KR20200058299A - Biomarkers for predicting prognosis of thyroid cancer - Google Patents

Abstract

The present invention relates to a kit for predicting prognosis of thyroid cancer, comprising at least one of a primer, probe, or antibody which specifically binds to at least one nucleic acid selected from the group consisting of CXCL16, AHNAK2, and THBS2 or protein encoded therefrom.

Description

Biomarkers for predicting prognosis of thyroid cancer

The present invention relates to a biomarker capable of predicting thyroid cancer prognosis.

Thyroid cancer is a common disease with a prevalence of up to 5%, and recently targeted treatments for thyroid cancer have been developed, for example, Sorafenib (Sorafenib) as a drug targeting the RET / PTC-RAS-MEK-ERK pathway. Farnesyl protein transferase inhibitors, and MEK inhibitors, drugs targeting tyrosine kinase, motesanib, axitinib, and sunitinib , Vandetanib, Gefitinib, and c-Met inhibitors, and VEGF and bFGF inhibitors Thalidomide, bone marrow matrix inhibitor Lenalidomide approved or clinically approved Research is ongoing.

Thyroid cancer has a very good prognosis with the rapid growth of small-sized cancers through recent early diagnosis, but still has some cases of metastasis and recurrence, leading to death. Reduced, high-risk groups are clinically needed to be actively treated and managed.

BRAFV600E gene mutation, TERT promoter gene mutation, etc. are known as prognostic factors for thyroid cancer, but BRAFV600E mutation is common enough to be found in 60-70% of thyroid cancer patients, but occurs at an early stage of cancer formation. It is believed that the direct link to death is low. In addition, the TERT promoter mutation is rare enough to be found in less than about 2-5% of patients with thyroid cancer, so there is a problem that does not greatly help predict prognosis.

Sun Wook Cho, et al., CXCL16 signaling mediated macrophage effects on tumor invasion of papillary thyroid carcinoma, Endocrine-Related Cancer, 2016, vol. 23, pp. 113-124

An object of the present invention is to provide a biomarker capable of predicting the prognosis of thyroid cancer.

In one embodiment of the present invention, it is an object to provide a panel for predicting the prognosis of thyroid cancer.

1. A kit for predicting prognosis of thyroid cancer, comprising a primer or probe that measures the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or an antibody that measures the level of a protein encoded by CXCL16, CXCL16, AHNAK2, and THBS2 .

2. In item 1, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF , PGF, and primers or probes that measure one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, Kit comprising SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF , further comprising an antibody that measures the level of a protein encoded by one or more selected from the group consisting of EGF .

3. A composition for predicting prognosis of thyroid cancer comprising a primer or probe for measuring the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or an antibody for measuring the level of protein encoded by CXCL16, CXCL16, AHNAK2, and THBS2 .

4. In item 3, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF , PGF, and primers or probes that measure one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, The composition further comprises an antibody that measures the level of a protein encoded by one or more selected from the group consisting of SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF .

5. Contact the sample with a composition comprising primers or probes that measure the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or contact the sample with a composition comprising an antibody that measures the level of protein encoded by CXCL16, AHNAK2, and THBS2 . , thyroid cancer comprising the step of: and, CXCL16, AHNAK2, and THBS2 the nucleic acid level, or CXCL16, AHNAK2, and when the protein level to be encoded by the THBS2 increased compared to a subject not having a thyroid cancer, which predicted to be the prognosis of the thyroid cancer poor Methods of providing information to predict prognosis.

6. In item 5, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF , PGF, and one or more nucleic acid levels selected from the group consisting of EGF , ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, A method of providing information, further comprising measuring the level of a protein encoded by one or more genes selected from the group consisting of THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF .

7. In item 6, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, and HBEGF . One or more nucleic acid levels selected from the group consisting of increased or compared to subjects without thyroid cancer, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1 , CCL2, MMP9, AREG, IL1B, and HBEGF , a method of providing information, predicting that the prognosis of thyroid cancer will be poor if the level of the protein encoded by one or more genes selected from the group consisting of is increased compared to a subject without thyroid cancer.

8. Item 6, wherein the level of one or more nucleic acids selected from the group consisting of RIMBP2, PGF , and EGF is reduced compared to a subject without thyroid cancer, or encoded by one or more genes selected from the group consisting of RIMBP2, PGF , and EGF . A method of providing information, predicting that the prognosis of thyroid cancer will be poor if the level of protein is reduced compared to a subject without thyroid cancer.

9. The method of claim 5, wherein predicting that the prognosis is not good is predicting that thyroid cancer will progress to a higher TNM (Tumor Node Metastasis) stage.

10. contacting a sample to be analyzed with cells comprising nucleic acid sequences of CXCL16, AHNAK2, and THBS2 ; And measuring the expression level of the nucleic acid, and when the expression level of the nucleic acid is decreased, the sample is a substance for preventing or treating thyroid cancer, and a screening method for a substance for treating thyroid cancer.

11. In item 10, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, RIMBP2 , PGF , and EGF contacting a sample to be analyzed with cells comprising one or more nucleic acid sequences selected from the group consisting of; And measuring the expression level of the nucleic acid.

12. In item 11, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, When the level of one or more nucleic acids selected from the group consisting of AREG, IL1B, and HBEGF is increased, the sample is a substance for preventing or treating thyroid cancer.

13. The screening method according to item 11, wherein when the sample to be analyzed is contacted with one or more nucleic acid levels selected from the group consisting of RIMBP2, PGF , and EGF , the sample is a substance for preventing or treating thyroid cancer.

The existing cancer gene panel focused on the accuracy of diagnosis, but the present invention relates to a gene analysis panel capable of predicting the prognosis at the time of initial diagnosis of thyroid cancer or after primary surgery. To increase the specificity, it can reduce the need for unnecessary treatment and follow-up from the initial diagnosis. Through this, it is possible to improve patient's quality of life and reduce medical expenses through patient-specific treatment.

FIG. 1 shows the expression of CXCL16 in various thyroid tissues: (A) is a representative image and results of immunohistochemical staining of CXCL16, quantifying white powder of CXCL16 + cells per nuclear area in normal thyroid tissue, benign tumor, and papillary thyroid cancer. , (B) is a graph showing mRNA expression of CXCL16 in normal thyroid tissue, benign tumor, and papillary thyroid cancer in the SNUH dataset, and (C) is mRNA expression of normal thyroid tissue in the TCGA dataset, and CXCL16 in papillary thyroid cancer. (D) is a graph showing the histochemical staining of CXCR6 in normal thyroid tissue and papillary thyroid cancer, and (E) is a graph showing mRNA expression of CXCR6 in normal thyroid tissue and papillary thyroid cancer.
2 is a diagram showing that high expression of CXCL16 in human papillary thyroid cancer tissues of the TCGA dataset is related to M2 macrophages and angiogenesis-related genes: (A) BRAF ^V600E- RAS score (BRS), M2 according to CXCL16 expression Results of heat map analysis of macrophage- and angiogenesis-related gene expression, (B) to (F) are M2 macrophages, angiogenesis, ERK pathway, PI3K / AKT pathway, in CXCL16High and CXCL16Low groups, respectively. And MAPK pathways through a single sample genome set amplification analysis.
3 is a graph showing that high expression of CXCL16, AHNAK2, and THBS2 from the TCGA dataset is associated with a poor prognosis in human papillary thyroid cancer. (A) CXCL16 alone; Or (B) CXCL16, AHNAK2, and THBS2; (C) Relapse-free survival Kaplan-Meier curves for CXCL16, AHNAK2, THBS2, and PLAU combinations.
4 is a diagram showing that cell survival can be reduced by inhibiting endogenous CXCL16 using shCXCL16. In the adaptive medium, ( A ) shows the concentration of CXCL16 in BHP10-3SCp ^shCXCL16 and BHP10-3Sp ^control cells, ( B ) shows the concentration of FRO ^shCXCL16 and ^{CXCL16 in} FRO ^control cells, and (C) shows BHP10-3SCp ^shCXCL16 And the survival rate of BHP10-3Sp ^control cells at 48 and 72 hours; ( D ) Survival rates of FRO ^shCXCL16 and FRO ^control cells were compared at 48 and 72 hours.
5 shows that inhibiting CXCL16 using shCXCL16 may inhibit tumor growth of papillary thyroid cancer in a xenograft mouse model. (A) is a representative image after subcutaneous transplantation of BHP10-3SCp ^shCXCL16 or BHP10-3Sp ^control cells into nude mice (7-9 animals per group) and tumor growth curves from subcutaneous cells, (B) of (A) Representative images (x400) of the results stained by IHC technique with anti-CXCL16, anti-ERG, and anti-F4 / 80 antibodies in the tumor tissues of the xenograft model and the percentage of CXCL16 +, ERG +, F4 / 80 + cells. , (C) will shown the M2 macrophages and mRNA expression of angiogenic genes in xenografts, (D) is in the FRO ^shCXCL16 or FRO ^control cells are subcutaneously implanted nude mice (8 mice per group) FRO ^shCXCL16 or FRO Tumor growth curve from ^control cells. * P <0.05 compared to the control.

The present invention will now be more fully described below with reference to the accompanying drawings, but only some, but not all, embodiments of the invention are illustrated. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments presented herein. The singular form used in this specification and the appended claims includes a plurality of objects unless expressly indicated otherwise.

Although various chemokines have pre-oncogenic activity in cancer, the action of CXCL16 is still controversial. In the present invention, the molecular characteristics of papillary acidic cancer expressing CXCL16 were investigated. CXCL16 was significantly higher in papillary thyroid cancer than in benign or normal thyroid tissue. In the TCGA dataset for papillary thyroid cancer, an increase in CXCL16 expression is associated with M2 macrophage- and angiogenesis-related genes and worse prognostic factors, including higher TNM stages, and BRAF ^V600E mutation. The papillary thyroid cancer group in which three genes including CXCL16, AHNAK2, and THBS2 were highly expressed showed poor relapse-free survival compared to the group with a lower expression level. Also, shCXCL16 was introduced into BPH10-3SCp cells, resulting in deletion of endogenous CXCL16 and subcutaneous injection of the cells into athymic mice. As a result, tumors from BHP10-3SCp ^shCXCL16 were ERG + endothelial cells and F4 / compared to tumors from BHP10-3SCp ^control . Tumor growth was delayed as the number of 80+ macrophages decreased. CXCL16 related genes including AHNAK2 and THBS2 were downregulated in tumors from BHP10-3SCp ^shCXCL16 compared to BHP10-3SCp ^control . In conclusion, increased CXCL16 expression is associated with more aggressive phenotypes in papillary thyroid cancer in connection with macrophage- and angiogenesis-related genes.

Among the cells, macrophages are cells that appear abundantly throughout the development of cancer. In clinical studies or studies using mice, macrophages have been shown to generally play a role in promoting cancer. In primary tumors, macrophages stimulate angiogenesis of tumors and increase cancer cell infiltration, mobility and intravascular invasion. During metastasis, macrophages prepare the site of metastasis and promote the extravasation, survival and continued growth of cancer cells. In addition, macrophages also act as immunosuppressive agents, such as preventing natural killer cells (NK cells) or T cells from attacking cancer cells, which occur during cancer progression or recovery after anticancer treatment.

Prognosis of thyroid cancer comprising an antibody that measures the level of a protein encoded by CXCL16, AHNAK2, and THBS2 , or a primer or probe that measures the nucleic acid level of CXCL16, AHNAK2, and THBS2 in one embodiment of the invention Provided is a composition for prediction. In another embodiment of the invention, the composition is ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG , IL1B, HBEGF, PGF, and primers or probes that measure one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, Antibodies that measure the level of a protein encoded by one or more selected from the group consisting of TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF may be further included. In one embodiment of the invention, comprising an antibody to measure the levels of the protein encoded by the CXCL16, AHNAK2, THBS2, and for measuring the nucleic acid level PLAU primer or probe, or CXCL16, AHNAK2, THBS2, and PLAU, Provided is a composition for predicting prognosis of thyroid cancer.

In another embodiment of the present invention, a thyroid cancer, comprising a primer or probe measuring the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or an antibody measuring the level of a protein encoded by CXCL16, CXCL16, AHNAK2, and THBS2 , Provide a kit for predicting prognosis. In one embodiment of the invention, the kit is ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG , IL1B, HBEGF, PGF, and primers or probes that measure one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, Of thyroid cancer, further comprising an antibody measuring the level of protein encoded by one or more selected from the group consisting of TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF Further, a kit for predicting prognosis is provided. The kit may further include instructions describing a method for using the kit.

The terms “biomarker”, “gene marker”, “prognostic prediction marker”, “prognostic marker”, “diagnostic marker”, “diagnostic marker” or “diagnostic marker” are used interchangeably. Possible, biomarker present in the living body, which means a substance capable of predicting or predicting disease diagnosis, prognosis, response to treatment, etc., and showing an increased pattern in cells derived from high-risk group thyroid cancer patients compared to cells from low-risk group thyroid cancer patients. Visible polypeptides or nucleic acids (eg, DNA, mRNA, etc.), lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like may be included. In, the markers for predicting thyroid cancer prognosis are CXCL16, and AHNAK2, THBS2, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1 Any one or more genes selected from the group consisting of CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF , CXCL16, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, in cells from high-risk group thyroid cancer patients CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, and HBEGF show increased expression, and RIMBP2, PGF , and EGF show reduced expression. They may use mRNA for either gene or any protein encoded by the gene, or a combination thereof.

When the kit of the present invention is a microarray, the probe is immobilized on the solid surface of the microarray, and when the kit of the present invention is a gene amplification kit, a primer is included.

“Primer” of the present invention means a nucleic acid sequence capable of forming a base pair with a template and serving as a starting point for copying the template strand, complementarily binding to a specific position on the template.

Primers can initiate DNA synthesis in the presence of a nucleoside triphosphate and reagents for polymerization at appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific to each marker gene. Primers can incorporate additional features that do not change the basic properties of the primer, which serves as the starting point for DNA synthesis.

Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art.

The term "probe" of the present invention refers to a linear oligomer of natural or modified monomers or linkages, which includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to the target nucleotide sequence, and is present naturally or Or artificially synthesized.

When using a probe, the probe is hybridized with a cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. These procedures are carried out in a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).

For example, among the above stringent conditions, the high stringency conditions were hybridized with 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), and 1 mM EDTA at 65 ° C., and 0.1 x SSC (standard saline citrate) /0.1% SDS. It means washing under 68 ℃ condition. Alternatively, high stringency means washing at 6 ° SSC / 0.05% sodium pyrophosphate at 48 ° C. conditions. A low stringency condition means, for example, washing at 42 DEG C in 0.2 x SSC / 0.1% SDS.

In the present invention, "antibody" or "binding protein" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. It is not only in full antibody form, but also includes functional fragments of antibody molecules. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected by a heavy chain and a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and includes Fab, F (ab '), F (ab') 2, and Fv. Among antibody fragments, Fab has a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain, and has one antigen-binding site. Fab 'differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombination techniques for generating Fv fragments are described in international publications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88 / 09344. In the double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are connected by disulfide bonds, and in the single-chain Fv (scFv), the variable region of the heavy chain and the variable region of the light chain are commonly linked through a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes, and can preferably be produced through genetic recombination technology. The antibody in the present invention is preferably in Fab form or in whole antibody form. In the present invention, the antibody may be an antibody conjugated with microparticles. In addition, the microparticles may be colored latex or colloidal gold particles. In the present invention, the antibody can be any antibody capable of measuring the expression level of a protein encoded by a known mRNA gene for the gene marker described above, for example, but not limited to, the kit Is a kit for immunoassay, and in one embodiment, the kit is a Luminex assay kit, a protein microarray kit, or an ELISA kit.

The Luminex assay kit, protein microarray kit, and ELISA kit are polyclonal and monoclonal antibodies to the protein encoded from the gene marker according to the present invention, and the polyclonal and monoclonal antibodies bound to the marker. Secondary antibody.

Examples of types of kits in the present invention may include, but are not limited to, immunochromatography strip kits, Luminex assay kits, protein microarray kits, ELISA kits, or immunological dot kits.

The diagnostic kit may additionally include essential elements necessary to perform a reverse transcriptase reaction. The reverse transcriptase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and may be, for example, about 7 bp to 50 bp long and about 10 bp to 30 bp long. In addition, primers specific to the nucleic acid sequence of the control gene may be included. Other reverse transcriptase reaction kits include test tubes or other suitable containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNAse, RNAse inhibitors DEPC-water, And sterilized water.

In addition, the kit may additionally include essential elements necessary to perform a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, and the like for preparing a fluorescent marker probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, the kit may additionally include essential elements necessary for performing ELISA. The ELISA kit contains antibodies specific to the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the ELISA kit may include antibodies specific to the control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, for example, labeled secondary antibodies, chromophores, enzymes, and other substances capable of binding substrates or antibodies thereof.

In addition, the kit may additionally include essential elements necessary to perform protein microarray to simultaneously analyze the complex marker. The microarray kit contains antibodies specific to the marker protein bound to the solid phase. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the protein microarray kit may include antibodies specific to the control protein. Other protein microarray kits may include reagents capable of detecting bound antibodies, for example, labeled secondary antibodies, chromophores, enzymes, and other substances capable of binding substrates or antibodies thereof. In a method of analyzing a sample using a protein microarray, a protein is separated from a sample, and the isolated protein is hybridized with a protein chip to form an antigen-antibody complex, and read and read to confirm the presence or expression level of the protein. , It can provide information necessary to predict thyroid cancer prognosis. The protein microarray of the present invention may include a polyclonal antibody to the protein bound to the slide, a monoclonal antibody to the protein, and an enzyme-coupled secondary antibody to the polyclonal antibody and monoclonal antibody.

ELISA can be used to measure the protein expression level. ELISA is a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled antibody recognizing a capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen from the complex of the antibody and antigen, labeled with the antibody attached to the solid support, and reacted with another antibody that recognizes the antigen from the complex of the antigen It includes various ELISA methods, such as indirect sandwich ELISA using a secondary antibody. More preferably, for attaching an antibody to a solid support and reacting a sample, then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex to enzymatically develop an antibody or to recognize an antigen of the antigen-antibody complex It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and colored enzymatically. The prognosis of thyroid cancer can be predicted by confirming the degree of complex formation between the thyroid cancer marker protein and the antibody.

Western blots using one or more antibodies to the thyroid cancer marker may be used. After separating the whole protein from the sample, electrophoresis is performed to separate the protein according to size, and then it is transferred to a nitrocellulose membrane to react with the antibody. The prognosis of thyroid cancer can be predicted by confirming the amount of protein produced by gene expression by confirming the amount of the generated antigen-antibody complex using a labeled antibody. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cell having thyroid cancer. mRNA or protein levels can be expressed as absolute (eg, μg / ml) or relative (eg, relative intensity of signals) differences of the marker proteins described above.

Immunohistostaining may be performed using one or more antibodies to the thyroid cancer marker. After collecting and fixing normal liver tissue and tissue suspected of thyroid cancer, paraffin embedded blocks are prepared by methods well known in the art. After making them into sections with a thickness of several μm and attaching them to a glass slide, they are reacted with one selected from the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the detection labels mentioned above to read whether the antibody is labeled on the microscope.

One or more antibodies to the thyroid cancer marker may be used in a protein chip, which is arranged at a predetermined position on a substrate and immobilized at a high density. The method of analyzing a sample using a protein chip, isolates the protein from the sample, hybridizes the separated protein with the protein chip to form an antigen-antibody complex, and reads it to check the presence or expression level of the protein to confirm thyroid cancer Can predict the prognosis.

The term “diagnosis” herein refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a specific disease or condition, and the prognosis of an object with a specific disease or condition It may include determining, or therametrics.

The term “prognosis” of the present invention means a prediction of a patient's condition or treatment result of a disease, and is used when a subject with “good prognosis” is predicted as a low-risk group, and if recurrence is low, metastatic cancer If not, or if the likelihood of metastasis is low, stage 1 or 2 thyroid cancer, or if the likelihood of developing stage 3 or 4 thyroid cancer is low, it is used when the likelihood of death from thyroid cancer is low. "Prognosis is poor" is used when the subject is predicted as a high-risk group, and if the likelihood of relapse is high, metastatic cancer or metastatic cancer, stage 3 or 4 thyroid cancer or stage 3 or 4 thyroid cancer If you have this, it means that you are more likely to die from thyroid cancer. In one embodiment of the present invention, a patient with poor prognosis refers to a patient who is predicted to progress to a higher TNM (Tumor Node Metastasis) stage of the patient over time.

The term “prognostic diagnosis” of the present invention can be used interchangeably with “prognostic prognosis”, and the prognosis of a subject with a particular disease or condition (eg, identification of a pre-metastatic or metastatic cancer state, stage of cancer) Determining, determining a cancer's responsiveness to treatment, or determining a high-risk group and a low-risk group, or monitoring the condition of an object to provide information on therapeutics (e.g., treatment efficacy) ).

In the TNM stage, T refers to the site where the tumor occurred and its size, and is divided into stages 1 to 4: T0 means no evidence for the tumor, and T1 to a has a tumor size of 0.5 cm or less , b is the tumor size of 0.5 to 1 cm, c is the tumor size of 1 cm to 2 cm, T2 means that the tumor is larger than 2 cm but not larger than 5 cm, T3 is the tumor It means that the size is 5 cm or more, and T4 means that the tumor is very large. In the TNM stage, N means that the tumor spreads to the surrounding lymphoid tissue, and is divided into stages 0 to 3, and the higher the number, the more the tumor spreads to the surrounding lymphoid tissue and the farther it is distributed: N0 is It means that the tumor has not been infiltrated into the lymphoid tissue, and N1 to N3 mean that the tumor has been infiltrated into the lymphoid tissue. In the TNM stage, M indicates whether metastasis has occurred to other organs, and is classified into stages 0 to 1, and is said to have metastasized when cells in the region where the tumor has occurred and cells in the other region have the same metastasis: M0 Means that it has not metastasized, and M1 means that the tumor has spread to nearby organs and areas.

The term "metastasis" of the present invention refers to a state in which cancer cells migrate from a cancerous lesion occurring in an organ or tissue to bind to and proliferate to another organ or tissue.

The method for predicting the prognosis of thyroid cancer according to the present invention is based on the association between tumor-associated macrophage (TAM) infiltration and bad prognosis of thyroid cancer, and is expressed with CXCL16 , which mediates the contribution of TAM to cancer progression. The AHNAK2, and THBS2 genes are analyzed based on the target nucleic acid sequence, for example, DNA sequence or RNA sequence, or based on the target protein sequence, so that the CXCL16 expression level is compared to the expression level of CXCL16 measured in normal human thyroid tissue. If it is increased and the expression of one or more of AHNAK2, and THBS2 is increased, it is diagnosed as a high risk group.

The kit further comprises an antibody or aptamer that specifically binds to an AFP nucleotide sequence, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded in the nucleotide sequence, most preferably the kit is AFP And an antibody or aptamer specifically binding to a CRP nucleotide sequence, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded in the nucleotide sequence, to analyze the diagnosis or prognosis of thyroid cancer. When thyroid cancer is diagnosed by adding antibodies or aptamers specifically binding to nucleotides of AFP and CRP or a protein encoded therein, the specificity and accuracy of thyroid cancer diagnosis is very high.

The kit can further analyze the diagnosis or prognosis of thyroid cancer by further comprising an antibody or aptamer that specifically binds a CRP nucleotide sequence, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded in the nucleotide sequence. have.

The level of gene expression in a biological sample can be confirmed by confirming the amount of mRNA or protein, and any one or more sequences selected from the genes used as markers in the present invention are nucleotide sequences or polypeptides encoded therefrom. It may include.

The term "expression" of the present invention refers to the biological production of products encoded by coding sequences. In most cases, the DNA sequence comprising the coding sequence is transcribed to form a messenger-RNA (mRNA). The messenger RNA is then translated to form a polypeptide product with relevant biological activity. In addition, the expression process can include additional processing steps for RNA transcription products (eg, splicing to remove introns), and / or post-translational processing of polypeptide products.

The biological sample in the present invention means tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid, or urine.

For nucleic acids, the term “substantial identity” of the present invention refers to at least about 80%, at least about 90% to 95% of a nucleotide with appropriate nucleotide insertions or deletions when two nucleic acids or their designated sequences are optimally aligned and compared. , Or at least about 98% to 99.5%.

For nucleotide and amino acid sequences, the term “identical” refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

The percent identity between two sequences is a function of the number of identical positions shared by the sequence (i.e.% identity = equal), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The number of locations / the total number of locations × 100). Comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm as described in the non-limiting examples below. Percent identity between two nucleotide sequences is NWSgapdna.CMP matrix and 40, 50 , 60, 70 or 80 gap weights and length weights of 1, 2, 3, 4, 5 or 6 can be determined using the GAP program of the GCG software package. Percent identity between two nucleotide or amino acid sequences is also incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4 [E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)). In addition, the percent identity between the two amino acid sequences is Blossum 62 matrix or PAM250 matrix, And in the GAP program of the GCG software package using gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 [Needleman and Wunsch (J .Mol. Biol. 48: 444-453 (1970).

By way of example, the polynucleotide sequence of the present invention may be identical to the reference sequence, that is, 100% identical, or it may include a nucleotide modification of a specific integer number or less compared to the reference sequence. The alteration is selected from the group consisting of substitution, or insertion including at least one nucleotide deletion, variation and displacement, the alteration being individually at the 5 'or 3' end position of the reference nucleotide sequence, or nucleotides within the reference sequence It may occur anywhere between these scattered end positions or within one or more contiguous groups within a reference sequence. The number of nucleotide changes is multiplied by the numerical number of each percent of identity (divided by 100) of the total number of nucleotides in the reference sequence, then subtract the product from the total number of nucleotides in the reference sequence, or by the formula Is determined:

_{n n ≤x n - (x n} · y).

In the above formula, n _n is the number of nucleotide changes, x _n is the total number of nucleotides in the reference sequence, y is 0.50 for 50% identity, 0.60 for 60%, 0.70 for 70%, 0.80 for 80% , 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and any non- _integer product of x _n and y is the most before subtracting it from x _n Rounded down to the nearest integer. Alteration of the polynucleotide sequence of the reference sequence can result in nonsense, missense, or shift mutations in this coding sequence, thereby altering the polypeptide encoded by the polynucleotide after the change.

Similarly, in another example, a polypeptide sequence of the invention can be identical to a reference sequence, i.e., 100% identical, or it comprises a specific integer number of amino acid changes or less relative to the reference sequence such that% identity is less than 100% can do. The alteration is selected from the group consisting of at least one amino acid deletion, substitution comprising conservative and non-conservative substitutions, or insertions, the alteration being within the amino-terminal or carboxy-terminal position of the reference polypeptide sequence, or within the reference sequence. It may occur anywhere between these terminal positions interspersed individually among amino acids or within one or more contiguous groups within a reference sequence. The number of amino acid changes for a given percent identity is multiplied by the numeric percentage of each percent identity (divided by 100) multiplied by the total number of amino acids in the polypeptide sequence encoded by the reference sequence, followed by the total number of amino acids in the polypeptide reference sequence. The product is subtracted from the number, or is determined by the formula:

_{n a ≤x a - (x a} · y).

Where n _a is the number of amino acid changes, x _a is the total number of amino acids in the polypeptide sequence, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc., Any non-integer product of x _a and y is rounded off from x _a to the nearest integer before subtracting it.

The term “or combinations thereof” as used herein refers to all permutations and combinations of the items listed before this term. For example, “A, B, C, or combinations thereof” means A, B, C, AB, AC, BC, or ABC, and when order is important in certain situations, also BA, CA, CB, CBA, BCA , ACB, BAC, or CAB.

In one embodiment of the invention, the sample composition comprising a primer or probe for measuring the nucleic acid level of CXCL16, AHNAK2, and THBS2, or the sample to an antibody for measuring the level of the protein encoded by the CXCL16, AHNAK2, and THBS2 after contact with a composition comprising, by measuring the level of protein encoded by the CXCL16, AHNAK2, THBS2 and nucleic acid level, or CXCL16, AHNAK2, THBS2 and the can predict the prognosis of the thyroid cancer. In one embodiment of the present invention , if the nucleic acid level of CXCL16, AHNAK2, and THBS2 or the protein level encoded by CXCL16, AHNAK2, and THBS2 is increased compared to a subject without thyroid cancer, it is predicted that the prognosis of thyroid cancer is poor. .

In addition, the information providing method according to the present invention is CXCL16 , and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9 , One or more nucleic acid levels selected from the group consisting of AREG, IL1B, HBEGF, PGF, and EGF , or CXCL16, and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8 , TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and may further comprise the step of measuring the level of the protein encoded by one or more genes selected from the group consisting of EGF , At this time, composed of CXCL16, and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, and HBEGF One or more nucleic acid levels selected from the group are increased compared to subjects without thyroid cancer, or CXCL16, and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU , Predicts that the prognosis of thyroid cancer will be poor if the level of a protein encoded by one or more genes selected from the group consisting of THBS1 , CCL2, MMP9, AREG, IL1B, and HBEGF is increased compared to subjects without thyroid cancer, and / or One or more nucleic acid levels selected from the group consisting of RIMBP2, PGF , and EGF are reduced compared to subjects without thyroid cancer, or RIMBP It is predicted that the prognosis of thyroid cancer will be poor if the level of the protein encoded by one or more genes selected from the group consisting of 2, PGF , and EGF is reduced compared to subjects without thyroid cancer.

In addition, in one embodiment of the present invention, comprising the step of contacting a sample to be analyzed with a cell containing a nucleic acid sequence of CXCL16, AHNAK2, and THBS2 and measuring the expression level of the nucleic acid, the expression level of the nucleic acid is reduced When possible, the sample provides a method for screening a substance for treating thyroid cancer, which is determined to be a substance for preventing or treating thyroid cancer. In one embodiment of the invention, the method comprises CXCL16, and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, One or more expression levels selected from the group consisting of MMP9, AREG, IL1B, HBEGF, PGF, and EGF can be further measured. In one embodiment of the present invention, CXCL16, and ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, When one or more nucleic acid levels or protein expression levels selected from the group consisting of CCL2, MMP9, AREG, IL1B, and HBEGF are increased, the sample may be determined as a substance for the prevention or treatment of thyroid cancer, and another embodiment of the present invention In an example, when one or more nucleic acid levels or protein expression levels selected from the group consisting of RIMBP2, PGF , and EGF are reduced when the sample to be analyzed is contacted, the sample may be determined as a substance for preventing or treating thyroid cancer.

CXCL16 (NM_022059) is a gene encoding the chemokine (CXC motif) ligand 16 (chemokine (CXC motif) ligand 16; CXCL16), the CXCL16 protein is a small cytokine belonging to the CXC chemokine family. It consists of a CXC chemokine domain, a mucin-like stalk, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation region capable of binding to SH2. Expression of CXCL16 is induced by inflammatory cytokines IFN-gamma and TNF-alpha.

MMP9 (NM_004994) is a gene encoding matrix metallopeptidase 9 (MMP9), the MMP9 protein is known as 92kDa type IV collagenase, or 92kDa gelatinase B, and is used for the degradation of extracellular matrix. It is an enzyme belonging to the associated zinc-metalloproteinase family. Proteins of the MMP family are involved in pathologic processes such as arthritis, brain hemorrhage and metastasis, as well as degradation of extracellular matrix in common physiological processes such as embryonic development, reproduction, angiogenesis, bone development, wound healing, and cell migration. .

XDH (NM_000379) is a gene encoding xanthine dehydrogenase (XDH), and XDH is a molybdenum-containing hydroxylase involved in the oxidative metabolism of purines and exists as a homodimer. XDH can be converted to xanthan oxidase by oxidation of irreversible sulfhydryl groups or irreversible proteolytic modification.

CYTIP (NM_004288) is a gene that encodes a cytohesin-interacting protein (CYTIP), contains two leucine zipper domains and a peptide presumed to be a C-terminal nuclear target signal, and has no hydrophobic region. . It is rarely expressed in resting NK and T cells.

IL8 (NM_000584, NM_001354840) is a gene encoding interleukine 8 (IL8), IL8 is a chemokine produced by macrophage and epidermal cells, airway smooth muscle cells, and endothelial cells. There are various receptors on the cell membrane that can bind IL8, such as CXCR1 and CXCR2. IL8, also known as neutrophil factor, induces chemotaxis in target cells, moves neutrophils or other granulocytes to the infected site and promotes phagocytosis when they arrive.

SERPINF1 (NM_002615, NM_001329903, NM_001329904, NM_001329905) is a gene that encodes serpin F1 (Pigment epithelium-derived factor) or PEDF, multifunctional with anti-angiogenesis, anti-tumor formation and neuromuscular function It is a secreted protein. It is found in mammals and has a size of 50 kDa and is being studied as a therapeutic agent for diseases such as choroidal neovascularization, heart disease and cancer.

PLAU (NM_001145031, NM_002658, NM_001319191) is a gene that encodes a serine protease, urokinase or urokinase-type plasminogen activator (uPA). Serine proteases are involved in the degradation of extracellular matrix and tumor cell migration and proliferation, and convert plasminogen to plasmin through Arg-Val specific cleavage of plasminogen. Certain polymorphisms of the PLAU gene are expected to be associated with late-onset Alzheimer's,

THBS2 (NM_003247) is a gene encoding thrombospondin-2, which is a disulfide linked homotrimeric glycoprotein that mediates cell-cell interaction and cell-matrix interaction. . It is controversial that it plays a positive role and a negative role with respect to the role in the development of cancer, but has been reported to regulate the cell surface properties of mesenchymal cells and is involved in cell adhesion and migration.

AHNAK2 ( NM_138420) is a gene encoding AHNAK nucleoprotein 2 (AHNAK2), AHNAK2 has a relatively short N-terminal and a relatively long C-terminal domain. The N-terminal PSD-95 / Discs-large / ZO-1 (PDZ) -like domain is expected to form a stable homodimer and will play a role in calcium signaling through calcium channel proteins.

ADAM8 (NM_001109, NM_001164489, NM_001164490) is a gene encoding A Disintegrin And Metalloproteinase domain-containing protein 8 (ADAM8), belongs to the ADAM family, and the family is a membrane protein structurally related to snake venom disintegrins to be. It is involved in a variety of biological processes, including cell-cell interactions and cell-matrix interactions, including fertilization, muscle development and neurogenesis. ADAM8 protein is involved in cell adhesion during neurogenesis.

MYO1F (NM_012335, NM_001348355) is a gene encoding myosin-If (myosin-If), which is mainly expressed in the immune system and is expected to be involved in cell adhesion and migration. The MYO1F gene is expected to be associated with non-syndrome hearing impairment.

TYMP (NM_001257989) was also known as ECGF1 , and is a gene encoding thymidine phosphorylase. Thymidine phosphorylase converts thymidine to 2-deoxyribose 1-phosphate and thymidine, promotes angiogenesis in vivo and stimulates in vitro growth of various endothelial cells. Mutations in the TYMP gene have been reported to be associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

C1QB (NM_000491.4) is a gene encoding the B-chain polypeptide of the serum complement subcomponent C1q associated with C1r and C1s that produces the first component of the serum complement system, C1q has 6 A-chains, It consists of 18 polypeptide chains comprising 6 B-chains and 6 C-chains. Each chain contains an N-terminal collagen-like region and a C-terminal C1q globular domain, and C1q deficiency is known to be associated with lupus erythematosus and glomerulonephritis.

CD68 (NM_001251, NM_001040059) is a gene encoding CD68 (Cluster of Differentiation 68) and is expressed in large amounts by monocyte lineages, circulating macrophages and tissue macrophages. The CD68 protein is a transmembrane glycoprotein and is used as a marker in various cells of macrophages, including monocytes, tissue cells, giant cells, Cooper cells, and osteoclasts.

CTSK (NM_000396) is a gene encoding the cathepsin K (CTSK) enzyme, the CTSK protein is a lysosomal cysteine proteinase related to bone remodeling and resorption, and is mainly expressed in osteoclasts.

AREG (NM_001657) is a gene encoding Amphiregulin (AREG), and the AREG protein is included in the epidermal growth factor (EGF) family, and is associated with astrocytes, Schwann cells and fibroblasts. It is a mitogen for Korea and an autocrine growth factor. AREG interacts with epidermal growth factor receptor (EGFR) to promote normal epidermal cell growth.

C1QC (NM_172369) is a C-chain polypeptide of C1q, a serum complement subcomponent that interacts with C1r and C1s to form a first serum complement system. C1q consists of 18 polypeptides comprising 6 A chains, 6 B chains and 6 C chains.

GDF15 (NM_004864) is a growth / differentiation factor 15, belonging to the TGF-β superfamily. GDF-15 is expressed at low concentrations in most organs, but is upregulated due to organ damage such as the liver, kidneys, heart and lungs. (Zimmers TA, Jin X, Hsiao EC, McGrath SA, Esquela AF, Koniaris LG (June 2005). "Growth differentiation factor-15 / macrophage inhibitory cytokine-1 induction after kidney and lung injury". Shock. 23 (6): 543-8 .; Hsiao EC, Koniaris LG, Zimmers-Koniaris T, Sebald SM, Huynh TV, Lee SJ (May 2000). "Characterization of growth-differentiation factor 15, a transforming growth factor beta superfamily member induced following liver injury" Molecular and Cellular Biology. 20 (10): 3742-51 .; Ago T, Sadoshima J (February 2006). "GDF15, a cardioprotective TGF-beta superfamily protein" .Circulation Research. 98 (3): 294-7. )

CD163 (NM_004244, NM_203416, NM_001370145, NM_001370146) is a high affinity clearance receptor for hemoglobin-hemoglobin complex. It is used as a cell marker from the monocyte / macrophage family and serves as an endogenous immune sensor for Gram positive and Gram negative bacteria.

Diseases associated with RIMBP2 (NM_015347, NM_001351226, NM_001351227, NM_001351228, NM_001351229) include CD40 ligand deficiency and spinoselevelra ataxia 6.

INHBA (NP_002183, NP_002183.1) is a subunit of both activin and inhibin, both of which are closely related glycoproteins with opposite biological effects. The INHIBA A subunit binds the alpha subunit to form multiple FSH secretion inhibitors. Inhibin is known to act negatively on the differentiation of gonadoplastic cells and has activity as a tumor suppressor. In addition, the serum level of inhibin reflects the size of the granulocyte cell tumor, and thus can be used as a marker in early as well as recurrent disease.

THBS1 (NM_003246) is a disulfide-linked homotrimeric protein, which is a sticky glycoprotein that mediates intercellular and cell-matrix interactions. The protein can be bound to fibrinogen, fibronectin, laminin, type V collagen, and integrin alpha-V / veta-1.

CCL2 (NM_002982) is one of the cytokine genes clustered on the q-arm of chromosome 17. It is a superfamily of secreted proteins involved in immune regulation and inflammatory responses. The superfamily is divided into four superfamily based on the arrangement of the N-terminal cysteine residues of the mature peptide. This chemokine is a member of the CC subfamily characterized by two contiguous cysteine residues.

IL1B (NM_000576) is a cytokine protein also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, and lymphocyte activating factor. The IL-1β precursor is cleaved by cytoplasmic caspase 1 (or interleukin 1 beta convertase) to produce mature IL-1β, CAPS (Cryopyrin-) due to mutations in the inflammasome receptor NLRP3, induced by IL-1β. Associated Periodic Syndromes).

HBEGF (NM_001945) is a member of the EGF family, a growth factor that interacts with NRD1, zinc-finger and BTB domain containing protein 16, and BAG1. HBEGF is responsible for mediating cell cycle progression, molecular chaperone regulation, cell survival, cell function, adhesion and cell migration.

PGF (NM_002632, NM_001207012, NM_001293643) is a placental growth factor, a member of the VEGF subfamily, and plays an important role in angiogenesis. In atherosclerosis, it is associated with plaque inflammation and neovascular growth. PGF is a molecular marker for preeclampsia, and research is underway.

EGF is an epidermal growth factor, known as a secreted protein found in urine, and found in human tissue, including the submandibular gland and parotid gland. EGF is known to affect cell differentiation, proliferation and survival. In particular, recombinant human EGF is used in the treatment of diabetic foot ulcers.

The following examples are intended for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

Example 1. Method

1.1 Thyroid tissue microarray

Thyroid tissue microarray (Cho, SW et al . Therapeutic potential of Dickkopf-1 in wild-type BRAF papillary thyroid cancer via regulation of beta-catenin / Ecadherin signaling.J Clin Endocrinol Metab 99 , E1641- 1649 (2014)) was used in the present invention. Paraffin-embedded thyroid tissue blocks were obtained from patients undergoing thyroid surgery at Boramae Hospital, Seoul National University from January 1993 to December 2003.

1.2 Processing of genomic data from Seoul National University Hospital and TCGA (The Cancer Genome Atlas) dataset

The inventors obtained mRNA expression data of 77 papillary thyroid cancers, 25 thyroid vesicle adenomas, and 81 normal thyroid tissues from the Seoul National University Hospital cohort. RNA sequence analysis was performed according to a conventionally known method (Yoo, SK et al . Comprehensive Analysis of the Transcriptional and Mutational Landscape of Follicular and Papillary Thyroid Cancers.PLoS Genet 12 , e1006239 (2016).), MRNA of TCGA as a validation set Data were used; Clinical information and mRNA expression data obtained from RNA sequencing of 492 papillary thyroid cancer and 59 pairs of normal thyroid tissue were obtained from UCSC Cancer Browser ( https://genome-cancer.ucsc.edu ). Genome Biol 15 , 550 ( Genome Biol 15 , 550 (using the standardized raw coefficient of the sequence read by the adjusted log transformation method) (Love, MI, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 2014).) Differentially expressed genes (DEGs) as DESeq2 q-value <0.05, | Log ₂ (fold change) | It was defined to be ≥ 1, and baseMean ≥ 100. To adjust the results of several experiments, the q-value calculated by Benjamini-Hochberg correction was used. To create a heat map, a central rlog value was applied to hierarchical clustering using Cluster 3.0. BRAF ^V600E or, Cell 159 S-score (Cancer Genome Atlas Research, based on the mRNA developed by TCGA N. Integrated genomic characterization of papillary thyroid carcinoma. In order to quantify the degree of similarity between the gene expression profile of a given tumor to the RAS mutation, 676-690 (2014).). Tumors with a negative BRS score ( BRAF ^V600E - RAS score) were defined as BRAF ^{V600E -like} , and tumors with a positive BRS were defined as RAS -like.

In addition, single sample gene set enrichment analysis (ssGSEA) of GenePattern (http://software.broadinstitute.org/cancer/software/genepattern/) was used to activate several signal pathways for each sample. was used to evaluate (Barbie, DA et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108-112 (2009).). Genes with low expression levels across the entire sample were excluded according to the baseMean value (<100) from DESeq2 prior to analysis. For PI3K / AKT and MAPK pathways, Kyoto Encyclopedia of Genes and Genomes (KEGG) database provided by Molecular Signatures Database (MSigDB) was used (Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M . & Tanabe, M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44, D457-462 (2016) .; Liberzon, A. et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739- 1740 (2011).). For M2 macrophage and angiogenesis, existing studies (Landa, I. et al . Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest 126 , 1052-1066 (2016) .; Coates, PJ, Rundle , JK, Lorimore, SA & Wright, EG Indirect macrophage responses to ionizing radiation: implications for genotype-dependent bystander signaling.Cancer Res 68 , 450-456 (2008) .; Lin, SL et al . Stimulation of Interferon-Stimulated Gene 20 Gene list based on by Thyroid Hormone Enhances Angiogenesis in Liver Cancer. Neoplasia 20 , 57-68 (2018).) was used. ERK scores were also calculated with ssGSEA with a list of 52 genes from the TCGA study.

1.3 Progress prediction model

To predict relapse with expression of CXCL16 and M2 macrophage-related genes, and angiogenesis-related genes, each gene expression was divided into two groups (high and low groups) based on the median expression value of each gene. Differentiated and made a combination of 2 to 4 genes. All gene combinations were tested to find a significant association with relapse. The papillary thyroid cancer group that survived without recurrence with the highly expressed gene was compared with other groups using Kaplan-Meier survival analysis.

1.4 Immunohistochemical staining

Formalin-fixed, paraffin-embedded tissue sections from tissue microarrays or xenograft tumors were prepared using the BenchMark XT automated immunohistochemistry slide staining system (Ventata) according to the instructions CXCL16 (Abcam, Cambridge, MA, USA; dilution ratio 1: 100), ERG (Ventana Medical Systems, Tucson, AZ, USA), F4 / 80 (eBioscience, San Diego, CA, USA, dilution ratio 1: 200), and Ki-67 (Neomarkers, Fremont CA, USA, dilution ratio) 1: 500) using immunohistochemistry. Dead cells were detected in xenograft tumor sections using the TUNEL assay (Millipore, MA, USA). To analyze the immune activity, the central region of the tumor was divided into four equal parts and five regions were randomly selected from each equal and central region. Cells that were immunoactive at 400x magnification were counted by two clinicians and expressed as a percentage of positive cells per area.

1.5 Animal Research

A 6-week-old female BALB / c nude mouse (Orient Bio, Seongnam-si, Korea) was purchased and used. In order to produce thyroid cancer xenograft model, cells having ^{shCXCL16 (5 × 10 6/100} μL PBS) or FRO cells ^{(2 × 10 6/100 μL} PBS) or a control group growth factor is minimized Mart rijel (70 μL , 4 ° C; BD Biosciences, San Jose CA, USA) and implanted in the skin folded on the back of nude mice. With respect to the macrophage-containing ectopic tumor, THP-1 cells ^{(1.25 × 10 6/100 μL} PBS) were implanted with BHP10-3SCp cells. After transplanting tumor cells, anti-CXCL16 antibody or IgG (R & D Systems, Minneapolis, MN, USA) was injected once weekly by intraperitoneal injection. Tumor size was measured with a caliper and tumor volume was calculated as follows:

Volume = ½ × a × b ²

a = intestinal tumor diameter

b = tumor diameter.

After 5-6 weeks, mice were euthanized and tumors were excised and fixed with 10% formalin.

1.6 Cell culture

A human papillary thyroid cancer cell line BHP10-3SCp, a tumor-forming clone of the BHP10-3M cell line containing RET / papillary thyroid cancer rearrangement, was prepared (Dr. Soon-Hyun Ahn (Seoul National University College of Medicine, Seoul, Korea) and Dr. Gary L. Clayman (MD Anderson Cancer Center, Houston, TX, USA). FRO cells (malignant thyroid cancer cells) with BRAF V ^600E and TERT C ^250T mutations and THP-1 cells (human monocytes / macrophages) with CDKN2A , RAS , and TP53 mutations, respectively. June-Key Chung and Dr. Obtained from Hyo-Soo Kim (Seoul National University College of Medicine, Seoul, Republic of Korea). All cells were cultured in Roswell Park Memorial Institute (RPMI) -1640 medium containing 10% fetal calf serum (FBS).

1.7 CXCL16 shRNA transfection

To eliminate the expression of endogenous CXCL16 in thyroid cancer cells, CXCL16 shRNA was transduced into BHP10-3SCp and FRO cells. Specifically, human CXCL16-specific shRNA (Mission TRCN0000057990 and TRCN0000057991, 1 × 106 TU / mL, pLKO.1 vector) and non-specific shRNA control (SHC003) lentiviral transduction particles were stained with Sigma-Aldrich (St. Louis , MO, USA). Cells were dispensed into a 96-well plate at a concentration of 1.6 x 10 ⁴ cells / well and lentiviral particles expressing specific shRNA were transfected with MOI 5. After 2 days, untransfected cells were screened with puromycin (Sigma-Aldrich) until complete death.

1.8 RNA extraction and RT-PCR analysis

Trizol (Trizol, (Invitrogen, Carlsbad, CA, USA)) was used to extract mRNA from xenograft tumors and cells, and RT-PCR was performed using Perkin-Elmer GeneAmp PCR System 9600 (Waltham, MA, USA). Was performed. PCR primer sets are listed in Table 1 below.

gene Primer sequence (5 '→ 3') IL-8 ForwardReverse TGT GAA GGT GCA GTT TTG CCA AGG
GTT GGC GCA GTG TGG TCC ACT C MMP9 ForwardReverse TCG AAC TTT GAC AGC GAC AAG
GCA CTG AGG AAT GAT CTA AGC PLAU ForwardReverse CTC ATC CTA CAC AAG GAC TAC
CAG GCA GAT GGT CTG TAT AGT ADAM8 ForwardReverse CGA TGA TGC TGC CTG CGA TTG
CGC AGG TGG AGG GTG AAG TT THBS2 ForwardReverse TTA CCG CTT CGT GCG CTT TGA C
AAC AGC GTG CCC CTG GAC TTG AHNAK2 ForwardReverse TCC TGG TGG AAG CGA GAT TCA G
ACC ACC TGT GAC ACT GTA GCC A XDH ForwardReverse GTG GAT GCT GTG GAG GAG AT
TGC TTC CGA GGA GTG TCT TT GAPDH ForwardReverse TGC ACC ACC AAC TGC TTA GC
GGC ATG GAC TGT GGT CAT GAG

1.9 Statistical Analysis

In this specification, data are expressed as mean and standard deviation. Continuous variables were analyzed by Kruskal-Wallis test or Mann-Whitney U test. Survival curves that did not recur were prepared using the Kaplan-Meier method and compared using the log rank test. For statistical analysis, SPSS version 22.0 software for Windows (SPSS, Chicago, IL, USA) was used, and P <0.05 was considered statistically significant.

Example 2. Results

2.1 CXCL16 expression is increased in thyroid cancer tissue compared to normal thyroid tissue or benign tumors.

To compare the expression of CXCL16 in various thyroid tissues, immunohistochemical staining was performed on tissue microarrays consisting of 21 normal thyroid tissues, 40 positive tumors, and 148 papillary thyroid pressures using anti-CXCL16 antibodies. CXCL16 protein expression was significantly increased in papillary thyroid cancer compared to benign tumors (P <0.001) or normal thyroid tissue (P <0.001) (FIG. 1A). In addition, through RNA sequencing results of the SNUH cohort comprising 81 normal thyroid tissues, 25 thyroid benign tumors, and 77 papillary thyroid cancers, CXCL16 was significantly upregulated in papillary thyroid cancer compared to normal thyroid tissue or benign tumors. It was confirmed (P <0.001, Figure 1B). In addition, similar trends were confirmed in the TCGA dataset including 50 normal thyroid tissues and 492 papillary thyroid cancers. CXCL16 was significantly upregulated in papillary thyroid cancer compared to normal thyroid tissue (P <0.001, Figure 1C). Expression of CXCL16 receptor CXCR6 in papillary thyroid cancer tissue has been described in a previous study (Cho, SW et al. CXCL16 signaling mediated macrophage effects on tumor invasion of papillary thyroid carcinoma.Endocr Relat Cancer 23, 113-124 (2016) ).). CXCR6 was expressed in both cancer and stromal cells in the papillary thyroid cancer tumor microenvironment. We compared CXCR6 expression in normal and papillary thyroid cancer tissues. Through immunohistochemical staining, it was confirmed that the expression level was similar between normal thyroid epithelial cells and papillary thyroid cancer cells (FIG. 1D). In addition, in the RNA sequencing results of the SNUH cohort, it was confirmed that CXCR6 was similarly expressed in normal thyroid tissue and papillary thyroid cancer (FIG. 1E).

2.2 Increased mRNA expression of CXCL16 is associated with a worse prognosis in human papillary thyroid cancer.

To confirm the clinical and pathological features of papillary thyroid cancer expressing CXCL16, the TCGA dataset was further analyzed. 492 papillary thyroid cancers were classified into the CXCL16low group and the CXCL16high group based on the median CXCL16 expression value, and the clinical pathological characteristics between the two groups were compared (Table 2).

Compared to the CXCL16low group, the CXCL16high group showed a malignant pathologic phenotype including large cell variant, thyroid envelope extension, lymph node metastasis, and higher TNM stage (P <0.001; Table 2). . The CXCL16high group showed more BRAF ^V600E mutations than the CXCL16low group (63.3% vs 31.2%, P <0.001; Table 2). In addition, CXCL16 expression showed a significant negative association with the BRAF ^V600E -RAS score (BRS) (r = 0.772, P <0.001), meaning that the tumor in the CXCL16high group had a BRAF ^V600E -like transcription profile. do. Taken together, it can be seen that high expression of CXCL16 is associated with a poor clinical pathological prognostic factor.

2.3 High mRNA expression of CXCL16 is associated with angiogenesis-related genes in M2 macrophages and human papillary thyroid cancer.

To confirm the molecular characteristics of the CXCL16high group, 55 M2 macrophage-related genes and 65 blood vessels are known because increased infiltration of M2 macrophages and increased angiogenesis in papillary thyroid cancer are known to be the cause of tumor aggressiveness. 120 genes were analyzed, including genes related to formation. A total of 120 genes were observed and 26 genes were differently expressed in the CXCL16low group and the CXCL16high group (FIG. 2A). As a result of analyzing the differently expressed genes (differential expression genes), 11 M2 macrophage-related genes and 12 blood vessel formation-related genes were significantly upregulated, 1 M2 macrophage-related gene ( RIMBP2 ) and 2 blood vessels Formation-related genes ( PGF and EGF ) were significantly downregulated in the CXCL16high group compared to the CXCL16low group (FIG. 2A, and Table 3). In addition, a single sample gene set amplification analysis (single sample gene set enrichment analysis, ssGSEA) through M2 macrophage gene characteristics including 68 genes (Fig. 2B) and angiogenesis-related gene characteristics including 65 genes (Fig. 2C) ) Was confirmed to be highly expressed in the CXCL16high group compared to the CXCL16low group. Pathway analysis showed that the ERK score (P <0.001; FIG. 2D), PI3K / AKT (P = 0.03; FIG. 2E) and MAPK (P = 0.03; FIG. 2F) pathway-related genes from ssGSEA were significantly higher in the CXCL16high group than in the CXCL16low group. It was confirmed that it is up-regulated.

baseMean Fold change
(log2) SE of log2 fold change p -value p- adj CXCL16 3367.9 1.07 0.04 4.9229E-149 9.9212E-145 M2 macrophage-related genes ADAM8 421.3 1.53 0.09 2.35457E-64 6.87705E-62 MYO1F 787.8 1.31 0.09 1.81998E-49 1.23912E-47 AHNAK2 6327.5 1.82 0.13 2.09689E-45 1.0257E-43 C1QB 4223.1 1.47 0.11 1.19167E-43 4.90119E-42 CTSK 1686.3 1.45 0.10 1.76627E-43 7.14771E-42 C1QC 3234.7 1.35 0.10 3.15713E-41 1.04134E-39 CYTIP 464.6 1.54 0.12 1.33812E-37 3.42657E-36 XDH 167.8 1.79 0.21 4.09276E-18 2.527E-17 GDF15 6349.5 1.07 0.13 6.87204E-17 3.88368E-16 CD68 3755.0 1.46 0.09 6.65452E-57 8.82293E-55 CD163 1500.1 1.24 0.10 1.39156E-32 2.43861E-31 RIMBP2 217.3 -1.19 0.12 4.84637E-23 4.20987E-22 Genes related to angiogenesis IL8 496.8 1.94 0.14 3.53832E-43 1.38731E-41 THBS2 2601.0 1.79 0.13 6.31159E-42 2.17804E-40 TYMP 2210.1 1.13 0.09 9.34866E-34 1.77238E-32 INHBA 203.5 1.64 0.14 1.93868E-32 3.36522E-31 SERPINF1 2210.7 1.35 0.11 3.36127E-32 5.71162E-31 PLAU 10500.5 1.50 0.13 1.77974E-31 2.86021E-30 THBS1 5572.3 1.32 0.12 3.13788E-30 4.59243E-29 CCL2 849.6 1.10 0.12 1.2055E-21 9.62918E-21 MMP9 2366.6 1.37 0.14 2.16025E-21 1.69663E-20 AREG 200.2 1.49 0.16 6.94046E-21 5.28014E-20 IL1B 166.7 1.00 0.12 7.57222E-16 3.97094E-15 HBEGF 3440.9 1.01 0.13 2.43054E-15 1.22212E-14 PGF 4565.0 -1.48 0.12 2.58213E-37 6.48849E-36 EGF 114.7 -1.29 0.14 6.22895E-21 4.7514E-20

2.4 The combination of CXCL16 and related genes predicted relapse-free survival in patients with papillary thyroid cancer.

Because the high expression of CXCL16 alone was not significantly associated with cancer recurrence (Table 2 and Figure 3A), a panel of genes for prediction was prepared by combining CXCL16 and its associated differential expression genes. Interestingly, the combination of the three genes CXCL16 , AHNAK2 , and THBS2 was able to significantly predict the recurrence of the disease (Figure 3B). When the three genes of CXCL16 , AHNAK2 , and THBS2 were highly expressed, they showed shorter relapse-free survival compared to those not (p = 0.047). Also, including C1QC , TYMP , PLAU , MMP9 , CYTIP , or ADAM8 Adding another fourth gene to the gene panel could enhance the predicted value. 3C shows a representative Kaplan-Meier curve using a panel of 4 genes. Papillary thyroid cancers with higher expression of CXCL16 , AHNAK2 , THBS , and PLAU genes showed shorter relapse-free survival compared to the non-group (P = 0.019).

2.5 Therapeutic Effect of CXCL16 Inhibition in Papillary Thyroid Cancer Tumors) in vivo )

To confirm the therapeutic usefulness of inhibiting CXCL16 in advanced thyroid cancer, animal experiments were performed using a rat metastatic tumor xenograft model. First, the endogenous CXCL16 was genetically deleted by transforming shCXCL16 into two cancer cell lines BHP10-3SCp and FRO. Through ELISA analysis, secreted CXCL16 concentrations were 67% and 96 in BHP10-3SCp ^control and FRO ^control cells (meaning untransformed cells) in conditioned medium of BHP10-3SCp ^shCXCL16 and FRO ^shCXCL16 , respectively. % Decreases (Figure 4A, B). Viability 72 hours cell material was reduced significantly from 79.2%, FRO ^shCXCL16 cells BHP10-3SCp ^shCXCL16 cells by 57.4% compared to the respective control cells (Fig. 4C, D). Through the metastasis tumor model, tumors from immune histochemically stained BHP10-3SCp ^shCXCL16 cells are not only CXCL16 positive but also ERG positive epithelial cells and F4 / 80 positive macrophages from BHP10-3SCp ^shCXCL16 cells compared to BHP10-3SCp ^control cells. Significantly decreased in the tumor (Fig. 5B).

Total RNA was obtained from metastatic tumors of BHP10-3SCp ^control and BHP10-3SCp ^shCXCL16 cells to confirm that the differential expression between the CXCL16low and CXCL16high groups in the TCGA dataset was directly regulated by CXCL16. Interestingly, M2 macrophages and / or angiogenesis-related differential expression genes such as IL-8, MMP9, PLAU, ADAM ± ¡¾8, THBS2, AHNAK2 , and XDH are BHP10-3SCp ^shCXCL16 compared to BHP10-3SCp ^control cells. Cells were significantly downregulated in the tumor (FIG. 5C). Likewise, tumor growth was significantly delayed in tumors from FROshCXCL16 cells compared to FROcontrol cells (FIG. 5D).

Through accumulated evidence, it was confirmed that various cytokines mediate pre-oncogenic activity in a tissue-specific manner. In the present invention, the TCGA dataset analysis confirmed that increased CXCL16 expression was associated with M2 macrophage and angiogenesis-related gene expression, and a poor prognostic factor including BRAF ^V600E mutation and higher TNM stage. CXCL16 alone but do not predict the disease outcome, the relapse-free survival was predicted using a combination of three genes including a gene differentially expressed in two related AHNAK2, and THBS2 with CXCL16. In the rat metastasis tumor model, papillary thyroid cancer cells lacking CXCL16 significantly reduced tumor growth, macrophage infiltration, and tumor angiogenesis. In addition, CXCL16-related differential expression genes up-regulated from human study tumors were significantly reduced in tumors from CXCL16-deficient cells, suggesting that CXCL16 can actively regulate the conditions of pre-oncology of the papillary thyroid cancer tumor microenvironment. Taken together, a panel of three genes, including CXCL16, can predict poor prognosis for papillary thyroid cancer.

In prior studies, CXCL16 was described as tumor associated macrophage (TAM), supporting the potential for TAM-mediated cell migration and invasion of papillary thyroid cancer. In the present invention, it was confirmed that the expression of CXCL16 is associated with TAM- related and BRAF ^V600E -like representative genes in human papillary thyroid cancer. TAM is known to have a pre-oncogenic role in various human cancers, including thyroid cancer (Riabov, V. et al. Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis.Front Physiol 5, 75 (2014) .), Targeting TAM in anti-cancer therapy has been studied in depth at the cellular and molecular level. The first approach was to target the macrophages themselves through systemic deficiencies. However, prolonged systemic deficiency of macrophages can lead to host immune suppression and susceptibility to opportunistic infections. Therefore, targeting downstream mediators of TAM may be a good alternative strategy. Several angiogenic factors such as VEGF-A and MMP9 mediate the activity of TAM depending on the tissue or organ. Therefore, it is necessary to identify tissue-specific TAM mediators and evaluate in each specific organ. The CXCL16-related differential gene from the TCGA dataset, M2 macrophages and / or angiogenesis-related genes, was down-regulated in xenograft tumors from CXCL16-deficient cells. This suggests that CXCL16 plays an important role in TAM-rich tumor microenvironment.

One of the strengths of the present invention is that the clinical significance of CXCL16 expression has been identified as a prognostic marker for human papillary thyroid cancer. The tumor's microenvironment is a very heterogeneous complex that contains numerous cytokines and various cell types that secrete growth factors. Therefore, there is a limit to predicting a value for a single factor. The present invention provides reliable predictive values of a panel of genes for predicting papillary thyroid cancer, comprising two genes associated with CXCL16. In addition, the present invention needs to apply this panel of genes to human papillary thyroid cancer.

In conclusion, higher CXCL16 expression is associated with macrophages, angiogenesis- and BRAFV600-like representative genes in human papillary thyroid cancer. A panel of three genes associated with CXCL16 can be used to predict disease prognosis.

For example, for purposes of claim construction, the claims set forth below should not be interpreted in any way narrower than their literal language, and therefore, exemplary embodiments from the specification should not be read as claims. Accordingly, it should be understood that the invention has been described by way of illustration and not limitation on the scope of the claims. Accordingly, the invention is limited only by the following claims. All publications, patents, patent applications, books and journal articles cited in this application are hereby incorporated by reference in their entirety.

Claims (13)

A kit for predicting the prognosis of thyroid cancer, comprising a primer or probe that measures the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or an antibody that measures the level of a protein encoded by CXCL16, CXCL16, AHNAK2, and THBS2 . The method according to claim 1, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF , And primers or probes measuring one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, A kit further comprising an antibody that measures the level of a protein encoded by one or more selected from the group consisting of PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF . A composition for predicting the prognosis of thyroid cancer, comprising a primer or probe for measuring the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or an antibody for measuring the level of protein encoded by CXCL16, CXCL16, AHNAK2, and THBS2 . The method according to claim 3, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF , And primers or probes measuring one or more nucleic acid levels selected from the group consisting of EGF , or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, The composition further comprises an antibody that measures the level of a protein encoded by one or more selected from the group consisting of PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF . Contacting a sample with a composition comprising a primer or probe that measures the nucleic acid level of CXCL16, AHNAK2, and THBS2 , or contacting the sample with a composition comprising an antibody that measures the level of a protein encoded by CXCL16, AHNAK2, and THBS2 . Including,
If the CXCL16, AHNAK2, and nucleic acid level, or CXCL16, AHNAK2, and protein levels encoded by the THBS2 of THBS2 increased compared to a subject not having a thyroid cancer, which predicted to be the prognosis of the thyroid cancer is not good, information for predicting thyroid cancer prognosis How to provide. The method according to claim 5, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, PGF , And measuring one or more nucleic acid levels selected from the group consisting of EGF , ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, RIMBP2, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, A method of providing information, further comprising measuring the level of a protein encoded by one or more genes selected from the group consisting of CCL2, MMP9, AREG, IL1B, HBEGF, PGF, and EGF . The method according to claim 6, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, and HBEGF One or more nucleic acid levels selected from are increased compared to subjects without thyroid cancer, or ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2 , MMP9, AREG, IL1B, and HBEGF , a method for providing information, predicting that the prognosis of thyroid cancer is poor when the level of a protein encoded by one or more genes selected from the group consisting of is increased compared to a subject without thyroid cancer. The method of claim 6, wherein the level of one or more nucleic acids selected from the group consisting of RIMBP2, PGF , and EGF is reduced compared to a subject without thyroid cancer, or of a protein encoded by one or more genes selected from the group consisting of RIMBP2, PGF , and EGF . A method of providing information, predicting that the prognosis of thyroid cancer will be poor if the level is reduced compared to a subject without thyroid cancer. The method according to claim 5, wherein predicting that the prognosis is poor is predicting that the thyroid cancer will progress to a higher TNM (Tumor Node Metastasis) stage. Contacting a sample to be analyzed with cells comprising nucleic acid sequences of CXCL16, AHNAK2, and THBS2 ; And
Measuring the expression level of the nucleic acid,
When the expression level of the nucleic acid is reduced, the sample is a substance for preventing or treating thyroid cancer, and a screening method for a substance for treating thyroid cancer. The method according to claim 10, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, IL1B, HBEGF, RIMBP2, PGF And contacting a sample to be analyzed with cells comprising one or more nucleic acid sequences selected from the group consisting of EGF ; And measuring the expression level of the nucleic acid. The method according to claim 11, ADAM8, MYO1F, C1QB, CTSK, C1QC, CYTIP, XDH, GDF15, CD68, CD163, IL8, TYMP, INHBA, SERPINF1, PLAU, THBS1, CCL2, MMP9, AREG, When the level of one or more nucleic acids selected from the group consisting of IL1B, and HBEGF is increased, the sample is a substance for preventing or treating thyroid cancer. The method according to claim 11, When the level of one or more nucleic acids selected from the group consisting of RIMBP2, PGF , and EGF is reduced when the sample to be analyzed is contacted, the sample is a substance for preventing or treating thyroid cancer.

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