WO2006062118A1 - Novel markers for predicting prognosis of papillary carcinoma of the thyroid - Google Patents

Abstract

A marker for predicting the prognosis of papillary carcinoma of the thyroid which comprises at least one gene selected from the group consisting of SUV39H2, CRLF1, TMPRSS2, FXYD3, MYCN, NMU, TREX1, KCNV1, CAPN6, PAPPA, SLC7A5(hLAT1), RASGRF1, SFTPB, CYP4B1, C8orf4, EHF and CYP1B1, a transcription product thereof or a translation product thereof.

Description

 Specification

 A novel marker for predicting prognosis of papillary thyroid cancer

 Technical field

 [0001] The present invention relates to markers such as genetic markers, mRNA markers, and protein markers for diagnosing or predicting malignancy, risk, or prognosis of papillary thyroid cancer. The present invention also relates to an antibody against the protein marker, a method for diagnosing or predicting malignancy, risk or prognosis of papillary thyroid cancer, and a DNAZRNA chip for measuring the marker. Furthermore, the present invention relates to a kit for diagnosing or predicting malignancy, risk or prognosis of papillary thyroid cancer.

 Background art

 [0002] The number of patients with thyroid cancer is said to be 10,000 in Japan every year According to the American Cancer Society, the number of new thyroid cancer patients in the United States in 2005 was 25,690 Similarly, there are 3100 (2005) new cases per year in Canada and 1431 new thyroid cancer cases in 2001 in the UK.

 [0003] There are five types of thyroid cancer: papillary cancer, follicular cancer, medullary cancer, undifferentiated cancer, and malignant lymphoma. In Japan, papillary cancer accounts for 85-90% of thyroid cancer.

 [0004] The proportion of nipple cancer varies depending on the odor intake rate in the country, and it is considered that the frequency increases as the odor intake increases. In Europe and the United States, almost no seaweed is consumed, so the ratio of papillary cancer is low. In the United States, the power of the 1920s began to add salt to salt and bread, and recently, the proportion of papillary thyroid cancer is almost the same as Japan. It is the same. In Europe, on the other hand, in order to improve the deficiency of odors, we began to add odor to the salt. After 1973, papillary cancer increased by 47%, and around 1983, nipple cancer became nearly 70%, and odor was added to the salt. With the exception of Germany, it is approaching the frequency of Japan. The presence or absence of papillary thyroid cancer can be diagnosed by palpation, cervical soft x-ray, ultrasound (echo) examination, cytology, and cervical chest CT examination.

[0005] In general, papillary thyroid cancer rarely causes cancer death with a good prognosis. However, about 10% of cases of papillary thyroid cancer have a poor prognosis, and distant metastases such as lung, brain, and bone also lead to cancer death. Thus, papillary thyroid cancer is said to be classified into a low-risk group with good prognosis and a high-risk group with poor prognosis. Cancer mortality classification for papillary thyroid cancer is performed at the time of surgery based on the size of the primary lesion, age, presence / absence of metastasis, presence / absence of cancer invasion outside the thyroid, etc.

 [0006] Even if it is determined to be papillary thyroid cancer in the low-risk group at the time of surgery, not only if it is determined to be papillary thyroid cancer in the high-risk group, then cancer death due to local recurrence or hematogenous metastasis It is necessary to see the postoperative course. As papillary thyroid cancer progresses more slowly than other cancers such as lung cancer, the prognosis is not known until 5 to 10 years have passed. Although the possibility of metastasis is currently evaluated by the blood thyroglobulin level in order to observe the course, the measured value is not accurate when the anti-thyroglobulin antibody is present, and it increases when the cells are destroyed by inflammation. It is difficult to say that it has excellent specificity.

[0007] ^m I whole body scintigraphy with CT scan is very useful for diagnosing the presence or absence of papillary thyroid cancer, especially the progress of metastasis and recurrence after surgery. Since scan uses radiation, it has a drawback that frequent inspections cannot avoid affecting the living body.

 [0008] Patent document 1 describes a thyroid tumor marker, but this marker is insufficient as a tumor marker for papillary thyroid cancer.

 Patent Document 1: Japanese Unexamined Patent Application Publication No. 2004-283074

 Disclosure of the invention

 Problems to be solved by the invention

Accordingly, an object of the present invention is to provide markers such as genetic markers, mRNA markers, and protein markers for diagnosing or predicting malignancy, risk, or prognosis of papillary thyroid cancer.

 Another object of the present invention is to provide an antibody against the protein marker, a method for diagnosing or predicting malignancy, risk or prognosis of papillary thyroid cancer, and a DNAZ RNA chip for measuring the marker. To do.

[0011] Furthermore, an object of the present invention is to provide a kit for diagnosing or predicting malignancy, risk or prognosis of papillary thyroid cancer. Means for solving the problem

 [0012] The inventor classified papillary thyroid cancer patients into a low-risk group and a high-risk group according to age, distant metastasis, and presence / absence of invasion. In addition, the present inventor found that the high-risk group (Group A: elderly patients 51 years of age or older who had cancer metastasis invasion outside the thyroid by pathological findings) and the low-risk group (Group C) ： A young patient under 30 years old who suffered from localized thyroid gland due to pathological findings) and compared the gene expression levels in the tissues of the primary lesion of papillary thyroid cancer in detail, and conducted extensive studies As a result, it was found that the expression levels of 11 genes are significantly different.

 [0013] As a result of further intensive studies, the present inventor has found that 3 out of the 11 genes described above have a high risk group (Group A) and a low risk group (Group B: 51 years old) The above-mentioned elderly patients with pathological findings found that there was a marked difference in the expression level of the primary lesion of papillary thyroid cancer between the thyroid and the thyroid gland.

 [0014] Furthermore, there are 6 genes (RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, CYP1B1) between group A (the elderly and poor prognosis group) and group B (the elderly and good prognosis group). We found that the difference in the expression level was large.

 That is, the present invention relates to the invention shown in the following items:

 1. At least one gene selected from SUV39H2, CRLF1, TMPRSS2, FXYD3, MYCN, NMU, TREX1, KCNV1, CAP N6, PAPPA, SLC7A5 (hLATl), RASGRF1, SFTPB, CYP4B1, C8orf4, EHF and CYP1B1 Alternatively, a marker for predicting the prognosis of papillary thyroid cancer consisting of a transcription or translation product thereof.

 2. SUV39H2, CRLF1, TMPRSS2, FXYD3, MYCN, NMU, TREX1, KCNV1, CAP N6, PAPPA and SLC7A5 (hLATl) At least one gene selected from the group consisting of transcription or translation products The described marker.

 3. The marker according to item 1, comprising at least one gene selected from the group consisting of KCNV1, PAPPA and SLC7A5 (hLATl), or a transcription or translation product thereof.

4. The marker consists of mRNA which is a transcription product of the gene, Ma ¹ ~~ force according to claim 1 '~~.

5. The marker according to item 1, wherein the marker is also a protein force that is a translation product of the gene. Marker

 6. A method for predicting the prognosis of papillary thyroid cancer, comprising quantifying the transcription product or translation product of the gene according to item 1.

 7. The method according to item 6, wherein the translation product of the gene according to item 1 is quantified.

 8. The method according to item 6, wherein the translation product of the gene according to item 1 in a blood, plasma or serum sample is quantified.

 9. The method according to item 6, wherein the translation product is quantified by an antigen-antibody reaction.

 10. The method according to item 9, wherein an ELISA method is used.

 11. A method for predicting prognosis of papillary thyroid cancer, comprising a step of quantifying thyroglobulin and a step of quantifying a translation product of the gene according to item 1.

 12. A kit for quantifying the protein according to Item 5, comprising a primary antibody against the protein according to Item 5 and a labeled secondary antibody against the primary antibody.

 13. The kit according to item 13, for predicting the prognosis of papillary thyroid cancer.

 The invention's effect

 [0016] According to the tumor marker of the present invention, the malignancy, risk or prognosis of papillary thyroid cancer, which is the most frequent among thyroid cancers, can be diagnosed or predicted. It can be performed.

 [0017] For example, in patients diagnosed with papillary thyroid cancer, the prognosis is predicted to be poor, and after surgery, the primary papillary cancer tissue is removed by surgery, and recurrence or metastasis takes place every 6 months to 1 year. It is possible to prevent cancer death by conducting a precise examination in consideration.

 [0018] On the other hand, in cases where the prognosis is predicted to be good, if the size of the primary tumor is less than 1 cm, the size of the cancer is regularly monitored for increase, and even if surgery is performed Subsequent examinations can be omitted once every 2-3 years, eliminating unnecessary examinations, and by performing frequent CT scans and whole body scintigraphy to examine whether there is recurrence or metastasis. Medical exposure can be kept to a minimum.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

 [0020] (1) Gene marker, mRNA marker and protein marker for predicting prognosis of papillary thyroid cancer:

 In one of the preferred embodiments of the present invention, the genetic markers for predicting the prognosis of papillary thyroid cancer of the present invention include the following genes, and these genetic markers are used alone or in combination of two or more types of markers. In combination, it can predict the prognosis of papillary thyroid cancer:

 SUV39H2,

 CRLF1,

 TMPRSS2,

 FXYD3,

 MYCN,

 NMU,

 TREX1,

 KCNV1,

 CAPN6,

 PAPPA and

 SLC7A5 (hLATl).

 [0021] hLATl and SLC7A5 have different gene names and GenBank accession numbers! It is an amino acid transporter gene that has a common salt and a common salt.

[0022] The gene marker of the present invention is more preferably KCNV1, PAPPA and SLC7A5 (hLA

T1) Power is a group power that is chosen.

[0023] In another embodiment, the gene marker for predicting the prognosis of papillary thyroid cancer of the present invention includes the following genes, and these gene markers are used alone or in two kinds: Combining these markers can predict the prognosis of papillary thyroid cancer:

RASGRF1 SFTPB

 CYP4B1

 C8orf4

 EHF

CYP1B1.

 [0024] The base sequence of the gene marker of the present invention can be confirmed by searching a corresponding database accession number (Accession Number) described in Table I in a known database (NCBI).

 [0025] [Table 1] Table I

[0026] The mRNA marker for predicting the prognosis of papillary thyroid cancer of the present invention also comprises at least one mRNA force expressed by the gene marker gene of the present invention.

[0027] The protein marker for predicting the prognosis of papillary thyroid cancer of the present invention is the present invention. At least one protein expressed by the gene marker gene.

[0028] In the present specification, the gene marker, mRNA marker and protein marker for predicting the prognosis of papillary thyroid cancer of the present invention may be simply referred to as a tumor marker.

 [0029] (2) Make a note of the sign:

 In the present specification, “predicting the prognosis of papillary thyroid cancer” includes diagnosing the malignancy of thyroid cancer before surgery, predicting the course of postoperative papillary thyroid cancer, and the like. Papillary thyroid cancer is a slow-growing cancer that requires complete removal of the cancer tissue by surgery. Therefore, by knowing the possibility of metastasis before surgery, it is possible to determine an appropriate policy for the type and frequency of post-surgery examinations. For example, by measuring the tumor marker of papillary thyroid cancer of the present invention, it becomes possible to predict the progress of 5 to 10 years after the operation in advance. As a result, it is possible to sufficiently care for a high-grade patient as much as possible to reduce the burden on the low-grade patient.

 [0030] In one embodiment, the method for predicting the prognosis of papillary thyroid cancer of the present invention is characterized by quantifying at least one mRNA expressed by a gene of the gene marker of the present invention. Examples of specimens for quantifying mRNA include primary lesion surgical specimens, leukocytes in blood, and the like.

[0031] In one preferred embodiment of the present invention, the method for predicting prognosis of papillary thyroid cancer of the present invention comprises quantifying at least one protein expressed by a gene of the gene marker of the present invention. To do. The protein can be quantified by, for example, HPLC, gel electrophoresis, isoelectric focusing, antigen-antibody reaction and the like. The ELISA method is particularly preferred as an antigen-antibody reaction. Conventionally, thyroglobulin has been used to predict the prognosis of papillary thyroid cancer metastasis. Thyroglobulin was thought to decrease in measured values due to surgery for papillary thyroid cancer and then to increase when metastasis occurred, but its accuracy was not satisfactory. However, if necessary, combine the measured values of at least one of the tumor markers of the present invention with a thyroglobulin sample (blood sample such as tissue, serum, plasma, lymph fluid, urine, saliva, etc.) if necessary. Thus, more accurate diagnosis of papillary thyroid cancer may be possible. [0032] The risk diagnosis of papillary thyroid cancer using the gene marker of the present invention is, for example, in the thyroid tissue, blood or urine (preferably in the blood) of the normal control group with the infomed'concentration. Based on the amount of the tumor marker of the present invention, it can be carried out by comparing this standard with the amount of the tumor marker of the present invention in papillary thyroid cancer tissue, blood or urine (preferably in blood).

 [0033] When predicting prognosis using the tumor marker of papillary thyroid cancer of the present invention, the quantification of the tumor marker (mRNA, protein) is, for example, absolute per cell in the case of mRNA. It may be measured as a relative amount or as a relative amount. In the case of protein, it can be measured as the concentration in a sample such as blood. It is preferable to measure the mRNA gene expression level as a relative amount from the viewpoint that the measurement operation is simple and accurate. The relative amount can be determined, for example, by setting a gene as an internal control and using the gene expression level. The gene serving as the internal control is not particularly limited as long as it is a gene expressed in normal thyroid tissue cells and thyroid tumor cells, but for example, β-cuttin (GenBank Ac No. X00351) gene, GAPDH gene, etc. A housekeeping gene is preferably exemplified. When the tumor marker is a protein, it can be preferably measured by an immunoassay such as ELISA using an antibody (monoclonal antibody, polyclonal antibody, particularly monoclonal antibody) against the protein.

[0034] The method for quantifying the mRNA marker is not particularly limited as long as it can quantitate the amount of specific mRNA in the cell. For example, the mRNA of the mRNA marker or the base sequence of the cDNA or Examples thereof include a method using a primer or probe that is an oligonucleotide that also serves as a partial force of the complementary base sequence and contains an oligonucleotide that binds site-specifically to the mRNA or cDNA of the mRNA marker. The primers and probes described above have various modifications for detecting and quantifying the mRNA if the oligonucleotide forms site-specific base pairs with the mRNA of the marker mRNA or its cDNA. It may be. Further, as the method, a simple method with a small amount of required sample and a high accuracy and sensitivity is preferable. Specifically, for example, a real-time PCR method, a competitive PCR method, a method for directly measuring mRNA, etc. Can be given. Among these, for example, the same tube or well More preferred is a method in which the mRNA of the gene marker of the present invention and the mRNA of the internal control gene can be measured simultaneously by the above reaction.

 [0035] As the real-time PCR method, for example, cDNA is synthesized from intracellular total RNA or mRNA using reverse transcriptase, and the target region is amplified by PCR using this cDNA as a cage, and a reagent for real-time monitoring. A method for monitoring and analyzing the production process of amplification products in real time using the. Examples of the real-time monitoring reagent include SYBR (registered trademark: MolecularProbes) Greenl, TaqMan (registered trademark: Applied Biosystems) probe, and the like.

 [0036] The competitive PCR method includes, for example, a method of synthesizing cDNA from intracellular total RNA or mRNA using reverse transcriptase, and reacting this cDNA with a DNA competitor in the same tube, Further, there may be mentioned a method in which an RNA competitor is added together with mRNA during the reverse transcription reaction. Further, the internal sequence other than the competitor primer sequence may be, for example, a sequence homologous to the sequence of the mRNA for amplification or a non-homologous sequence.

 [0037] Furthermore, examples of the method for directly measuring the mRNA include Invader (registered trademark: ThirdWave Technologies) RNA assembly. However, the marker gene mRNA quantification method for predicting the prognosis of papillary thyroid cancer using the gene marker of the present invention is not limited to these methods, and the oligonucleotide, primer or probe is used. Various quantification methods used can be applied.

[0038] As a specific method for quantifying the protein marker for quantifying the genetic marker for predicting the prognosis of papillary thyroid cancer of the present invention, any method can be used as long as it can quantitate a specific protein in a cell. For example, a method using an antibody specific for the protein of the protein marker can be mentioned, and among them, a simple method with a small amount of necessary cells and a high accuracy and sensitivity is preferable. Specific examples include various enzyme immunoassays (EIA) and radio immunoassays (RIA). Among these, the enzyme-linked immunosorbent assay (ELISA) is used because it is more sensitive and simple. ) And Sandwich ELISA are preferred. The antibody used in these methods may be a monoclonal antibody or a polyclonal antibody, depending on the protein quantification method. . However, the protein marker quantification method for diagnosis of papillary thyroid cancer of the present invention is not limited to these methods.

 [0039] In one preferred embodiment, the antibody of the present invention is, for example, an antibody produced by immunizing a mouse with a protein expressed by the gene marker gene of the present invention, an antibody produced from a hybridoma, particularly a monoclonal antibody. An antibody is mentioned. The animal used for immunization is particularly preferably a mouse, such as mice, rats, rabbits, goats and the like. In order to obtain the antibody of the present invention, it is necessary to prepare a hybridoma using the protein expressed by the gene of the gene marker of the present invention as an immunogen, and then select an hybridoma that produces an antibody that reacts with the protein. There is. Induction of immunity can usually be performed by dividing the immunogen in an amount of lng to 10 mg into 1 to 5 times over 10 to 14 days. After sufficient immunization, organs capable of producing antibodies (spleen and lymph nodes) are aseptically removed from the animal and used as the parent strain at the time of cell fusion. The spleen is most preferable as an organ to be removed. Myeloma cells are used as cell fusion partners. Myeloma cells are derived from mice

, Rat origin, human origin, etc., preferably mouse origin. Examples of cell fusion include methods using polyethylene glycol, cell electrofusion, and the like, but methods using polyethylene glycol are simple and preferred. Selection of spleen cells, myeloma cells, and hybridomas that did not undergo cell fusion can be performed, for example, by culturing in a serum medium supplemented with HAT supplement (hypoxanthine monoaminopterin-thymidine). The selection of the hyperidoma is preferably direct ELISA on an EIA plate in which the above-mentioned culture supernatant is collected and the protein expressed by the gene marker gene of the present invention is immobilized. Select a well that showed strong color development as a result of direct ELISA, and subject the cell of the well to clawing. The hybridoma corresponding to the strongly colored culture supernatant is selected as a hybridoma that produces an antibody that reacts with the protein expressed by the gene marker gene of the present invention.

[0040] Cloning is an operation for selecting and unifying antibody-producing hyperpridoma. There are a limiting dilution method, a fibrin gel method, a method using a cell sorter, etc., but the limiting dilution method is preferred. As a result, a hybridoma that produces the desired monoclonal antibody can be obtained. By culturing the hyperidoma obtained by the above method, in the culture supernatant Monoclonal antibodies can be obtained. Furthermore, in order to obtain a large amount of monoclonal antibodies, there are in vivo and in vitro methods, but in vivo methods are preferred, particularly in mouse ascites. Purification of monoclonal antibodies from the culture supernatant and mouse ascites is performed by ammonium sulfate salt folding, affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, etc. Considering simplicity, affinity chromatography is the most preferred. In order to obtain a higher purity monoclonal antibody, it is preferable to perform gel filtration chromatography or ion exchange chromatography as final purification after affinity chromatography. In order to use purified monoclonal antibodies for sandwich ELISA, antibody combinations must be determined. A sandwich ELISA can measure a small amount of antigen by sandwiching the antigen between two different antibodies, but each antibody preferably reacts with a different epitope. In order to determine the combination, it is preferable that a part of the purified monoclonal antibody is immobilized on an EIA plate and a part thereof is labeled with piotin or the like. However, if the antibody class is different, the labeling is not necessarily performed. Not necessary. A serially diluted protein expressed by the gene marker gene of the present invention is added to an EIA plate on which an antibody is immobilized, and an antibody labeled or not labeled is added to examine the combination. Finally, it is preferred to select a combination that can measure at least 10 ng / ml protein, preferably lng / ml protein.

 [0041] (3) Kit for predicting prognosis of papillary thyroid cancer, DNAZRNA / chip:

 The DNAZRNA chip of the present invention comprises DNAZRNA capable of hybridizing with mRNA expressed by the gene marker gene of the present invention.

 [0042] The kit of the present invention includes a primary antibody against a protein expressed by the gene marker gene of the present invention, and a labeled secondary antibody against the primary antibody.

[0043] The kit used for quantifying the mRNA marker for predicting the prognosis of papillary thyroid cancer using the gene marker of the present invention is for amplifying the cDNA of the mRNA marker in a quantifiable manner. A specific mRNA in a cell comprising the primer and polymerase, and the probe to be paired with the amplification product for detection. Other consumable reagents included in the kit of the present invention are not particularly limited. Examples include enzymes, knockers, reaction reagents, and (d) NTP mixes necessary for quantifying mRNA.

 [0044] Further, a kit used for quantifying a protein marker for predicting prognosis of papillary thyroid cancer using the gene marker of the present invention comprises a first antibody specific for the protein of the protein marker, A kit for quantifying a specific protein in a cell containing a second antibody specific to the first antibody, for example, an antibody labeled with an appropriate enzyme or chemical substance. Other consumable reagents contained in the kit of the present invention are not particularly limited, and examples thereof include enzymes, buffers, reaction reagents and the like necessary for quantifying proteins.

 [0045] Further, the DNAZRNA chip used for quantifying the mRNA level for the determination of papillary thyroid cancer using the gene marker of the present invention is the nucleotide sequence of the mRNA of the mRNA marker or its cDNA or the This is a DNAZRNA chip equipped with an oligo DNAZRNA that also serves as part of the complementary base sequence.

 [0046] Further, the DNAZRNA chip used for quantifying the mRNA marker for the determination of papillary thyroid cancer using the gene marker of the present invention, the mRNA of the mRNA marker or its nucleotide sequence or its complementary nucleotide sequence This is a DNAZRNA chip with oligo DNAZRNA.

 Example

 [0047] The present invention will be described in more detail in the following examples, but the present invention is not limited to the following examples.

[0048] (1) Primary screening:

 (sample)

 The following samples were used in the primary screen:

 • High risk group (Group A: elderly patients over 51 years old with pathological findings of metastasis of cancer outside the thyroid gland) 2 cases

 • Low risk group (Group C: young patients under 30 years old with pathological findings and localized thyroid cancer) 2 cases.

[0049] (Tissue sample and RNA preparation) Tissue of thyroid papillary carcinoma (TPC) was obtained by surgery after obtaining informed consent of the patient. All tissues were quenched in liquid nitrogen and stored at –80 ° C until use. Tumors were classified by skilled pathologists according to the WHO histological classification of thyroid tumors, and the tissues were of high purity (> 90% neoplastic cells in the tumor).

 [0050] Total RNA was obtained from the method of Chmczynski and Sacchi (Single-Step Methods of RNA Isolation by Acid Guanidinium Thyocyanate-Phenol— Cnloroform Extraction. Chomczy nski P and Sacchi N. Anal. Biochem. 162: 156-159, 1987. ) And purified using RN easy Mini kit (Qiagen, Valencia, CA).

 [0051] (Microarray analysis)

 Microarray analysis was performed as detailed at http: 〃 www.afiVmetrix.com! cRNA is prepared from 10 μg of total RNA, hybridized to HG-U133A Gene chip, Affimetrix oligonucleotide arrays (containing over 20,000 human genes), scanned, and analyzed according to the Aifymetrix (Santa Clara, CA) protocol did. Scanned image files were visually inspected for artifacts and standardized using GCOS software (Affimetrix). The fold change values, which show relative changes in gene expression levels, between young and old TPC tumors are compared and expressed differently between these two conditions by using the GCOS software (Affimetrix). Identified genes

[0052] (Data analysis)

Using these cDNA samples as a gene chip manufactured by Affinmetrix, a gene group with a different expression between two groups of papillary cancer that is considered to have a good prognosis and a papillary cancer that is thought to have a poor prognosis is known 20,000 genes Elected the middle power. The j8 actin gene was used as an endgenous control. As a result, the top 95 genes were selected from genes with significantly different expression levels that had a gene expression relative ratio of 2.0 or more between the two groups. The results are shown in Table II below. The 95 genes listed in Table II have a relative gene expression ratio between the two groups of 3.6 or more, and may be useful as genetic markers for predicting the prognosis of papillary thyroid cancer. Next, secondary screening was performed using these 95 genes.

表 4] 表 II

[表 5] 表 II (続き) (Fine) [^ 0053 Table 3] Table II (continued)

Table 4] Table II

[Table 5] Table II (continued)

(2) Secondary screening

 (sample)

 The following samples were used in the secondary screening, which are different from the primary screening:

• High-risk group (Group A: elderly patients over 51 years old with pathological findings of metastasis of cancer outside the thyroid gland) 4 cases • Low risk group (Group B: Elderly people over 51 years old with pathological findings and localized thyroid cancer) 4 cases.

 [0058] · Low risk group (Group C: young patients under 30 years old with pathological findings of cancer with limited thyroid gland).

 [0059] (Preparation of tissue samples and RNA)

 Tissue samples and RNA were prepared and cDNA was synthesized in the same manner as the primary screening. A sample of papillary thyroid cancer different from the primary screening (Group C: young people (under 30 years old) with cancer remaining in the thyroid gland. Group A: Old people (51 years old and over) with distant cancer Using 4 samples of metastatic or extrathyroidal invasion as a sample, the expression level of these genes was quantified by TaqMan PCR using ABI ABI 7900HT and Microfludic card (Distinctive gene expression of human lung adenocarcinoma carrying LKBl mutations. Oncogene 23, 5084-5091, 2004).

 [0060] (Micro Fluidic Cards)

 The Micro Fluidic Card (Applied Biosystems, Foster City, Calif.) Containing Applied Biosystems fluorogenic 5 nuclease assays was used to detect gene expression differences among the 95 targets selected by the gene chip. The relative level of gene expression was determined from fluorescence data generated during PCR real-time quantitative RT-PCR (TaqMan PCR) using the ABI PRISM 7900 HT Sequence Detection (Applied Biosystems). An external endogenous control (FAM-GAPDH) was used as a standard in relative quantitation calculations. lOOng cDNA and TaqMan Universal Master Mix (Applied Biosystems) were used for the assembly.

 [0061] As a result, 11 genes (SUV39H2 24.9 fold, CRLF1 16.3 fold, TMPRSS2 12.96 fold, FXYD3 12.7 fold, MYCN 11.5 fold, NMU 11.1 fold, TREX1 10.4 fold, expressed differently between group A and group C by 9.0 fold or more. Times, KCNV1 9.9 times, CAPN6 9.9 times, PAPPA 9.0 times, SLC7A5 (hLA Tl) 13.7 times) (+ GAPDH gene was used as Endgenous control). The results are shown in Table III.

[0062] Among these 11 genes, the same year was used to exclude the effect of age on gene expression. Significant also in cases where the cancer stays within the thyroid gland at the age of age (in this case, 51 years or older) and between cases where the cancer is distantly metastasized or has extravasation outside the thyroid (ie, between groups A and B) In this study, genes with different gene expression levels were examined. Three of these genes (SLC7A5 (hLA T1) 137.57 times, PAPPA 70.57 times, KCNV1 23.6 times) showed cancer infiltration (prognosis) rather than age It was revealed that it significantly affected the difference in gene expression. The results are shown in Table III.

 [0063] That is, these three genes (hLATl and SLC7A5 are the same gene) are classified into group A (old people with poor prognosis !, group) and group C (young people) according to their gene expression levels. And a difference (over 9.0 times) between the A group (the elderly and the poor prognosis group) and the B group (the elderly and the good prognosis group). Significant differences were observed (more than 23.6 times). That is, these three genes are genes that showed a greater difference in prognosis (cancer malignancy) rather than age, and are considered to be very useful as markers for predicting the prognosis of papillary thyroid cancer.

 [0064] In addition, 23 genes listed in Table III (RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, CYP1 Bl, HS3ST1, PCDHA2, SCEL, CXCL2, KIAA1579, CH13L1, PR02533, CD2AP, PROSl, CMG1, EROl-L, OSF-2, AGR2, FOSB, ZNF surgery, SMC2L1, FLNB) were also measured for their gene expression levels. As a result, group A (the elderly had a poor prognosis! Group) and group C (the young Although the difference was not as strong as the above 11 genes (6.8 times or less), the group A (the elderly had a poor prognosis !, group) and the group B (the elderly had a prognosis) There was a greater difference (1.92 to 51.8 times). In other words, these 23 genes are also found to have differences in prognosis (cancer malignancy) rather than age, and are considered useful as markers for predicting the prognosis of papillary thyroid cancer. In particular, among these 23 genes, 6 genes (RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, CYP1B1) have large differences in expression levels between group A and group B (5.9 to 51.8 times), and papillary thyroid cancer It may be useful as a genetic marker for predicting the prognosis.

[0065] [Table 6] Table III. Results of secondary screening

 (“-” In the table indicates that there was no difference.) Gene name A / C A / B

SUV39H2 24.9 times 11.9 times

CRLF1 16.3 times 3.9 times

TMPRSS2 12.96 times 2.5 times

FXYD3 12.7 times 1.3 times

MYCN 11.5 times 2.9 times

NMU 11.1 times 5.9 times

TREX1 10.4 times 1

CAPN6 9.9 times 1.85 times

SLC7A5 (hLATl) 13.7 times 137.57 times

PAPPA 9.0 times 70.57 times

KCNV1 9.9 times 23.6 times

RASGRFl 2.0 times 51.8 times

SFTPB 6.8 times 12.4 times

CYP4B 1 1.57 times 11.4 times

C8orf4 2.23 times 10 times

EHF 3.5 times 7.9 times

CYP I BI 1.2 times 5.9 times

HS3ST1 5.2 times 4.6 times

PCDHA2 4.3 times 4.2 times

SCEL 1.5 times 4.0 times

CXCL2 1.9 times 3.8 times

KIAA1579 3.4 times 3.5 times

CH13L1 ― 3.4 times

PR02533 1.7 times 3.3 times

CD2AP ― 3.2 times

PROS1 ― 3.1 times

CMG1 3.0 times 2.9 times

EROl-L 1.1 times 2.6 times

OSF-2 1.3 times 2.6 times

AGR2 1.2 times 2.2 times

FOSB 2.5 times 2.2 times

ZNF407 3.0 times 2.1 times

SMC2L1 1.2 times 1.98 times

FLNB ― 1.92 times [0066] In addition, blood thyroglobulin levels before surgery were measured for groups A, B, and C used in the secondary screening. The results are shown in Table IV along with the pathological findings.

[0067] [Table 7]

 Table IV. Results of blood thyroglobulin measurement

産業上の利用可能性

Industrial applicability

 Based on the above results, these 11 genes (especially 3 genes) are considered to be useful in clinical examination as markers for predicting the prognosis of papillary thyroid cancer. In addition, a combination of these genes or a combination with thyroglobulin is new and may be useful because it increases the diagnosis rate as a thyroid cancer tumor. The expression level of these genes can be quantified at the blood level by using leukocytes in the blood as well as the surgical tissue, or the antigen-antibody method can be used at the protein level, which is convenient at the serum level. It can be measured, predicts the prognosis of patients, and can be useful for searching for recurrence and metastasis after surgery, which is very useful in outpatient practice.

Claims

 The scope of the claims  [I] SUV39H2, CRLF1, TMPRSS2, FXYD3, MYCN, NMU, TREX1, KCNV1, CAPN6, PAPPA, SLC7A5 (hLATl), RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, and CYPl Bl A marker for predicting the prognosis of papillary thyroid cancer comprising a gene or a transcription or translation product thereof.  [2] Claim 1 consisting of at least one gene selected from the group consisting of SUV39H2, CRLF1, TMPRSS2, FXYD3, MYCN, NMU, TREX1, KCNV1, CAPN6, PAPPA and SLC7A5 (hLATl), or a transcription or translation product thereof. Marker described in.  [3] The marker according to claim 1, which also has at least one gene selected from the group that also has KCNV1, PAPPA, and SLC7A5 (hLATl) power, or a transcription or translation product power thereof. [4] The mouse ¹ ~~ force` ~~ according to claim 1, wherein the marker comprises mRNA which is a transcription product of the gene.  [5] The marker according to claim 1, wherein the marker also has a protein power that is a translation product of the gene.  [6] A method for predicting the prognosis of papillary thyroid cancer, comprising quantifying the transcription product or translation product of the gene according to claim 1. [7] The method according to claim 6, wherein the translation product of the gene according to claim 1 is quantified.  [8] The method according to claim 6, wherein the translation product of the gene according to claim 1 in a blood, plasma or serum sample is quantified. [9] The method according to [6], wherein the translation product is quantified by an antigen-antibody reaction.  [10] The method according to claim 9, wherein ELISA is used.  [II] A method for predicting prognosis of papillary thyroid cancer, comprising a step of quantifying thyroglobulin and a step of quantifying a translation product of the gene according to claim 1.  [12] A kit for quantifying the protein according to claim 5, comprising a primary antibody against the protein according to claim 5 and a labeled secondary antibody against the primary antibody. [13] The kit according to claim 13, for predicting the prognosis of papillary thyroid cancer.