JP7602238B2 - Probe set for detecting TERT promoter mutations - Google Patents

Description

The present invention relates to a probe set for detecting TERT promoter mutations and a method for detecting TERT promoter mutations using the same.

The incidence of papillary thyroid cancer (PTC) is increasing worldwide. Although PTC generally has a good prognosis, approximately 10-15% of patients experience recurrence in local lesions or distant organs, some of which are refractory to treatment (e.g., Non-Patent Document 1). Therefore, to assess the risk of recurrence, active research is being conducted on biomarkers to predict the aggressiveness and prognosis of PTC.

In recent years, mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene have been found in many types of cancer, including thyroid cancer. Two mutually exclusive hotspots, called C250T (chr5: 1,295,250C>T) and C228T (chr5: 1,295,228C>T), are known. In PTC, many studies have reported that the coexistence of BRAF ^V600E mutations and TERT promoter mutations is strongly associated with aggressive characteristics and poor prognosis (e.g., Non-Patent Document 2). Furthermore, TERT promoter mutations may also be associated with neoplastic transformation (Non-Patent Document 3). Therefore, TERT promoter mutations are one of the most important molecular markers in PTC. To detect such TERT promoter mutations, the Sanger method and PCR using a probe are used.

Ito Y. et al., World J Surg., 42(3):615-622 (2018) Matsuse M. et al., Sci Rep., 7:41752, (2017) Oishi N., et al., Mod Pathol., 30(11):1527-1537 (2017)

However, when detecting mutations in the TERT promoter, the Sanger sequencing method cannot detect mutations in samples with trace amounts or low allele frequencies, and in PCR methods using probes, it is usually necessary to use different probes to distinguish between two mutations. Furthermore, even with probe-based methods, there are problems such as difficulty in detection in samples with trace amounts or low allele frequencies, and insufficient sensitivity, making them practically unusable for clinical cancer diagnosis. Therefore, the objective of the present invention is to provide a probe that can distinguish between two mutations in a single assay and can detect even very trace amounts of samples or mutations with low allele frequencies.

The present inventors have been developing a probe capable of detecting two mutations in the TERT promoter at once, and as a result of extensive research, have discovered that the use of a probe having a specific sequence allows the above-mentioned two mutations to be detected with high sensitivity. Based on these findings, the present inventors have conducted further research, and as a result have completed the present invention.

That is, the present invention is as follows.
[1] A probe set for detecting TERT promoter mutations, comprising the following two types of probes:
(1) A probe consisting of a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence complementary to said sequence, and (2) a probe consisting of a nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary to said sequence. [2] The probe set according to [1], characterized in that a labeling substance is bound to each of the above probes.
[3] The probe set according to [2], wherein the labeling substance is a fluorescent dye, and a quencher is bound to each of the probes.
[4] The probe set according to any one of [1] to [3], wherein each probe comprises one or more modifications of the sugar-phosphate backbone.
[5] The probe set according to [4], wherein 40% or more of the nucleotide residues in each probe are modified.
[6] The probe set according to [4] or [5], wherein the modification is a bridge between the oxygen atom at the 2' position of the sugar and the carbon atom at the 4' position via a methylene.
[7] A method for detecting a mutation in a TERT promoter, comprising the following steps (1) to (4):
(1) contacting the probe set according to any one of [3] to [6] with a nucleic acid containing a target region of a TERT promoter;
(2) amplifying a region including the target region by PCR;
(3) measuring the fluorescence intensity of a solution containing the PCR product amplified in the step (2); and (4) detecting a mutation in the TERT promoter based on the measurement result of the step (3). [8] The method according to [7], wherein the PCR is digital PCR.
[9] A kit for detecting a TERT promoter mutation, comprising the probe set according to any one of [1] to [6], and two types of PCR primer sets capable of amplifying a target region of the TERT promoter.
[10] One of the primers is
(1) a nucleotide sequence represented by SEQ ID NO:5; or (2) a sequence in which one or more nucleotides are deleted, substituted or added in the sequence of (1), and the other of the primers is
(3) A nucleotide sequence represented by SEQ ID NO: 6; or (4) a sequence in which one or more nucleotides have been deleted, substituted, or added in the sequence of (3).

The probe of the present invention makes it possible to distinguish between two mutations (C250T and C228T) in the TERT promoter in a single assay, and also makes it possible to detect and identify even very small amounts of samples or mutations with low allele frequency.

1 is a schematic diagram of the principle of the primer of the present invention. When there is a mutation at position 250, the FAM probe binds to position 250 and its vicinity, and the Hex probe binds to position 228 and its vicinity. When there is a mutation at position 228, the FAM probe binds to position 228 and its vicinity, and in the case of Wt, the HEX probe binds to position 228 and its vicinity, allowing two types of probes to detect mutations at two locations. 2 shows the results of two-dimensional plotting using the probe of the present invention. When the C228T mutation is present, each of mut and wt appears as a single positive droplet. When the C250T mutation is present, each appears as a double positive droplet. Figure 3 shows the results of the detection limit of ddPCR. The detection limit of ddPCR was determined using serial dilutions of PCR products of TERT promoter mutations (C228T or C250T) in wild-type PCR products. The TERT promoter region (163 bp) was amplified and purified using DNA extracted from cell lines with only wild-type, C228T, and C250T. A total of 3,000 copies of a mixture of mutant and wild-type products were prepared (0%, 0.125%, 0.25%, and 0.5%), and mutant copy numbers were measured using ddPCR. The results were compared with those using 100% wild-type products, and the differences were analyzed by Mann-Whitney U test using GraphPad Prism software. Significant differences were only observed between samples with ≥0.25% mutant alleles (both C228T and C250T) and wild-type samples. Figure 4 shows the low allele frequency of TERT promoter mutation in three samples (PTC A, PTC B, and PTC C). (A) Droplet digital PCR results. Each dot represents a positive droplet of mutant or wild-type allele. NTC: no template control, Wt: wild-type control, hetMut: both mutant and wild-type controls (heterozygous). (B) Sanger sequencing chromatograms. The hotspots of TERT promoter mutations are indicated by arrows. PTC A, PTC B, and PTC C all harbored the C228T mutation. Figure 5 shows an overview of the TERT mutation and expression status in the present cases. (A) Flow chart of TERT mutation/expression screening. (B) Pie chart of results. (C) Relative TERT expression levels in each group classified with the above status. (D) Correlation between TERT expression levels and patient age. Mutation-negative/expression-positive cases are plotted. Solid line represents linear regression model with 95% confidence interval shown in dotted line. Figure 6 shows Kaplan-Meier curves of different classifications based on TERT mutation/expression status. The vertical scale corresponds to censored data. The p-values of the log-rank test are displayed. The cases were separated based on mutation status. Figure 7 shows Kaplan-Meier curves of recurrence-free survival. The vertical scale corresponds to censored data. The p-values of the log-rank trend test are displayed. FIG. 8 shows the results of sensitivity, specificity, positive predictive value, and negative predictive value in mutation detection using the probe of the present invention when a fine needle aspiration cytological washing fluid sample is used.

1. Probe set for detecting TERT promoter mutation The present invention provides a probe set for detecting a mutation in a TERT promoter (hereinafter sometimes referred to as the "probe set of the present invention"), which includes a probe for detecting a mutant sequence (also referred to as a "mutant probe" or "mut probe") and a probe for detecting a wild-type sequence (also referred to as a "wild-type probe" or "wt probe"). Hereinafter, the mutant probe and the wild-type probe may be collectively referred to as the "probe of the present invention".

The mutant probe consists of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence complementary to said sequence. This probe can detect either of two hot spots called C250T (chr5: 1,295,250C>T) (this site is also simply called "position 250") and C228T (chr5: 1,295,228C>T) (this site is also simply called "position 228"). The above probe hybridizes to the region of chr5: 1,295,246 to chr5: 1,295,255 and the region of chr5: 1,295,223 to chr5: 1,295,232. On the other hand, the wild-type probe consists of the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary to said sequence. This probe hybridizes to the region chr5:1,295,246 to chr5:1,295,255 only when the region is wild-type. In this specification, unless otherwise specified, the nucleotide sequence and position are described based on the sequence of the human TERT promoter region, but the corresponding nucleotide sequence and position in the ortholog of a non-human mammal (e.g., rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey) are also included in the description.

As used herein, detection of a TERT promoter mutation means detecting whether or not the TERT promoter region in the nucleic acid of the subject has a specific mutation, i.e., detecting whether the specific mutation is wild-type or mutant, and, if it is mutant, detecting or identifying the type of mutation (i.e., whether it is C250T or C228T).

The probe of the present invention may be bound to a labeling substance. The labeling substance is not particularly limited, and may be, for example, a fluorescent dye, an isotope, etc., with a fluorescent dye being preferred. Various types of such fluorescent dyes are commercially available, and examples of the fluorescent dye include 6-FAM (fluorescein), HEX, TE, Quasar 670, Quasar 570, Quasar 705, Pulsar 650, TET, HEX, VIC, JOE, CAL Fluor Orange, CAL Fluor Gold, CAL Fluor Red, Texas Red, Cy, and Cy5. Examples of the isotope include stable isotopes and radioisotopes, and stable isotopes are preferred. Examples of the stable isotope include ² H, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³³ S, ³⁴ S, and ³⁶ S.

When a fluorescent dye is bound to the probe of the present invention, it is preferable that the probe is further bound to a quencher capable of quenching the fluorescence from the fluorescent substance. In addition, it is preferable that the fluorescent dye bound to the mutant probe and the fluorescent dye bound to the wild-type probe are different. With this configuration, when a nucleic acid molecule having a mutation at position 250 of the TERT promoter is used, the mutant probe hybridizes to position 250 and its vicinity, and the wild-type probe hybridizes to position 228 and its vicinity, and two types of fluorescence derived from the fluorescent dye of each probe are detected. On the other hand, when a nucleic acid molecule having a mutation at position 228 is used, the mutant probe hybridizes to position 228 and its vicinity, and the wild-type probe cannot hybridize, so only the fluorescence derived from the fluorescent dye of the mutant probe is detected. In addition, when a nucleic acid molecule having a wild-type TERT promoter is used, the wild-type probe hybridizes to position 228 and its vicinity, and the mutant probe cannot hybridize, so only the fluorescence derived from the fluorescent dye of the wild-type probe is detected. This schematic diagram is shown in Figure 1.

Various quenchers are commercially available for use in the present invention, and are not particularly limited as long as they can quench the fluorescence from the fluorescent dye. As the quencher, either a fluorescent dye or a non-fluorescent dye may be used, but from the viewpoint of detection accuracy, a non-fluorescent dye is preferable. Specific examples of quenchers include 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), Eclipse Dark Quencher, minor groove binder (MGB), and non-fluorescent quencher (NFQ).

Examples of the constituent units of the probe of the present invention include ribonucleotides and deoxyribonucleotides. The nucleotide residues contain sugars, bases, and phosphates as components. Ribonucleotides have ribose residues as sugars and adenine (A), guanine (G), cytosine (C), and uracil (U) (which can also be replaced with thymine (T)) as bases, while deoxyribonucleotide residues have deoxyribose residues as sugars and adenine (dA), guanine (dG), cytosine (dC), and thymine (dT) (which can also be replaced with uracil (dU)) as bases.

These nucleotides may be modified (modified nucleotide residues may be referred to as "modified nucleotide residues") or unmodified (unmodified nucleotide residues may be referred to as "unmodified nucleotide residues"). By including modified nucleotide residues, nuclease resistance may be improved, and as a result, stability may be improved. Furthermore, by including modified nucleotide residues, binding affinity with target DNA may be improved. When modified nucleotide residues are included, the proportion of modified nucleotide residues in the probe of the present invention is not particularly limited, but it is preferable that at least one-third (e.g., 40%, 50%, 60%, 70%, 80%, 90% or 100%) of all nucleotide residues are modified nucleotide residues. Furthermore, the probe of the present invention may include a linker region composed of molecules different from unmodified nucleotide residues and modified nucleotide residues. Furthermore, the position of the modified nucleotide in the probe is not particularly limited. In a preferred embodiment, at least the nucleotide residues at positions 2, 4 to 6, and 8 of each probe of the present invention are modified, but the modifications are not limited to these positions.

The modified nucleotide residue may be, for example, any of the components of the unmodified nucleotide residue modified. In the present invention, "modification" includes, for example, substitution, addition, and/or deletion of the components, and substitution, addition, and/or deletion of atoms and/or functional groups in the components. The modified nucleotide residue includes, for example, naturally occurring nucleotide residues and artificially modified nucleotide residues. For the naturally occurring modified nucleotide residues, see, for example, Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196). In addition, the modified nucleotide residue includes, for example, a residue that is a substitute for a nucleotide.

The modification of the nucleotide residue can be, for example, a modification of the sugar-phosphate backbone (which backbone also includes a base) (hereinafter, the sugar-phosphate backbone). In the sugar-phosphate backbone, when the sugar is ribose, for example, the ribose residue can be modified. For example, the 2' carbon of the ribose residue can be modified, specifically, for example, the hydroxyl group attached to the 2' carbon can be modified with a methyl group, or the hydroxyl group can be substituted with hydrogen or a halogen such as fluoro. In addition, the ribose residue can be substituted with deoxyribose by substituting the hydroxyl group of the 2' carbon with hydrogen. The ribose residue can be substituted with, for example, a stereoisomer, for example, an arabinose residue. Hereinafter, a nucleic acid in which the hydroxyl group attached to the 2' carbon of the sugar is modified with a methyl group as described above may be referred to as a 2'-O-methyl modified nucleic acid. In addition, in the present invention, "nucleic acid" includes nucleic acid monomers such as nucleotides.

The sugar phosphate backbone may be replaced with, for example, a non-ribose phosphate backbone having a non-ribose residue (including non-deoxyribose residues) and/or a non-phosphate, and such replacement is also included in the modification of the sugar phosphate backbone. The non-ribose phosphate backbone may be, for example, an uncharged form of the sugar phosphate backbone. The nucleotide substitute replaced with the non-ribose phosphate backbone may be, for example, morpholino, cyclobutyl, pyrrolidine, etc. Other examples of the substitute include artificial nucleic acids. Specific examples include PNA (peptide nucleic acid) and bridged artificial nucleic acid (BNA: Bridged Nucleic Acid). Examples of BNA include locked artificial nucleic acid (LNA) (also called 2'-O, 4'-C-methylene bridged nucleic acid) and 2'-O, 4'-C-ethylene bridged nucleic acid (ENA: 2'-O, 4'-C-Ethylene bridged Nucleic Acid), with LNA being preferred. In a preferred embodiment, each probe of the present invention has at least LNA at nucleotide residues 2, 4 to 6, and 8.

2. Method for detecting TERT promoter mutation using the probe of the present invention In another aspect, the present invention provides a method for detecting a TERT promoter mutation using the probe set of the present invention described in 1 above (hereinafter, sometimes referred to as the "detection method of the present invention"). The method for detecting or measuring the mutation is not particularly limited, and may be, for example, a method using a PCR method. In addition, in this specification, the method for detecting a TERT promoter mutation also includes a method for assisting in the detection of a TERT promoter mutation.

Methods using such PCR include a method in which a target nucleic acid is amplified by PCR and mutations in the amplified product are detected by fluorescence or luminescence, PCR-RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2766-2770 (1989) etc.), PCR-SSO (specific sequence oligonucleotide) method, ASO (allele specific oligonucleotide) hybridization method which combines PCR-SSO method and dot hybridization method (Saiki, Nature, 324, 163-166 (1986) etc.), TaqMan (registered trademark, Roche Molecular Systems)-PCR method (Livak, KJ, Genet Anal,14,143(1999), Morris, T. et al., J. Clin. Microbiol.,34,2933(1996)) and digital PCR (e.g. droplet digital PCR, etc.).

Other methods include the cycling probe method, the Invader (registered trademark, Third Wave Technologies) method (Lyamichev V et al., Nat Biotechnol, 17, 292 (1999)), a method using FRET (Fluorescence Resonance Energy Transfer) (Heller, Academic Press Inc, pp. 245-256 (1985), Cardullo et al., Proc. Natl. Acad. Sci. USA, 85, 8790-8794 (1988), International Publication No. 99/28500, JP 2004-121232 A, etc.), a method using a DNA chip or microarray (Wang DG et al., Science 280,1077 (1998) etc.), the Southern blot hybridization method, the dot hybridization method (Southern, E., J. Mol. Biol. 98, 503-517 (1975)) or other known methods can be used.

From the viewpoint of sensitivity, a method using PCR is preferable. Accordingly, in one embodiment, the present invention provides a method for detecting a mutation in a TERT promoter, comprising the following steps (1) to (4):
(1) contacting the probe set of the present invention with a nucleic acid containing a target region of the TERT promoter;
(2) amplifying a region including the target region by PCR;
(3) measuring the fluorescence intensity of a solution containing the PCR product amplified in the step (2); and (4) detecting a mutation in the TERT promoter based on the measurement results of the step (3).

In the step (1), the method of contacting the probe set of the present invention with a nucleic acid (hereinafter also referred to as a "test nucleic acid") containing a target region of the TERT promoter (hereinafter sometimes simply referred to as a "target region") is not particularly limited, but can be carried out, for example, by adding the probe set and the test nucleic acid to the same solution. In addition, the "target region of the TERT promoter" refers to a region to which a mutant probe and/or a wild-type probe can bind, i.e., the region from chr5:1,295,223 to chr5:1,295,255 (shown in SEQ ID NO:9), and the test nucleic acid is not particularly limited as long as it contains the region.

The solution also typically contains a PCR primer and DNA polymerase. The PCR primers include two types of primer sets (i.e., a forward primer and a reverse primer) that have a sequence complementary to a chromosomal region containing a target region and are designed to specifically amplify the region containing the target region. Specific primers include, for example, one of the primers is (A) a nucleotide sequence shown in SEQ ID NO: 5, or (B) a sequence in which one or more (e.g., 2, 3, 4, 5) nucleotides are deleted, substituted, or added in the sequence of (A), and the other primer is (C) a nucleotide sequence shown in SEQ ID NO: 6, or (D) a sequence in which one or more (e.g., 2, 3, 4, 5) nucleotides are deleted, substituted, or added in the sequence of (C), but are not limited to these primers, and known primers can also be used as appropriate. For example, the forward primer may be (E) a primer having a nucleotide sequence shown in any one of SEQ ID NOs: 10 to 13, or (F) a primer having a sequence in which one or more (e.g., 2, 3, 4, 5) nucleotides have been deleted, substituted, or added in the sequence of (E). The reverse primer may be (G) a primer having a nucleotide sequence shown in any one of SEQ ID NOs: 14 to 17, or (H) a primer having a sequence in which one or more (e.g., 2, 3, 4, 5) nucleotides have been deleted, substituted, or added in the sequence of (G). The forward primer and reverse primer may be used in any combination. The DNA polymerase may also be any DNA polymerase used in PCR.

The test nucleic acid can be prepared from the blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair, lesions, etc. of the test mammal by using known extraction and purification methods. For example, when extracting nucleic acid from a sample derived from a lesion (e.g., a thyroid cancer site, etc.), it can also be extracted from cells contained in the needle washings of a cytological diagnosis, which is useful because a sample for cytological diagnosis and a sample for genetic testing can be obtained simultaneously. In addition, the test animal may be an animal known to have a mutation in the TERT promoter, an animal suspected of having a mutation in the TERT promoter, or an animal for which it is unknown whether or not it has a mutation in the TERT promoter.

In the step (2), the method of amplifying the target region by PCR is not particularly limited, but it is preferable to amplify the target region by a digital PCR method. Digital PCR is performed, for example, by the following procedure. A reaction solution containing the probe set of the present invention, a test nucleic acid, a PCR primer set, and DNA polymerase is set in a digital PCR device, and the reaction solution is distributed to a large number of reaction wells, ranging from several thousand to several tens of thousands, provided on a chip by the device. The reaction wells are, for example, minute wells with an opening size of several tens of μm or nanoliter-sized droplets. When the reaction mix is added to the chip, the reaction mix is distributed to the reaction wells so that some of the reaction wells on the chip contain, preferably, about one copy of the target region derived from the extracted nucleic acid, and the other reaction wells do not contain the target region derived from the extracted nucleic acid. When PCR is carried out using the extracted nucleic acid as a template in a reaction mix distributed among multiple reaction wells, at least a portion of the nucleic acid is amplified in the reaction well in which the nucleic acid containing the target region is present, and during this process, the nuclease activity of the DNA polymerase cleaves the probe bound to the target region, dissociating the fluorescent dye and emitting fluorescence.

The measurement of the fluorescence intensity in the step (3) can be carried out, for example, using a known fluorescence reader. When the PCR is carried out by digital PCR, the fluorescence intensity can be measured, for example, by counting the number of reaction wells in which fluorescence is detected using a reader of a digital PCR device. When multiple probes with different fluorescent dyes are used, multiple fluorescence intensities can be measured at once by detecting the fluorescence derived from each fluorescence.

In the step (4), a mutation in the TERT promoter can be detected based on the measurement results in the step (3). The principle of detecting a mutation in the TERT promoter using the probe set of the present invention is as described in 1 above. Figure 2 shows an example of a two-dimensional plot of the measurement results, with the horizontal axis representing different fluorescence intensities. As is clear from Figure 2, by using the probe set of the present invention, it is possible to distinguish between mutations at two locations in a single assay.

3. Kit for detecting TERT promoter mutation In another aspect, the present invention provides a kit for detecting a mutation in a TERT promoter (hereinafter, sometimes referred to as the "kit of the present invention"), comprising the probe set of the present invention and two types of PCR primer sets capable of amplifying a target region of the TERT promoter. Specific examples of such PCR primers are as described in 2. above. The kit of the present invention may contain other reagents necessary for PCR. When the reagent is a reagent that does not adversely affect the reaction when stored in the presence of the probe set and/or primer set of the present invention, it can be mixed with the probe set and/or primer set and included in the kit. Alternatively, the reagent and the probe and/or primer set of the present invention may be provided separately without being mixed. The reagents include a DNA extraction reagent, a DNA polymerase, dNTPs, a reaction buffer, a nucleic acid containing a target region serving as a positive control for PCR, a container, an instrument, an instruction manual, and the like.

4. Method for Treating or Preventing Thyroid Cancer When a mutation is detected in the TERT promoter in a test mammal as a result of the detection method of the present invention described in 2. above, a therapeutic or preventive drug for thyroid cancer (hereinafter collectively referred to as "therapeutic drug") to be administered to the test mammal can be selected (or determined) and a therapeutically or prophylactically effective amount of the therapeutic drug can be administered to the test mammal to treat or prevent thyroid cancer. Alternatively, thyroid cancer can be treated by surgically removing the whole or part of the thyroid gland or by radiation therapy. In this specification, the term "therapeutic drug" includes not only drugs intended for the radical treatment of thyroid cancer, but also drugs intended for inhibiting the progression of these diseases or for alleviating symptoms. Examples of thyroid cancer to be treated or prevented include papillary cancer, follicular cancer, medullary cancer, undifferentiated cancer, and malignant lymphoma.

Examples of such therapeutic agents include, but are not limited to, thyroid hormone drugs, molecular targeted therapeutic agents (e.g., lenvatinib, sorafenib, vandetanib, etc.), and cytotoxic anticancer agents (e.g., doxorubicin, paclitaxel, etc.). The above therapeutic agents may be used in appropriate combinations depending on the symptoms of the patient or test mammal.

The above-mentioned therapeutic agents may be administered orally or parenterally as a pharmaceutical composition in an appropriate dosage form, either as the active ingredient alone or by mixing with a pharma- ceutical acceptable carrier, excipient, diluent, etc. Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. On the other hand, compositions for parenteral administration include, for example, injections and suppositories, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. The dosage of the therapeutic agent may be appropriately set depending on various conditions such as the type of compound, symptoms of the subject, age, body weight, and drug tolerance.

The present invention will be further explained below with reference to examples, but the present invention is not limited to these.

In the examples described below, experiments were carried out as follows.
<Procedure>
1. PTC sample and patient information
We collected 159 PTC samples from operations performed at Nagasaki University Hospital (Nagasaki Prefecture) and Kuma Hospital (Kobe City) between November 2001 and December 2017. Clinicopathological data were collected from the patients' medical records. The age of the patients at the time of surgery ranged from 14 to 81 years (median age: 54 years, 17.0% male). The AJCC/TNM staging system (8th edition) was used for staging. Their histological subtypes were as follows: 146 were classical Nippon Gene PTCs (25 were microcarcinomas), 10 were follicular variant PTCs (4 were microcarcinomas), 2 were diffuse sclerosing variant PTCs, and 1 was tall cell variant PTC. The study protocol was approved by the Institutional Review Boards of Nagasaki University and Kuma Hospital. Written informed consent was obtained from each patient. Fresh tumor tissue samples were collected during surgery, flash frozen in liquid nitrogen, and stored at −80° C. DNA and total RNA were simultaneously extracted using ISOGEN reagent (Nippon Gene) according to the manufacturer's instructions.

2. Direct DNA sequencing
The mutation status of BRAF (near c.1799A>T) and the promoter region of TERT were analyzed by direct DNA sequencing (Sanger sequencing) as previously described (Matsuse M. et al., Sci Rep, 7:41752 (2017)).

3. Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems). The following PCR reactions were performed with SYBR Premix Ex Taq II (Takara Bio) on a Thermal Cycler Dice real-time system (Takara Bio). Relative expression was calculated using cycle threshold (CT) values determined using the second derivative method. TERT mRNA levels were normalized using the mRNA expression of TATA binding protein (TBP) as a reference. The primer sequences are as follows: TERT ex 6-7 F; 5'-AGCCACGTCTCTACCTTGAC-3' (SEQ ID NO: 5) and TERT ex7-8 R; 5'-CTCATTCAGGGAGGAGCTCT-3' (SEQ ID NO: 6), TBP ex2 F; 5'-CCTGCCACCTTACGCTCAG-3' (SEQ ID NO: 7) and TBP ex3 R; 5'-TGGTGTTCTGAATAGGCTGTGG-3' (SEQ ID NO: 8).

4. Droplet Digital PCR (ddPCR)
ddPCR was performed using ddPCR Supermix for Probes (Bio-Rad) on a QX100 Droplet Generator (Bio-Rad), a C1000 Touch thermal cycler (Bio-Rad), and a QX100 droplet reader (Bio-Rad). The probes used for ddPCR were as follows: TERT mut; 5'-/56-FAM/C+CC+C+T+TC+CGG (SEQ ID NO: 3)/3IABkFQ/-3' and TERT wt 228; 5'-/5HEX/C+CC+C+C+TC+CGG (SEQ ID NO: 4)/3IABkFQ/-3' (the bases after + are locked nucleic acids). The primers were the same as those used for direct DNA sequencing. The TERT mut probe can bind to both C228T and C250T, whereas the wt probe can only bind to C228, making it possible to distinguish between C228T and C250T in a two-dimensional (2D) display using both of the above probes simultaneously (Figure 2). In the case of the C250T mutation, both FAM and HEX signals are detected, whereas in the case of the C228T mutation, only the FAM signal is detected. In the nucleotide sequence shown in SEQ ID NO:3, the unmodified sugar-phosphate backbone is shown as SEQ ID NO:1. Similarly, in the nucleotide sequence shown in SEQ ID NO:4, the unmodified sugar-phosphate backbone is shown as SEQ ID NO:2.

5. Recurrence as an outcome measure Recurrence was defined as local disease or local/distant metastasis, surgically resected and pathologically confirmed by ultrasound, scintigraphy, or other imaging, within 6 months of initial treatment. Time to recurrence was calculated based on the date of reoperation or the date of detection of recurrence by medical imaging.

6. Statistical analysis Univariate Fisher or Fisher-Freeman-Halton exact tests were used for categorical data analysis. Pairwise statistical comparisons of proportions of three or more groups were performed using the SAS COMPPROP macro (http://www2.sas.com/proceedings/sugi31/204-31.pdf). The characteristics of different PTC subgroups were compared using the nonparametric Mann-Whitney test or the Kruskal-Wallis test, followed by the Dwass, Steel, Critchlow-Fligner multiple comparison procedure for continuous data. The Kaplan-Meier method and log-rank test were used to analyze recurrence-free survival (RFS). Factors influencing RFS were evaluated with the Cox proportional hazards model. Hazard ratios (HRs) were calculated in 5 percentile increments to determine the threshold of TERT expression level. Multivariate logistic regression models were used to identify factors associated with extrathyroidal extension (ETE) or pT category. Analyses of very few outcomes (<5 per cell) or when quasi-perfect separation was observed were performed using Firth's approach to fit penalized maximum likelihood, which reduces bias. Non-automated model optimization was routinely performed using Akaike's information criterion. Stepwise variable selection was applied to models that were amenable to automatic optimization. Once the most appropriate model was determined, maximum likelihood estimates of each parameter and their Wald-type 95% confidence intervals were calculated. Statistical evaluations were performed using SAS Studio (3.71 release) for SAS version 9.4M5 (SAS Institute) or IBM SPSS Statistics version 24 software (IBM). Graphs were created using GraphPad Prism 6 (GraphPad). All p values were two-sided and were considered significant when p < 0.05.

Example 1: Analysis of TERT promoter mutation status and TERT expression First, 159 PTC samples were screened for mutations in the TERT promoter region (C228T and C250T) by direct DNA sequencing. TERT promoter mutations were found in 20 (12.6%) samples, all of which were C228T, and none of which were C250T in the ongoing series. Next, TERT mRNA expression was examined by real-time qRT-PCR. TERT expression was confirmed in all TERT promoter mutation-positive samples. Interestingly, even among the 139 mutation-negative samples, 56 (40.3%) showed TERT expression. Next, we explored the possibility that there are tumors with low allele frequencies of TERT promoter mutations that cannot be detected by conventional sequencing. To examine the presence of mutations with high sensitivity, ddPCR was used. First, the detection limit of ddPCR for TERT promoter mutations was determined using serial dilutions of PCR products of the TERT promoter region containing C228T or C250T in the PCR product of the wild-type promoter. The detection limit for the allele frequency of the mutations was approximately 0.25% (Fig. 3). Next, 56 TERT-expressing samples were analyzed using ddPCR. In three samples (5.4%), mutations with low allele frequency were identified, hereafter referred to as PTC A, B, and C (Fig. 4A). All had the C228T mutation according to the 2D representation. The allele frequencies of the mutations in PTC A, B, and C were 17%, 10%, and 5%, respectively (Table 1). Because tumor tissues contain not only tumor cells but also stromal cells, endothelial cells, and blood cells, the mutant allele frequency in tumor cells was corrected for tumor density based on the ratio of TERT promoter mutation to BRAF ^T1799A mutation. Because, although controversial, the BRAF ^T1799A mutation is considered to be a clonal heterozygous mutation in tumor cells. After correction, the allele frequencies of TERT promoter mutations in tumor cells were as follows: PTC A; 14%, PTC B; 4%, PTC C; 3%, which means that 28%, 8%, and 6% of tumor cells have TERT promoter mutations in PTC A, B, and C, respectively (Table 1). Next, we retrospectively confirmed the chromatograms of direct DNA sequencing of these samples. Although a very small peak of C228T was observed (Figure 4B), it was not possible to distinguish these signals from background signals with confidence. The results of direct DNA sequencing, expression analysis, and ddPCR are summarized in Figure 5A-C.

Example 2: Association between TERT mutation/expression status and clinicopathological features
The association between TERT promoter mutation/expression status and clinicopathological characteristics was analyzed. The 159 cases were classified into three groups: TERT promoter mutation-negative/mRNA expression-negative group (mut-/exp-), TERT promoter mutation-negative/mRNA expression-positive group (mut-/exp+), and TERT promoter mutation-positive group (mut+/exp+). As shown in Table 2, tumors with mutations (mut+/exp+) showed statistically significant differences in age, extrathyroidal capsule invasion, and stage II/III/IV compared with the other two groups (mut-/exp- and mut-/exp+) (1 vs. 3 and 2 vs. 3). These findings suggest that TERT promoter mutations confer aggressive properties to PTC.

The Kaplan-Meier method and Cox proportional hazards model were used to evaluate RFS. Four cases with distant metastasis at the time of surgery and an additional 20 cases followed up within 6 months were excluded in this analysis. The survival curves of the three groups were separated and statistically significant in trend (Fig. 7; log-rank trend, p<0.001). After adjusting for age, sex, tumor size, extrathyroidal capsule invasion, and lymph node metastasis, the HRs for recurrence in the mut+/exp+ group were 20.47 (95% CI: 4.54-114.1, p<0.001) and 5.38 (95% CI: 1.14-30.32, p=0.046) compared with the mut-/exp- and mut-/exp+ groups, respectively (Table 3, first vs second comparison). In the best fit model, the HRs for the mut+/exp+ group were 23.39 (95% CI: 4.49-121.85, p<0.001) and 6.24 (95% CI: 1.44-27.13, p=0.015) compared with the mut-/exp- and mut-/exp+ groups, respectively (Table 3, comparison between first and second rounds).

Kaplan-Meier curves and HRs in the two-group analysis based on mutation status (mut-/exp- and mut-/exp+ vs. mut+/exp+) and expression status (mut-/exp- vs. mut-/exp+ and mut+/exp+) are shown in Figure 6 and Table 4, respectively.

Example 3: Analysis of sensitivity and specificity of TERT mutation detection by the probe of the present invention Next, the results of TERT mutation detection when using the probe of the present invention and the fine needle aspiration cytology needle washing solution were compared with the results of evaluation of surgical specimens to analyze the sensitivity, specificity, positive predictive value, and negative predictive value of the mutation detection by the probe. First, the cytology needle was washed with RNAlater (registered trademark) (Thermo Fisher Scientific), and the cell extract taken from the patient's thyroid gland was spun down to precipitate the cells. Next, DNA was extracted using ISOGEN reagent (Nippon Gene), and ddPCR was performed. The probe and ddPCR primers used in this example, as well as the ddPCR method, were the same as those in Example 1.

Sensitivity was calculated by dividing the number of samples determined to be positive by both ddPCR (Fine needle aspiration cytology) and resected specimens by the total number of samples determined to be positive by resected specimens. Specificity was calculated by dividing the number of samples determined to be negative by both ddPCR and resected specimens by the total number of samples determined to be negative by resected specimens. Positive predictive value was calculated by dividing the number of samples determined to be positive by both ddPCR and resected specimens by the total number of samples determined to be positive by ddPCR. Negative predictive value was calculated by dividing the number of samples determined to be negative by both ddPCR and resected specimens by the total number of samples determined to be negative by ddPCR. The results are shown in Figure 8. Figure 8 shows that the sensitivity, specificity, positive predictive value, and negative predictive value of the probe for mutation detection were all high.

By using the probe set of the present invention, it is possible to distinguish between two mutations (C250T and C228T) in the TERT promoter in a single assay, and it is also possible to detect and identify even very small amounts of samples or mutations with low allele frequency. By using the probe set of the present invention, mutations can be detected with high sensitivity, and therefore it can be used for detecting mutations in cytological specimens for thyroid nodules or their needle washings.

Claims (5)

A probe set for detecting a TERT promoter mutation, comprising the following two types of probes:
(1) a probe consisting of the nucleotide sequence shown in SEQ ID NO: 3, and (2) a probe consisting of the nucleotide sequence shown in SEQ ID NO: 4,
The probe set is characterized in that a labeling substance is bound to each of the probes, the labeling substance being a fluorescent dye, and a quencher is bound to each of the probes. A method for detecting a mutation in the TERT promoter, comprising the following steps (1) to (4):
(1) contacting the probe set according to claim 1 with a nucleic acid comprising a target region of the TERT promoter;
(2) amplifying a region including the target region by PCR;
(3) measuring the fluorescence intensity of a solution containing the PCR product amplified in the step (2); and (4) detecting a mutation in the TERT promoter based on the measurement results of the step (3). The method of claim 2, wherein the PCR is digital PCR. The method according to claim 2 or 3, wherein the nucleic acid containing the target region of the TERT promoter is derived from a fine needle aspiration cytology specimen or a needle wash thereof. The method according to claim 4, wherein the fine needle aspiration cytology specimen or needle washings thereof are fine needle aspiration cytology specimens or needle washings thereof for diagnosing thyroid cancer.