFR inhibitors are described in, e.g., Puxeddu et al., Current Opinion in Oncology, 23:13-21 (201 1 ), and Sherman, Best Practice & Research Clinical Endocrinology & Metabolism, 23 713-722 (2009), which are incorporated herein by reference for their descriptions of EGFR and VEGFR inhibitors and inhibition.

[0104] Some embodiments comprise testing for bone metastasis in a thyroid cancer patient, wherein the thyroid cancer was determined to have an NTRK2-TERT fusion. Some embodiments comprise testing for lung metastasis in a thyroid cancer patient, wherein the thyroid cancer was determined to have an NTRK2-TERT fusion. Some embodiments comprise testing for lymph node metastasis in a thyroid cancer patient, wherein the thyroid cancer was determined to have an NTRK2-TERT fusion.

[0105] Some embodiments comprise a determination regarding an NTRK2- TERTfusion in a thyroid cancer, and pursuing a more aggressive treatment if the

is detected in the thyroid cancer or a less aggressive treatment if the NTRK2-TERT fusion is not detected in the thyroid cancer. For example, a more aggressive treatment may comprise administering an anti-cancer agent such as those discussed above, e.g., telomerase inhibitors, reverse transcriptase inhibitors, agents other than a telomerase or reverse transcriptase inhibitor, radioactive iodine, and/or a tyrosine kinase inhibitor. In some embodiments, the less aggressive treatment does not comprise administering an anti-cancer agent such as those discussed above, e.g., telomerase inhibitors, reverse transcriptase inhibitors, agents other than a telomerase or reverse transcriptase inhibitor, radioactive iodine, and/or a tyrosine kinase inhibitor. In some embodiments, the more aggressive treatment comprises a thyroidectomy or hemithyroidectomy or equivalent procedure and the less aggressive treatment does not comprise a thyroidectomy or

hemithyroidectomy or equivalent procedure. In some embodiments, the more aggressive treatment comprises a thyroidectomy and the less aggressive treatment does not comprise a thyroidectomy, but may optionally comprise a

hemithyroidectomy.

[0106] Some embodiments comprise monitoring a cancer in which an NTRK2- TERTiusion is present by monitoring an NTRK2-TERT iusion in a sample from the individual having the cancer. The monitoring may comprise one or more tests, or two or more tests separated by a period of time, e.g., at least 1 or 2 weeks or 1 , 2, 3, 4, 5, 6, 9, or 12 months. In some embodiments, the monitoring method comprises determining a level of tumor burden based on the amount of NTRK2- TEflTfusion detected. In some embodiments, the monitoring method comprises determining that tumor burden has changed (e.g., increased or decreased) based on a determining that the amount of NTRK2- TERT fusion has changed (e.g., increased or decreased) relative to a previous measurement. In some embodiments, the monitoring comprises determining a level of the NTRK2- TERT fusion before and after treatment, such as the treatments discussed above. A response to treatment can be monitored one or more times or over a course of time, e.g., two or more tests separated by a period of time, e.g., at least 1 or 2 weeks or 1 , 2, 3, 4, 5, 6, 9, or 12 months. Thus, some embodiments, including the response to treatment and/or tumor burden- monitoring embodiments, can comprise determining that the amount of NTRK2- TERTfusion has changed (e.g., increased or decreased) relative to a previous measurement by an amount greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or 99%, or increased by and amount greater than or equal to 100%, 150%, 200%, or 300%.

[0107] NTRK2-TERT fusion nucleic acids, polypeptides, primers, and probes for methods and uses of the invention

[0108] In some embodiments of the methods and uses discussed above concerning thyroid cancer, certain molecules related to an NTRK2-TERT fusion are used.

[0109] In some embodiments of the methods and uses discussed above concerning thyroid cancer, an NTRK2-TERT fusion polypeptide, which may be isolated, is used. In some embodiments, the NTRK2-TERT1us^'\or polypeptide comprises an amino acid sequence translated from exons 1-8 of NTRK2. In some embodiments, the NTRK2-TERT \us\on polypeptide comprises an amino acid sequence translated from exons 3-16 of TERT. In some embodiments, the NTRK2- TERTiuslon polypeptide comprises an amino acid sequence translated from exons 1-16 of TERT. In some embodiments, the NTRK2-TERT fusion polypeptide comprises a functional RNA-binding telomerase ribonucleoprotein complex domain, which can be a fragment of the wild-type domain, e.g., a fragment comprising amino acids 326-613 of wild-type hTERT, which was reported to bind telomerase RNA in Lai et al., Mol. Cell. Biol. 21 , 990-1000 (2001 ) (doi: 10.1 128/MCB.21.4.990- 1000.2001 ) (see, e.g., Fig. 5 therein). In some embodiments of the methods and uses discussed above concerning thyroid cancer, an oligopeptide comprising a subsequence of NTRK2 amino acids fused to a subsequence of TEf?7^"amino acids, for example, at least 10 amino acids from the sequence of NTRK2 fused to at least 10 amino acids from the sequence of TERT, or 10-50 amino acids from the sequence of NTRK2 fused to 10-50 amino acids from the sequence of TERT, is used.

[01 10] In some embodiments of the methods and uses discussed above concerning thyroid cancer, an NTRK2- TERT fusion nucleic acid, such as a DNA, cDNA, or RNA (primary transcript or mRNA), is used. In some embodiments, the NTRK2-TERT ius on nucleic acid is isolated. In some embodiments of the methods and uses discussed above concerning thyroid cancer, a recombinant vector comprising an NTRK2-TERT fusion, such as an NTRK2-TERT fusion nucleic acid, such as one or more of the NTRK2-TERT fusion nucleic acids listed above, is used. For example, the, or polypeptide can be used in a determining step as discussed above, e.g., as a reagent or control, or in the case of vectors and polynucleotides, as a source of reagents or controls, e.g., as starting material for production of reagents or controls. In some embodiments of the methods and uses discussed above concerning thyroid cancer, a host cell, such as a human, non-human, and/or immortalized host cell, comprising an NTRK2-TERT fusion, such as an NTRK2- TERTfusion nucleic acid, such as one or more of the NTRK2-TERT fusion nucleic acids listed above, e.g., on an extrachromosomal element such as a recombinant vector, or ectopically integrated into a chromosome of the host cell, or on an artificial chromosome, such as a bacterial artificial chromosome or yeast artificial chromosome, is used. For example, the host cell can be used as a source of nucleic acid and/or polypeptide for use in a determining step as discussed above, e.g., as a reagent or control.

[01 1 1] In some embodiments, the NTRK2-TERT ius\on is an in-frame fusion. Thus, in an NTRK2-TERT fusion mRNA or cDNA, codons from TERT can be in the same reading frame as codons from NTRK2. An unspliced NTRK2- TERT fusion nucleic acid can comprise an exon-intron structure that can be spliced to produce an NTRK2-TERT fusion nucleic acid with codons from TERT^'m the same reading frame as codons from NTRK2.

[01 12] In some embodiments, the NTRK2-TERT fusion nucleic acid is unspliced and comprises a junction point between NTRK2 and TERT intronic or untranslated region subsequences. For example, upon transcription and mRNA splicing, the 3'-terminal nucleotide of an exon of NTRK2 is joined to the 5'-terminal nucleotide of an exon of TERT or the TERT start codon. As a further example, the unspliced NTRK2-TERT fusion nucleic acid can comprise a junction point between a subsequence of the NTRK2 n\ron following NTRK2 exon 8 and a subsequence of the region 10-15 kb upstream of TERT exon 1 in gDNA, e.g., 1 1.5-13 kb upstream, 11 -13 kb upstream, or 1 1.5-12.5 kb upstream, which can be upstream of the TERT transcription start site. As a further example, the spliced NTRK2-TERT fusion nucleic acid can comprise a junction point between the 3'-terminal nucleotide of NTRK2 exon 8 and the 5'-terminal nucleotide of TERT exon 3. As discussed previously, sequences that function as exons in an unfused gene may become intronic in a fusion, e.g., an unspliced NTRK2-TERT us\on nucleic acid with a junction point between a subsequence of the intron following NTRK2 exon 8 and a subsequence of the TERT5' UTR can be spliced into an mRNA with a junction point between the 3'-terminal nucleotide of NTRK2 exon 8 and the 5'-terminal nucleotide of TERT exon 3. Thus, as a result of the splicing of the fusion primary transcript, TERT exons 1 and 2 may be omitted from the fusion mRNA. In some embodiments, the NTRK2-TERT fusion nucleic acid is an mRNA or cDNA comprising exons 3-16 of TERT. In some embodiments, the NTRK2-TERT fusion nucleic acid is an mRNA or cDNA comprising exons 1 -16 of TERT. In some embodiments, the NTRK2-TERT fusion nucleic acid is an mRNA or cDNA comprising a sequence encoding a functional RNA-binding telomerase ribonucleoprotein complex domain, which can be a fragment of the wild-type domain, e.g., a fragment comprising amino acids 326-613 of wild-type hTERT, which was reported to bind telomerase RNA in Lai et al., Mol. Cell. Biol. 21 , 990-1000 (2001 ) (doi: 10.1 128/MCB.21.4.990-1000.2001 ) (see, e.g., Fig. 5 therein).

[01 13] For exon designations for human TERT, see, e.g., the exon information under entry NX_014746 in the neXtprot™ database or RefSeq/GenBank accession nos. NM_198253.2 or NM_001 193376.1 , each of which is incorporated herein by reference. For exon designations for NTRK2, see, e.g., the exon information under entry NX_Q16620 in the neXtprot™ database or RefSeq/GenBank accession nos. N _001018066.2, NM_001.018065.2, NM_001018064.1 , NM_006180.3, or

NM_001007097.1 , each of which is incorporated herein by reference. Exon designations are similarly available for TERT and NTRK2 in other species, including other mammalian species. Where multiple possible transcripts exist, as for TERT, a reference to a specific exon encompasses that exon of each variant unless otherwise indicated. [01 14] In some embodiments, the NTRK2-TERT fusion nucleic acid comprises exons 1 -8 of NTRK2. In some embodiments, an NTRK2-TERT fusion nucleic acid comprises exons 3-16 of TERT.

[01 15] In some embodiments, the NTRK2-TERT fusion nucleic acid comprises a sequence spanning the

point chosen from

CCCAATTGTGGGGTTGGC, CCAATTGTGGGGTTGG, CAATTGTGGGGTTG, AATTGTGGGGTT, ATTGTGGGGT, TTGTGGGG, TGTGGG,

CCCAAUUGUGGGGUUGGC, CCAAUUGUGGGGUUGG, CAAUUGUGGGGUUG, AAUUGUGGGGUU, AUUGUGGGGU, UUGUGGGG, or UGUGGG.

[01 16] In some embodiments, the NTRK2- TERT fusion nucleic acid comprises a sequence 100%, 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 1. In some embodiments, the NTRK2-TERT^'fusion nucleic acid comprises a sequence 100%, 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 3 (the TERT sequence portion of SEQ ID NO: 1) and/or a sequence 100%, 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 2 (the NTRK2 sequence portion of SEQ ID NO: 1 ).

[01 17] In some embodiments, the NTRK2- TERT fusion nucleic acid is amplified from a biological sample, isolated from a biological sample, contained in a biological sample, a product of a primer extension reaction wherein the template for the extension reaction was isolated or derived (e.g., by a process comprising reverse transcription or cloning) from a biological sample. The biological sample may be any of the samples discussed previously.

[01 18] In some embodiments of the methods and uses discussed above concerning thyroid cancer, a primer pair comprising a first primer specific for NTRK2 and a second primer specific for TERTis used. In some embodiments, the primer pair can be used to amplify an NTRK2-TERT fusion, and/or the primer pair can be used for primer extension assays for NTRK2 and TERT. In some embodiments, the NTRK2 primer anneals to exon 1 , 2, 3, 4, 5, 6, 7, or 8 of NTRK2. In some embodiments, the TERT primer anneals to one of exons 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 of TERT. In some embodiments, the NTRK2 primer is a forward primer, meaning that it has the same sense as the NTRK2 coding strand. An NTRK2 forward primer can anneal to the non-coding strand and prime synthesis of a coding strand sequence or subsequence. In some embodiments, the TERT primer is a reverse primer, meaning that it has the same sense as the TERT non- coding strand. A TERT reverse primer can anneal to the TEftTcoding strand and prime synthesis of a non-coding strand sequence or subsequence. In some embodiments, one or both of the primers is labeled. The label may be any form of detectable label, e.g., a fluorescent label (e.g., FITC, FAM, HEX, etc.), radioactive label (e.g.,³²P,³H,¹ C, etc.), or affinity label (e.g., biotin, digoxygenin, etc.). In some embodiments, a primer is attached to a solid phase, such as a bead or surface. An exemplary primer pair is 5'-CTGCCTGAATGAAAGCAGCA-3' (forward primer, anneals to NTRK2 non-coding strand) and 5'-CAGGATCTCCTCACGCAGAC-3' (reverse primer, anneals to TEHTcoding strand). In some embodiments of the methods and uses discussed above concerning thyroid cancer, a primer set consisting of one or more primers specific for NTRK2 and one or more primers specific for TERT, which can be used to amplify one or more NTRK2- TERT fusions, is used.

[01 19] In some embodiments of the methods and uses discussed above concerning thyroid cancer, a composition comprising a primer pair comprising a first primer specific for NTRK2 and a second primer specific for TERT, such as the primers discussed above, is used. The primers can be synthetic oligonucleotides. One or both of the primers may be labeled as discussed above. A primer may be attached to a solid phase, such as a bead or surface. The composition can be an amplification mixture, e.g., comprising a polymerase (e.g,. a DNA polymerase, such as a thermostable DNA polymerase) and other amplification reagents, such as NTPs or dNTPs, magnesium ions, monovalent cations and anions, and buffer. The composition can further comprise an NTRK2-TERT fusion nucleic acid. In some embodiments, the composition comprises a complex of a primer annealed to an NTRK2-TERT fusion nucleic acid, such as an NTRK2 forward primer annealed to the non-coding strand of the NTRK2-TERT fusion nucleic acid and/or a TERT reverse primer annealed to the coding strand of the NTRK2-TERT fusion nucleic acid. In some embodiments, the composition comprises a complex of an extension product annealed to an NTRK2-TERT fusion nucleic acid, such as an extension product primed by an NTRK2 forward primer annealed to the non-coding strand of the NTRK2-TERT fusion nucleic acid (which may itself be an extension product, such as from an earlier round of amplification, or the starting material for the amplification) and/or an extension product primed by a TERT reverse primer annealed to the coding strand of the NTRK2-TERT fusion nucleic acid (which may itself be an extension product, such as from an earlier round of amplification, or the starting material for the amplification). In some embodiments, the primer pair or amplification mixture is contained in a reaction vessel, such as a tube (e.g., a plastic tube having a volume ranging from 0.09 ml to 2.2 ml) or microwell plate (e.g., a 96-well plate or 384-well plate).

[0120] In some embodiments of the methods and uses discussed above concerning thyroid cancer, a sequencing reaction mixture comprising a primer, such as an NTRK2 primer or a TERT primer as discussed above, is used. The sequencing reaction mixture can further comprise an NTRK2-TERT fusion nucleic acid and/or sequencing reagents such as a polymerase (e.g., DNA polymerase), dNTPs, magnesium ions, monovalent cations and anions, and buffer. In some embodiments, the dNTPs comprise one or more labelled dNTPs. In some embodiments, the primer is labeled as discussed above. In some embodiments, the sequencing reaction mixture comprises at least one ddNTP. In some embodiments, the sequencing reaction mixture is contained in a reaction vessel, such as a tube (e.g., a plastic tube having a volume ranging from 0.09 ml to 2.2 ml) or microwell plate (e.g., a 96-well plate or 384-well plate).

[0121 ] In some embodiments of the methods and uses discussed above concerning thyroid cancer, a junction probe that anneals to an NTRK2-TEHT fusion nucleic acid at a location comprising the junction between sequence from NTRK2 and sequence from TERT s used. The junction probe can anneal to the coding or non-coding strand. In some embodiments, the junction probe comprises a sequence chosen from CCCAATTGTGGGGTTGGC, CCAATTGTGGGGTTGG,

CAATTGTGGGGTTG, AATTGTGGGGTT, ATTGTGGGGT, TTGTGGGG, TGTGGG, CCCAAUUGUGGGGUUGGC, CCAAUUGUGGGGUUGG, CAAUUGUGGGGUUG, AAUUGUGGGGUU, AUUGUGGGGU, UUGUGGGG, or UGUGGG. In some embodiments, the junction probe comprises a sequence chosen from the reverse complement of CCCAATTGTGGGGTTGGC, CCAATTGTGGGGTTGG,

CAATTGTGGGGTTG, AATTGTGGGGTT, ATTGTGGGGT, TTGTGGGG, TGTGGG, CCCAAUUGUGGGGUUGGC, CCAAUUGUGGGGUUGG, CAAUUGUGGGGUUG, AAUUGUGGGGUU, AUUGUGGGGU, UUGUGGGG, or UGUGGG. In some embodiments, the junction probe is in solution, attached to a solid phase (e.g., a bead or surface), and/or hybridized to an NTRK2-TERT fusion nucleic acid. The junction probe can be labeled, e.g., chemically modified, with any form of detectable label, e.g., a fluorescent label (e.g., FITC, FAM, HEX, etc.), radioactive label (e.g.,³²P,³H,¹⁴C, etc.), enzymatic label (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, etc.), or affinity label (e.g., biotin, digoxygenin, etc.), or an equivalent.

EXAMPLE

[0122] RNA Isolation. A formalin fixed, paraffin embedded block of a follicular thyroid carcinoma and associated clinicopathological information were obtained from Asterand (Detroit, Ml). One hematoxylin- and eosin-stained slide was prepared and reviewed by an independent pathologist at Asuragen to confirm the histologic diagnosis. Five 10 micron sections were then cut for nucleic acids isolation. Total nucleic acids (TNA) were extracted using the Ambion RecoverAII Total Nucleic Acid isolation Kit for FFPE tissues (cat#AM1975; Life Technologies, Carlsbad, CA) according to the recommended protocol. Briefly, sections were deparaffinized with Xylene and digested with Proteinase K. TNA isolation was followed by a DNAse treatment according to protocol. RNA was then purified and eluted in H₂0. RNA concentration was determined using a NanoDrop ND1000 (NanoDrop Technologies, Waltham, MA).

[0 23] RNA-Seq Library Preparation and Sequencing. 100 to 500 ng of total RNA was depleted of rRNA using the RiboZero rRNA Removal Kit (Epicentre), following the manufacturer's protocol. rRNA depleted RNA was fragmented through hydrolysis by heat incubation with metal ions. First strand cDNA synthesis was primed with random hexamers and second strand synthesis was performed with dUTP incorporation. Following. end repair, cDNA was A-tailed and lllumina sequencing adapters were ligated. Adapter ligated cDNA was size selected with AMPure beads and treated with uracil-DNA glycosolase (UDG) to enable strand specificity. Libraries were enriched by PCR, assessed by KAPA quantification and pooled in equimolar ratios. Pooled libraries were sequenced on an lllumina HiSeq 2000 with 2x50 paired-end reads to a coverage depth of 50 million reads per sample.

[0124] RNA-Seq Data Analysis. Raw sequence reads were aligned to the human reference sequence (hg19, GRCh37) using Bowtie. Langmead, B., Trapnell, C, Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome biology 10, R25 (2009). Candidate gene fusions were identified by identifying read pairs that mapped discordantly (on two separate chromosomes or to distant regions on the same chromosome).

Breakpoint spanning reads were identified by aligning to breakpoints implicated by discordant read pairs. Fusions supported by breakpoint spanning reads were considered as candidates for confirmation by Sanger sequencing.

[0125] Identification and characterization of the NTRK2 TERT fusion. The whole transcriptome RNA-Seq of a formalin fixed paraffin embedded (FFPE) follicular thyroid carcinoma revealed a novel rearrangement involving TERT and associated with its overexpression. The RNA-Seq data revealed an in-frame fusion between neurotrophic tyrosine kinase receptor, NTRK2, and TERT (Figure 1 ). A fusion between exon 8 of NTRK2 and exon 3 of TERT was supported by RNA-Seq reads aligning to the predicted breakpoint sequence and confirmed by Sanger sequencing. Coverage of nascent transcription revealed the DNA breakpoint for TERT to be upstream of exons 1 and 2. However, these exons were excised from the mature fusion transcript. Levels of TERT expression estimated by RNA-Seq for exons 3 through 16 was found to be equivalent to the expression of exons 1 -8 of NTRK2, consistent with a model in which expression of these exons is under the control of the NTRK2 promoter. Relative to benign FFPE samples also profiled by RNA-Seq, TERT expression levels were upregulated over 100 fold, thus confirming a novel gene-fusion mechanism for ectopic expression of TERT^'m thyroid carcinomas.

[0126] Fusion Validation by PCR and Sanger Sequencing. The

' NTRK2ITERT gene fusion was confirmed by PCR and Sanger sequencing. The PCR used a target specific primer pair (Forward primer:

CTGCCTGAATGAAAGCAGCA; Reverse primer: CAGGATCTCCTCACGCAGAC) and template from the sample identified as positive for the fusion by RNA-Seq. A no template control (NTC) was included to confirm absence of contaminants (data not shown). An aliquot of the amplified PCR products and NTC were run on 2.2% Lonza Flashgel™& 50bp - 1.5kb Flashgel™ DNA marker to confirm the presence and the size of the PCR products as well as to determine the presence of non-specific PCR products. (Figure 2.) Bi-directional Sanger sequencing of the PCR products was performed (ACGT, Inc). DNA sequence was analyzed using ABI's Sanger

Sequencing Software v1 .0.

[0127] Discussion. The chimeric NTRK2-TERT polypeptide observed in this Example is likely capable of telomere elongation as its predicted peptide sequence retains the entire reverse transcriptase domain of Tert and 50% of the RNA-binding telomerase ribonucleoprotein complex domain. The fusion polypeptide also contains two leucine rich N-terminal domains of Ntrk2 which may facilitate protein-protein interactions between partners of Ntrk2 and the chimeric polypeptide product of NTRK2-TERT. The two Ntrk2 Immunoglobulin l-set domains are notably absent from the fusion polypeptide suggesting a loss of function with respect to cell-cell adhesion. The Ntrk2 C-terminal tyrosine kinase domain is also absent which could lead to potential dysregulation of MAPK and PI3K signaling pathways, both of which are key driver pathways of thyroid carcinogenesis.

* *_*

[0128] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. The citation of any references herein is not an admission that such references are prior art to the present invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0129] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. The recitation of series of numbers with differing amounts of significant digits in the specification is not to be construed as implying that numbers with fewer significant digits given have the same precision as numbers with more significant digits given. [0130] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0131 ] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.^' Those skilled in the art will recognize, or be able to ascertain using no more than routine

experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0132] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[01 33] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of characterizing the genotype of a thyroid cell, comprising determining the presence of an NTRK2-TERT fusion in the thyroid cell.

2. The method of claim 1 , wherein the thyroid cell is a malignant cell.

3. The method of claim 1 , wherein the thyroid cell is a premalignant cell.

4. A method of characterizing a biological sample obtained from a thyroid gland or a subject having thyroid cancer, comprising determining the presence or absence of an NTRK2-TERT fusion in the biological sample obtained from a thyroid gland or a subject having thyroid cancer.

5. The method of claim 4, wherein the biological sample obtained from a thyroid gland or a subject having thyroid cancer comprises at least one isolated cell.

6. The method of claim 4, wherein the biological sample obtained from a thyroid gland or a subject having thyroid cancer comprises isolated nucleic acid.

7. The method of claim 4, wherein the biological sample obtained from a thyroid gland or a subject having thyroid cancer comprises isolated polypeptide.

8. A method of distinguishing non-malignant thyroid cells from malignant thyroid cells, comprising determining the presence of an

in thyroid cells.

9. A method of stratifying a thyroid cancer, comprising determining the presence of an NTRK2-TERT fusion in one or more cells of the thyroid cancer.

10. The method of claim 9, wherein the stratifying comprises assigning a degree of progression or aggressiveness to the thyroid cancer.

11. The method of claim 9, further comprising administering or pursuing a more aggressive treatment if the NTRK2-TERT 1us\on is present.

12. A method of treating thyroid cancer, comprising pursuing a more aggressive treatment for thyroid cancer in a patient if an NTRK2-TERT fusion is determined to be present, or pursuing a less aggressive treatment if the NTRK2-TERT fusion is determined to be absent in the patient.

13. The method of claim 12, wherein the more aggressive treatment comprises thyroid resection, optionally radioactive iodine administration, and an additional intervention, and the less aggressive treatment comprises thyroid resection and optionally radioactive iodine administration without the additional intervention.

14. The method of claim 13, wherein the additional intervention comprises administration of at least one anti-cancer drug other than radioactive iodine.

15. The method of claim 12, wherein the more aggressive treatment comprises whole thyroid removal and the less aggressive treatment comprises

hemithyroidectomy.

16. The method of claim 12, wherein the more aggressive treatment comprises immunotherapy or chemotherapy and the less aggressive treatment does not comprise immunotherapy, or chemotherapy.

17. The method of any of claims 1 1 -16, wherein the more aggressive treatment comprises a telomerase-directed therapy.

18. The method of claim 12, wherein the more aggressive treatment comprises inhibiting telomerase activity and the less aggressive treatment does not comprise inhibiting telomerase activity.

19. The method of claim 18, wherein inhibiting telomerase activity comprises administering at least one reverse transcriptase inhibitor.

20. The method of claim 18, wherein inhibiting telomerase activity comprises administering Lamivudine, abacavir, zidovudine, emtricitabine, or tenofovir.

21. The method of claim 18, wherein inhibiting telomerase activity comprises administering GRN163L or GRNVAC1 .

22. A method of treating or monitoring thyroid cancer, comprising pursuing a treatment for thyroid cancer in a patient if an NTRK2-TERT fusion is determined to be present, or monitoring the thyroid cancer without treatment if the NTRK2-TERT fusion is determined to be absent in the patient.

23. A method of monitoring tumor burden in a thyroid cancer patient, comprising determining the level of an NTRK2-TERT fusion in a sample from the thyroid cancer patient.

24. A method of monitoring a response to treatment of thyroid cancer in a thyroid cancer patient, comprising determining the level of an NTRK2-TERT fusion in a sample from the thyroid cancer patient.

25. The method of any one of the preceding claims, wherein the determining step comprises an assay for the NTRK2-TERT fusion chosen from a sequencing, hybridization, amplification, affinity, antibody binding, proteomic, or nanostring assay.

26. The method of any one of the preceding claims, wherein the determining step comprises contacting a sample or separate aliquots of a sample with (i) a first primer that anneals to wild-type TERT but not the NTRK2-TERT fusion and (ii) a second primer that anneals to both wild-type TERT and the NTRK2-TERT fusion and determining extension of at least one of the primers.

27. The method of any one of the preceding claims, wherein the determining step is performed on a thyroid sample chosen from a fine needle aspirate of thyroid tissue, a formalin-fixed paraffin-embedded thyroid tissue sample, a thyroid biopsy, isolated thyroid cells, or biomolecules isolated from thyroid cells.

28. The method of any one of the preceding claims, wherein the determining step is performed on a biological sample chosen from a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, or a buccal swab sample.

29. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion is an in-frame fusion.

30. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion comprises an intronic junction point.

31. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion comprises exons 3 through 16 of TERT as an mRNA.

32. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion comprises exons 1 through 16 of TERT as a primary transcript.

33. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion comprises exons 1 through 8 of NTRK2 as an mRNA.

34. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion does not comprise exons 9 through 20 of NTRK2 as an mRNA.

35. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion is a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide shows telomere elongation activity in a telomere elongation assay.

36. The method of any one of the preceding claims, wherein cells having the NTRK2-TERT fusion have a telomerase upregulation phenotype.

37. The method of any one of the preceding claims, wherein the determining step is performed on a sample from a subject having follicular thyroid cancer.

38. The method of any one of the preceding claims, wherein the determining step determines the presence or absence of the NTRK2-TERT fusion in genomic DNA.

39. The method of any one of the preceding claims, wherein the determining step determines the presence or absence of the NTRK2-TERT fusion in mRNA.

40. The method of any one of the preceding claims, wherein the determining step determines the presence or absence of an NTRK2-TERT fusion polypeptide.

41. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion, as an mRNA, has a sequence comprising a G as the most 3' base from NTRK2 and a G as the most 5' base from TERT.

42. The method of any one of the preceding claims, wherein the NTRK2-TERT fusion, as an mRNA, has a sequence comprising AUUGUGGGGUUG from -6 to +6 relative to the fusion junction point.