JP5629894B2 - A novel marker for diagnosing papillary thyroid cancer - Google Patents
A novel marker for diagnosing papillary thyroid cancer Download PDFInfo
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Description
本発明は、甲状腺乳頭癌を診断するための遺伝子マーカー、ｍＲＮＡマーカー、蛋白質マーカー、抗体、方法、ＤＮＡチップおよびキットに関する。 The present invention relates to a genetic marker, mRNA marker, protein marker, antibody, method, DNA chip and kit for diagnosing papillary thyroid cancer.
甲状腺癌には乳頭癌、濾胞癌、髄様癌、未分化癌、悪性リンパ腫の５種類が存在し、日本では甲状腺癌の85〜90％を乳頭癌が占めている。 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 to 90% of thyroid cancer.
甲状腺乳頭癌は、一般的に、予後が良く、癌死することはほとんどない。しかし、甲状腺乳頭癌の約10％の症例は予後が悪く、肺、脳、骨などの遠隔転移から癌死に至る。このように、甲状腺乳頭癌は、予後の良い低リスク群と予後の悪い高リスク群に分類されるといわれている。甲状腺乳頭癌の癌死危険度分類は、手術の時点で、原発巣の大きさ、年齢、転移の有無、癌の甲状腺外への浸潤の有無等によって行われる。 Papillary thyroid cancer generally has a good prognosis and rarely dies. However, about 10% of cases of papillary thyroid cancer have a poor prognosis, leading to cancer death from distant metastases such as lung, brain, and bone. 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. The cancer death risk classification of 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 invasion of cancer outside the thyroid, and the like.
高リスク群の甲状腺乳頭癌であると判定された場合だけでなく、手術の時点で低リスク群の甲状腺乳頭癌と判定された場合も、その後、局所再発あるいは血行性転移で癌死することがあるので、術後の経過をみる必要がある。甲状腺乳頭癌は肺癌等の他の癌に比し進行が遅いため、5年から10年の経過を見ないとその予後はわからないのが現状である。経過を見る上で転移の可能性は現在血中サイログロブリン値で評価しているが、抗サイログロブリン抗体を持つ際には測定値は正確ではなく、また炎症によって細胞が破壊される際にも上昇が認められるため、特異性に優れているとは言いがたい。 Not only if it is determined to be papillary thyroid cancer in the high-risk group, but also if it is determined to be papillary thyroid cancer in the low-risk group at the time of surgery, the cancer may subsequently die due to local recurrence or hematogenous metastasis. Because there is, it is necessary to see the progress after surgery. As papillary thyroid cancer progresses slower than other cancers such as lung cancer, the prognosis is not known unless 5 to 10 years have passed. Although the possibility of metastasis is currently evaluated by the blood thyroglobulin level, the measured value is not accurate when having anti-thyroglobulin antibodies, and it increases when cells are destroyed by inflammation. Because it is recognized, it is difficult to say that it has excellent specificity.
特許文献１は甲状腺腫瘍マーカーを記載しているが、このマーカーは甲状腺乳糖癌の腫瘍マーカーとしては不十分なものである。
従って、本発明は、甲状腺乳頭癌を診断するための新規の遺伝子マーカー、ｍＲＮＡマーカー、蛋白質マーカー、抗体、方法、ＤＮＡチップおよびキットを提供することを目的とする。 Accordingly, an object of the present invention is to provide a novel gene marker, mRNA marker, protein marker, antibody, method, DNA chip and kit for diagnosing papillary thyroid cancer.
本発明者は、甲状腺乳頭癌患者を、年齢および遠隔転移・浸潤の有無により、低リスク群と高リスク群に分類した。そして、本発明者は、高リスク群（Ａ群：50歳以上の老年者であって、病理所見で甲状腺外に癌の転移・浸潤が見られた患者）および低リスク群（Ｃ群：30歳以下の若年者であって、病理所見で癌が甲状腺内限局していた患者）において、甲状腺乳頭癌の原発巣の組織における遺伝子発現量を詳細に比較し、鋭意検討を重ねた結果、１１個の遺伝子について著しく発現量に差があることを見出した。 The inventor classified papillary thyroid cancer patients into a low-risk group and a high-risk group according to age and presence / absence of distant metastasis / invasion. Then, the inventor of the present invention has a high risk group (Group A: elderly patients over 50 years old who have metastasis / invasion of cancer outside the thyroid by pathological findings) and a low risk group (Group C: 30 As a result of extensive comparison of gene expression levels in tissues of the primary lesion of papillary thyroid cancer in a young patient under the age of a patient whose cancer was localized in the thyroid by pathological findings, It was found that there was a significant difference in the expression level for each gene.
更に鋭意検討を重ねた結果、本発明者は、上記１１遺伝子のうち３遺伝子について、同年齢（老年者）における高リスク群（上記Ａ群）と低リスク群（Ｂ群：50歳以上の老年者であって病理所見で癌が甲状腺内限局していた患者）間の甲状腺乳頭癌の原発巣の組織における発現量においても著しく差があることを見出した。これらの知見から、本発明者は本発明を完成した。 As a result of further intensive studies, the present inventor has found that about 3 genes out of the 11 genes described above, the high-risk group (Group A) and the low-risk group (Group B: old age over 50 years old) in the same age (elderly) We found that there was also a significant difference in the expression level of papillary thyroid cancer in the tissues of the primary lesion among patients who had been diagnosed with pathological findings. Based on these findings, the present inventor completed the present invention.
即ち、本発明は、以下の各項に示す発明に関する：
項１． 以下の少なくとも１種の遺伝子からなる、甲状腺乳頭癌を診断するための遺伝子マーカー：
SUV39H2
CRLF1
TMPRSS2
FXYD3
MYCN
NMU
TREX1
KCNV1
CAPN6
PAPPA
SLC7A5（ｈLAT1）。
項２． 以下の少なくとも１種の遺伝子からなる、上記項１に記載の遺伝子マーカー：
KCNV1
PAPPA
SLC7A5（ｈLAT1）。
項３． 以下の少なくとも１種の遺伝子からなる、甲状腺乳頭癌を診断するための遺伝子マーカー：
RASGRF1
SFTPB
CYP４B１
C8orf4
EHF
CYP１B１。
項４． 上記項１〜３のいずれかに記載の遺伝子によって発現される少なくとも１種のｍＲＮＡからなる、甲状腺乳頭癌を診断するためのｍＲＮＡマーカー。
項５． 上記項１〜３のいずれかに記載の遺伝子によって発現される少なくとも１種の蛋白質からなる、甲状腺乳頭癌を診断するための蛋白質マーカー。
項６． 上記項１〜３のいずれかに記載の遺伝子によって発現される少なくとも１種のｍＲＮＡを定量することを特徴とする、甲状腺乳頭癌を診断する方法。
項７． 上記項１〜３のいずれかに記載の遺伝子によって発現される少なくとも１種の蛋白質を定量することを特徴とする、甲状腺乳頭癌を診断する方法。
項８． 抗原抗体反応により前記蛋白質を定量することを特徴とする、上記項７に記載の方法。
項９． ＥＬＩＳＡ法を用いることを特徴とする、上記項８に記載の方法。
項１０． サイログロブリンを定量する工程と項１〜３のいずれかに記載の遺伝子によって発現される少なくとも１種の蛋白質を定量する工程を含むことを特徴とする、甲状腺乳頭癌を診断する方法。
項１１． 上記項４に記載のｍＲＮＡとハイブリダイズし得るＤＮＡを備える、ＤＮＡチップ。
項１２． 上記項５に記載の蛋白質に対する抗体。
項１３． 上記項５に記載の蛋白質に対する一次抗体と、該一次抗体に対する標識化された二次抗体とを含む、キット。
項１４． 甲状腺乳頭癌を診断するための項１３に記載のキット。
That is, the present invention relates to the invention shown in the following items:
Item 1. A genetic marker for diagnosing papillary thyroid cancer comprising at least one of the following genes:
SUV39H2
CRLF1
TMPRSS2
FXYD3
MYCN
NMU
TREX1
KCNV1
CAPN6
PAPPA
SLC7A5 (hLAT1).
Item 2. Item 2. The gene marker according to Item 1, comprising at least one of the following genes:
KCNV1
PAPPA
SLC7A5 (hLAT1).
Item 3. A genetic marker for diagnosing papillary thyroid cancer comprising at least one of the following genes:
RASGRF1
SFTPB
CYP4B1
C8orf4
EHF
CYP1B1.
Item 4. 4. An mRNA marker for diagnosing papillary thyroid cancer, comprising at least one mRNA expressed by the gene according to any one of items 1 to 3.
Item 5. A protein marker for diagnosing papillary thyroid cancer, comprising at least one protein expressed by the gene according to any one of Items 1 to 3.
Item 6. A method for diagnosing papillary thyroid cancer, comprising quantifying at least one mRNA expressed by the gene according to any one of Items 1 to 3.
Item 7. A method for diagnosing papillary thyroid cancer, characterized by quantifying at least one protein expressed by the gene according to any one of Items 1 to 3.
Item 8. Item 8. The method according to Item 7, wherein the protein is quantified by an antigen-antibody reaction.
Item 9. Item 9. The method according to Item 8, wherein an ELISA method is used.
Item 10. A method for diagnosing papillary thyroid cancer, comprising a step of quantifying thyroglobulin and a step of quantifying at least one protein expressed by the gene according to any one of Items 1 to 3.
Item 11. A DNA chip comprising DNA that can hybridize with the mRNA according to Item 4.
Item 12. 6. An antibody against the protein according to item 5.
Item 13. 6. A kit comprising a primary antibody against the protein according to item 5 above, and a labeled secondary antibody against the primary antibody.
Item 14. Item 14. The kit according to Item 13, for diagnosing papillary thyroid cancer.
以下、本発明をより詳細に説明する。 Hereinafter, the present invention will be described in more detail.
（１）甲状腺乳頭癌を診断するための遺伝子マーカー、ｍＲＮＡマーカーおよび蛋白質マーカー：
本発明の甲状腺乳頭癌を診断するための遺伝子マーカーは、以下の少なくとも１種の遺伝子からなる：
SUV39H2
CRLF1
TMPRSS2
FXYD3
MYCN
NMU
TREX1
KCNV1
CAPN6
PAPPA
SLC7A5（ｈLAT1）。
(1) Genetic marker, mRNA marker and protein marker for diagnosing papillary thyroid cancer:
The genetic marker for diagnosing papillary thyroid cancer of the present invention comprises at least one of the following genes:
SUV39H2
CRLF1
TMPRSS2
FXYD3
MYCN
NMU
TREX1
KCNV1
CAPN6
PAPPA
SLC7A5 (hLAT1).
hLAT1とSLC7A5は、遺伝子名およびGene Bank のaccession numberは異なっているが、同一遺伝子であり、共通の塩基配列を有するamino acid transporter geneである。 hLAT1 and SLC7A5 are amino acid transporter genes having the same gene and a common base sequence, although the gene name and the accession number of Gene Bank are different.
本発明の遺伝子マーカーは、より好ましくは、以下の少なくとも１種の遺伝子からなる：
KCNV1
PAPPA
SLC7A5（ｈLAT1）。
More preferably, the genetic marker of the present invention comprises at least one of the following genes:
KCNV1
PAPPA
SLC7A5 (hLAT1).
また、別の実施形態において、本発明の遺伝子マーカーは、以下の少なくとも１種の遺伝子からなる：
RASGRF1
SFTPB
CYP４B１
C8orf4
EHF
CYP１B１。
In another embodiment, the genetic marker of the present invention consists of at least one of the following genes:
RASGRF1
SFTPB
CYP4B1
C8orf4
EHF
CYP1B1.
本発明の遺伝子マーカーの塩基配列は、公知のデータベース（NCBI）で、表１に記載の対応のアクセッション番号（Accession Number）を検索することによって確認できる。 The base sequence of the gene marker of the present invention can be confirmed by searching the corresponding accession number described in Table 1 in a known database (NCBI).
本発明の甲状腺乳頭癌を診断するためのｍＲＮＡマーカーは、本発明の遺伝子マーカーの遺伝子によって発現される少なくとも１種のｍＲＮＡからなる。 The mRNA marker for diagnosing papillary thyroid cancer of the present invention comprises at least one mRNA expressed by the gene of the gene marker of the present invention.
また、本発明の甲状腺乳頭癌を診断するための蛋白質マーカーは、本発明の遺伝子マーカーの遺伝子によって発現される少なくとも１種の蛋白質からなる。 The protein marker for diagnosing papillary thyroid cancer of the present invention comprises at least one protein expressed by the gene marker gene of the present invention.
本明細書において、本発明の甲状腺乳頭癌を診断するための遺伝子マーカー、ｍＲＮＡマーカーおよび蛋白質マーカーを合わせて、単に腫瘍マーカーと呼ぶことがある。 In the present specification, the genetic marker, mRNA marker, and protein marker for diagnosing papillary thyroid cancer of the present invention may be simply referred to as a tumor marker.
（２）甲状腺乳頭癌を診断する方法：
本明細書において、「甲状腺乳頭癌を診断する」とは、術前に甲状腺癌の悪性度を診断すること、甲状腺乳頭癌の予後を予測すること、術後の甲状腺乳頭癌の経過を予測すること等を含む。甲状腺乳頭癌は、癌組織の手術による全摘出が必要であるが、進行が遅い癌である。従って、手術前に転移の可能性を予め把握しておくことで、手術後の検査の種類、頻度などについて適切な方針を決定することができる。例えば、本発明の甲状腺乳頭癌の腫瘍マーカーを測定することで、手術後５〜１０年の経過を事前に予測することが可能になる。これにより、悪性度の低い患者の負担を軽減できるだけでなく、悪性度の高い患者のケアを十分に行うことが可能になる。
(2) Method for diagnosing papillary thyroid cancer:
In this specification, “diagnosing papillary thyroid cancer” means diagnosing the malignancy of thyroid cancer before surgery, predicting the prognosis of papillary thyroid cancer, and predicting the course of postoperative papillary thyroid cancer. Including things. Papillary thyroid cancer is a slow-growing cancer that requires complete removal of the cancer tissue by surgery. Therefore, by grasping in advance the possibility of metastasis before surgery, it is possible to determine an appropriate policy for the type and frequency of examination after surgery. 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. This not only reduces the burden on patients with low malignancy, but also makes it possible to sufficiently care for patients with high malignancy.
１つの実施形態において、本発明の甲状腺乳頭癌を診断する方法は、本発明の遺伝子マーカーの遺伝子によって発現される少なくとも１種のｍＲＮＡを定量することを特徴とする。ｍＲＮＡを定量するための検体としては、原発巣の手術検体、血液中の白血球等が挙げられる。 In one embodiment, the method for diagnosing papillary thyroid cancer of the present invention is characterized by quantifying at least one mRNA expressed by the gene marker gene of the present invention. Examples of specimens for quantifying mRNA include primary specimens of surgical specimens, leukocytes in blood, and the like.
本発明の１つの好ましい実施形態において、本発明の甲状腺乳頭癌を診断する方法は、本発明の遺伝子マーカーの遺伝子によって発現される少なくとも１種の蛋白質を定量することを特徴とする。該蛋白質は抗原抗体反応により定量することができる。抗原抗体反応としてはＥＬＩＳＡ法が特に好ましい。甲状腺乳頭癌の転移等の予後を予測するために、従来サイログロブリンが使用されてきた。サイログロブリンは、甲状腺乳頭癌の手術により測定値が低下し、その後転移が起こると測定値が上昇すると考えられていたが、その精度は満足のいくものではなかった。しかしながら、本発明の腫瘍マーカーの少なくとも１種とサイログロブリンのサンプル（組織、血清、血漿等の血液サンプル、リンパ液、尿、唾液等）の測定値を組み合わせることで、より精度の高い甲状腺乳頭癌の診断が可能になり得る。 In one preferred embodiment of the present invention, the method for diagnosing papillary thyroid cancer of the present invention is characterized by quantifying at least one protein expressed by the gene marker gene of the present invention. The protein can be quantified by antigen-antibody reaction. The ELISA method is particularly preferred as the antigen-antibody reaction. Conventionally, thyroglobulin has been used to predict the prognosis such as metastasis of papillary thyroid cancer. Thyroglobulin was thought to decrease in measured values due to surgery for papillary thyroid cancer and then increase in measured values when metastasis occurred, but the accuracy was not satisfactory. However, more accurate diagnosis of papillary thyroid cancer by combining at least one of the tumor markers of the present invention and a measured value of a thyroglobulin sample (blood sample such as tissue, serum, plasma, lymph fluid, urine, saliva). Can be possible.
本発明の遺伝子マーカーを用いた甲状腺乳頭癌の診断は、例えば、低リスク群（30歳以下の若年者であって、病理所見で癌が甲状腺内に限局していた患者）からの甲状腺組織、血液または尿中（好ましくは、血液中）における本発明の腫瘍マーカーの量を基準とし、この基準と甲状腺乳頭癌組織、血液または尿中（好ましくは、血液中）における本発明の腫瘍マーカーの量とを比較することによって行われ得る。 Diagnosis of papillary thyroid cancer using the gene marker of the present invention is, for example, a thyroid tissue from a low-risk group (a patient younger than 30 years whose pathological findings are limited to cancer in the thyroid), Based on the amount of the tumor marker of the present invention in blood or urine (preferably in blood), this amount and the amount of the tumor marker of the present invention in papillary thyroid cancer tissue, blood or urine (preferably in blood). And can be done by comparing
本発明の甲状腺乳頭癌の腫瘍マーカーを用いた診断を行う場合、腫瘍マーカー（ｍＲＮＡ、蛋白質）の定量は、例えば、ｍＲＮＡの場合には１細胞あたりの絶対量として測定してもよく、相対量として測定してもよい。タンパク質の場合には、血液等のサンプル中の濃度として測定することができる。測定操作が簡便であり正確である点等から、ｍＲＮＡの遺伝子発現量は、相対量として測定することが好ましい。前記相対量は、例えば、内部対照となる遺伝子を設定し、その遺伝子発現量を用いて求めることができる。前記内部対照となる遺伝子としては、正常甲状腺組織細胞および甲状腺腫瘍細胞で発現する遺伝子であれば特に限定されないが、例えば、βアクチン（ＧｅｎＢａｎｋ Ａｃ Ｎｏ． Ｘ００３５１）遺伝子、GAPDH遺伝子が好ましく例示できる。腫瘍マーカーがタンパク質の場合、当該タンパク質に対する抗体（モノクローナル抗体、ポリクローナル抗体、特にモノクローナル抗体）を使用して、ＥＬＩＳＡ等の免疫学的測定法により好ましく測定できる。 When the diagnosis using the tumor marker of papillary thyroid cancer of the present invention is performed, the quantification of the tumor marker (mRNA, protein) may be measured as an absolute amount per cell in the case of mRNA, for example. May be measured. In the case of protein, it can be measured as the concentration in a sample such as blood. From the viewpoint that the measurement operation is simple and accurate, the gene expression level of mRNA is preferably measured as a relative amount. 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. For example, β-actin (GenBank Ac No. X00351) gene and GAPDH gene can be preferably exemplified. When the tumor marker is a protein, it can be preferably measured by an immunoassay such as ELISA using an antibody against the protein (monoclonal antibody, polyclonal antibody, particularly monoclonal antibody).
本発明の甲状腺乳頭癌を診断するための遺伝子マーカーを定量するための前記ｍＲＮＡマーカーを定量する方法としては、細胞内の特定ｍＲＮＡ量を定量できる方法であれば、特に制限されず、例えば、前記ｍＲＮＡマーカーのｍＲＮＡもしくはそのｃＤＮＡの塩基配列またはそれらの相補塩基配列の一部からなるオリゴヌクレオチドであって、前記ｍＲＮＡマーカーのｍＲＮＡまたはｃＤＮＡに部位特異的に結合するオリゴヌクレオチドを含むプライマーやプローブを用いた方法が挙げられる。前記プライマーやプローブは、前記オリゴヌクレオチドが前記マーカーｍＲＮＡのｍＲＮＡまたはそのｃＤＮＡと部位特異的塩基対を形成するものであれば、前記ｍＲＮＡを検出・定量するための様々な修飾がされたものであってよい。また、前記方法としては、必要試料量が少なく、精度および感度がよく、簡便な方法が好ましく、具体的には、例えば、リアルタイムＰＣＲ法やコンペティティブＰＣＲ法、または、ｍＲＮＡを直接測定する方法等があげられる。これらの中でも、例えば、同一チューブまたはウェル内の反応で、本発明の遺伝子マーカーのｍＲＮＡと内部対照遺伝子のｍＲＮＡとを同時に測定できる方法がより好ましい。 The method for quantifying the mRNA marker for quantifying the genetic marker for diagnosing papillary thyroid cancer of the present invention is not particularly limited as long as it is a method capable of quantifying the amount of specific mRNA in the cell. An oligonucleotide consisting of a part of the base sequence of the mRNA marker mRNA or its cDNA or a complementary base sequence thereof, and a primer or probe containing an oligonucleotide that binds site-specifically to the mRNA or cDNA of the mRNA marker Method. The primers and probes may be modified in various ways to detect and quantify the mRNA as long as the oligonucleotide forms a site-specific base pair with the mRNA of the marker mRNA or its cDNA. You can. In addition, the method is preferably a simple method that requires a small amount of sample, has high accuracy and sensitivity, and is specifically a real-time PCR method, a competitive PCR method, a method for directly measuring mRNA, or the like. can give. Among these, for example, a method that can simultaneously measure mRNA of the gene marker of the present invention and mRNA of the internal control gene in a reaction in the same tube or well is more preferable.
前記リアルタイムＰＣＲ法としては、例えば、細胞内のトータルＲＮＡやｍＲＮＡから逆転写酵素を用いてｃＤＮＡを合成し、このｃＤＮＡを鋳型に目的領域をＰＣＲで増幅し、リアルタイムモニタリング用試薬を用いて増幅産物の生成過程をリアルタイムでモニタリングし、解析する方法があげられる。前記リアルタイムモニタリング試薬としては、例えば、ＳＹＢＲ（登録商標：ＭｏｌｅｃｕｌａｒＰｒｏｂｅｓ社）ＧｒｅｅｎＩや、ＴａｑＭａｎ（登録商標：Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）プローブ等があげあられる。 As the real-time PCR method, for example, cDNA is synthesized from intracellular total RNA or mRNA using reverse transcriptase, the target region is amplified by PCR using this cDNA as a template, and amplified product using a reagent for real-time monitoring. A method for monitoring and analyzing the generation process of the worm in real time. Examples of the real-time monitoring reagent include SYBR (registered trademark: Molecular Probes) Green I, TaqMan (registered trademark: Applied Biosystems) probe, and the like.
また、前記コンペティティブＰＣＲ法としては、例えば、細胞内のトータルＲＮＡやｍＲＮＡから逆転写酵素を用いてｃＤＮＡを合成し、このｃＤＮＡとＤＮＡコンペティターとを同一チューブ内で反応させる方法や、さらに前記逆転写反応時にｍＲＮＡとともにＲＮＡコンペティターを加えて反応させる方法等があげられる。またコンペティターのプライマー配列以外の内部配列としては、例えば、増幅目的ｍＲＮＡの配列と相同配列でもよく、非相同な配列でもよい。 Examples of the competitive PCR method include, 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. For example, a method of reacting by adding an RNA competitor together with mRNA during the reaction may be mentioned. The internal sequence other than the competitor primer sequence may be, for example, a sequence homologous to the sequence of the amplification-target mRNA or a non-homologous sequence.
またさらに、前記ｍＲＮＡを直接測定する方法としては、例えば、Ｉｎｖａｄｅｒ（登録商標：ＴｈｉｒｄＷａｖｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）ＲＮＡアッセイ等があげられる。ただし、本発明の遺伝子マーカーを用いて甲状腺乳頭癌を診断する方法のためのマーカー遺伝子のｍＲＮＡの定量方法としては、これらの方法に限られず、前記オリゴヌクレオチド、プライマーまたはプローブを用いた種々の定量方法を適用できる。 Furthermore, examples of the method for directly measuring the mRNA include Invader (registered trademark: Third Wave Technologies) RNA assay. However, the method for quantifying the mRNA of the marker gene for the method of diagnosing papillary thyroid cancer using the gene marker of the present invention is not limited to these methods, and various quantifications using the oligonucleotides, primers or probes. Method can be applied.
本発明の甲状腺乳頭癌を診断するための遺伝子マーカーを定量するための前記蛋白質マーカーを定量する具体的方法としては、細胞内の特定蛋白質を定量できる方法であれば、特に制限されず、例えば、前記蛋白質マーカーの蛋白質に特異的な抗体を用いた方法があげられ、その中でも、必要な細胞量が少なく、精度および感度がよく、簡便な方法が好ましい。具体的には、例えば、各種のエンザイムイムノアッセイ（ＥＩＡ）やラジオイムノアッセイ（ＲＩＡ）等があげられ、これらの中でも、より感度がよく、簡便という点から、固相酵素免疫検定法（ＥＬＩＳＡ）やサンドウィッチＥＬＩＳＡが好ましい。これらの方法に使用する抗体としては、前記蛋白質の定量方法に応じて、モノクローナル抗体であっても、ポリクローナル抗体であってもよい。ただし、本発明の甲状腺乳頭癌の診断のための蛋白質マーカーの定量方法は、これらの方法に限られない。 The specific method for quantifying the protein marker for quantifying the genetic marker for diagnosing papillary thyroid cancer of the present invention is not particularly limited as long as it is a method capable of quantifying a specific protein in a cell. A method using an antibody specific for the protein of the protein marker can be mentioned. Among them, a simple method is preferable because it requires a small amount of cells, has high accuracy and sensitivity. Specific examples include various enzyme immunoassays (EIA) and radioimmunoassays (RIA). Among these, solid-phase enzyme immunoassay (ELISA) and sandwiches are preferred because they are more sensitive and simple. ELISA is 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.
本発明の抗体は、例えば本発明の遺伝子マーカーの遺伝子によって発現される蛋白質をマウスに免疫して得られたハイブリドーマから産生される抗体、特にモノクローナル抗体である。免疫に用いられる動物には、マウス、ラット、ウサギ、ヤギ等があげられるが、マウスが特に好ましい。本発明の抗体を得るためには、本発明の遺伝子マーカーの遺伝子によって発現される蛋白質を免疫原としてハイブリドーマを作製した後、該蛋白質に反応する抗体を産生するハイブリドーマを選択する必要がある。免疫の惹起は、通常1ng〜10mgの量の免疫原を10〜14日の日数を開けて1〜5回に分けた操作で行うことができる。十分な免疫後、抗体産生能を有する器官（脾臓やリンパ節）を動物から無菌的に摘出し、細胞融合時の親株とする。なお、摘出する器官としては、脾臓が最も好ましい。細胞融合のパートナーとしては、ミエローマ細胞が用いられる。ミエローマ細胞には、マウス由来、ラット由来、ヒト由来等があるが、マウス由来が好ましい。細胞融合には、ポリエチレングリコールを用いる方法、細胞電気融合法等が挙げられるが、ポリエチレングリコールを用いる方法が簡便で好ましい。細胞融合しなかった脾臓細胞やミエローマ細胞とハイブリドーマとの選択は、例えばHATサプリメント（ヒポキサンチン−アミノプテリン−チミジン）を添加した血清培地で培養することで行うことができる。ハイブリドーマの選択は、前述の培養上清を採取し、本発明の遺伝子マーカーの遺伝子によって発現される蛋白質を固相化したEIAプレートでの直接ELISAが好ましい。直接ELISAの結果、強い発色がみられたウエルを選択し、そのウエルの細胞をクローニングに供する。その強く発色した培養上清に対応するハイブリドーマを、本発明の遺伝子マーカーの遺伝子によって発現される蛋白質に反応する抗体を産生するハイブリドーマとして選択する。 The antibody of the present invention is, for example, an antibody produced from a hybridoma obtained by immunizing a mouse with a protein expressed by the gene marker gene of the present invention, particularly a monoclonal antibody. Examples of animals used for immunization include mice, rats, rabbits and goats, with mice being particularly preferred. In order to obtain the antibody of the present invention, it is necessary to prepare a hybridoma using a protein expressed by the gene marker gene of the present invention as an immunogen, and then select a hybridoma that produces an antibody that reacts with the protein. Induction of immunity can usually be performed by an operation in which an immunogen in an amount of 1 ng to 10 mg is divided into 1 to 5 times with 10 to 14 days. After sufficient immunization, organs capable of producing antibodies (spleen and lymph nodes) are aseptically removed from the animals and used as parent strains for cell fusion. The spleen is most preferable as an organ to be removed. Myeloma cells are used as cell fusion partners. Myeloma cells include mouse origin, rat origin, human origin, etc., but mouse origin is preferred. 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 or myeloma cells that have not undergone cell fusion and hybridomas can be performed, for example, by culturing in a serum medium supplemented with HAT supplements (hypoxanthine-aminopterin-thymidine). The selection of the hybridoma is preferably direct ELISA on an EIA plate obtained by collecting the culture supernatant described above and immobilizing the protein expressed by the gene marker gene of the present invention. As a result of direct ELISA, a well showing strong color development is selected, and the cells in the well are subjected to cloning. The hybridoma corresponding to the strongly colored culture supernatant is selected as a hybridoma producing an antibody that reacts with the protein expressed by the gene marker gene of the present invention.
クローニングとは、抗体産生ハイブリドーマを選別し単一化する作業であり、限界希釈法、フィブリンゲル法、セルソーターを用いる方法等があるが限界希釈法が好ましい。これにより、目的とするモノクローナル抗体を産生するハイブリドーマを獲得することができる。上記方法により得られたハイブリドーマを培養することで、培養上清中にモノクローナル抗体を得ることができる。さらに、大量のモノクローナル抗体を得るには、in vivoおよびin vitroによる方法があるが、in vivoによる方法、特にマウス腹水で得る方法が好ましい。培養上清やマウス腹水からのモノクローナル抗体の精製は、硫酸アンモニウム塩折法、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、ハイドロキシアパタイトカラムクロマトグラフィー等により行われるが、精製純度や簡便性を考慮するとアフィニティークロマトグラフィーが最も好ましい。さらに高純度のモノクローナル抗体を得るためには、アフィニティークロマトグラフィーの後に最終精製としてゲルろ過クロマトグラフィーやイオン交換クロマトグラフィー等を行うのが好ましい。精製したモノクローナル抗体をサンドイッチELISAに利用するためには、抗体の組み合わせを決定しなければならない。サンドイッチELISAは異なる2種類の抗体で抗原を挟み込むことで微量な抗原を測定できるが、それぞれの抗体は異なるエピトープに反応することが好ましい。組み合わせを決定するためには、精製したモノクローナル抗体の一部をEIAプレートに固相化し、一部をビオチン等で標識化することが好ましいが、抗体のクラスが異なれば標識化は必ずしも必要ではない。抗体を固相化したEIAプレートに本発明の遺伝子マーカーの遺伝子によって発現される蛋白質を段階希釈したものを添加し、標識化した、あるいはしていない抗体を添加して組み合わせを検討する。最終的に、少なくとも10ng/ml、好ましくは1ng/mlの蛋白質まで測定できる組み合わせを選択することが好ましい。 Cloning is an operation for selecting and unifying antibody-producing hybridomas. There are a limiting dilution method, a fibrin gel method, a method using a cell sorter, etc., but the limiting dilution method is preferred. Thereby, the hybridoma which produces the target monoclonal antibody is acquirable. By culturing the hybridoma obtained by the above method, a monoclonal antibody can be obtained in the culture supernatant. Furthermore, in order to obtain a large amount of monoclonal antibody, there are in vivo and in vitro methods, but an in vivo method, particularly a method using mouse ascites is preferred. Monoclonal antibodies are purified from the culture supernatant or mouse ascites by ammonium sulfate salt folding, affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, etc., but affinity chromatography is used in consideration of purification purity and simplicity. Is most preferred. In order to obtain a higher-purity monoclonal antibody, gel filtration chromatography, ion exchange chromatography, or the like is preferably performed as a final purification after affinity chromatography. In order to use the purified monoclonal antibody in a sandwich ELISA, the antibody combination must be determined. A sandwich ELISA can measure a minute 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 to immobilize a part of the purified monoclonal antibody on an EIA plate and label a part with biotin or the like, but labeling is not necessarily required if the antibody class is different. . A serially diluted protein expressed by the gene marker gene of the present invention is added to an antibody-immobilized EIA plate, and a labeled or unlabeled antibody is added to examine the combination. Finally, it is preferable to select a combination that can measure at least 10 ng / ml, preferably 1 ng / ml of protein.
（３）甲状腺乳頭癌を診断するためのキット、ＤＮＡチップ：
本発明のＤＮＡチップは、本発明の遺伝子マーカーの遺伝子によって発現されるｍＲＮＡとハイブリダイズし得るＤＮＡを備える。
(3) Kit for diagnosing papillary thyroid cancer, DNA chip:
The DNA chip of the present invention comprises DNA that can hybridize with mRNA expressed by the gene marker gene of the present invention.
また、本発明のキットは、本発明の遺伝子マーカーの遺伝子によって発現される蛋白質に対する一次抗体と、該一次抗体に対する標識化された二次抗体とを含む。 Moreover, the kit of the present invention comprises 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.
本発明の遺伝子マーカーを用いての甲状腺乳頭癌を診断するためのｍＲＮＡマーカーを定量するために使用するキットは、前記ｍＲＮＡマーカーのｃＤＮＡを定量可能なように増幅するための前記プライマーおよびポリメラーゼと、検出のため前記増幅産物に対合させる前記プローブとを含む細胞内の特定ｍＲＮＡを定量できるキットである。本発明のキットに含まれるその他の消耗試薬としては、特に制限されず、例えば、ｍＲＮＡを定量するために必要な酵素、バッファー、反応試薬等があげられる。 A kit used for quantifying an mRNA marker for diagnosing papillary thyroid cancer using the genetic marker of the present invention comprises the primer and a polymerase for amplifying the mRNA marker cDNA in a quantifiable manner; A kit capable of quantifying specific mRNA in a cell containing 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, and examples thereof include enzymes, buffers, reaction reagents and the like necessary for quantifying mRNA.
また、本発明の遺伝子マーカーを用いての甲状腺乳頭癌を診断するための蛋白質マーカーを定量するために使用するキットは、前記蛋白質マーカーの蛋白質に特異的な第一の抗体と、前記第一の抗体に特異的な第二の抗体であって、例えば、適宜な酵素または化学物質で標識化された抗体とを含む細胞内の特定蛋白質を定量するためのキットである。本発明のキットに含まれるその他の消耗試薬としては、特に制限されず、例えば、蛋白質を定量するために必要な酵素、バッファー、反応試薬等があげられる。 Further, a kit used for quantifying a protein marker for diagnosing papillary thyroid cancer using the gene marker of the present invention comprises a first antibody specific for a protein of the protein marker, and the first antibody. A second antibody specific to an antibody, for example, a kit for quantifying a specific protein in a cell containing an antibody labeled with an appropriate enzyme or chemical substance. Other consumable reagents included 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.
また、本発明の遺伝子マーカーを用いた甲状腺乳頭癌の判定のためのｍＲＮＡマーカーを定量するために使用するＤＮＡチップは、前記ｍＲＮＡマーカーのｍＲＮＡもしくはそのｃＤＮＡの塩基配列またはその相補塩基配列の一部からなるオリゴＤＮＡを備えるＤＮＡチップである。 In addition, a DNA chip used for quantifying an mRNA marker for determination of papillary thyroid cancer using the gene marker of the present invention is a part of a base sequence of the mRNA marker mRNA or its cDNA or a complementary base sequence thereof. A DNA chip comprising an oligo DNA consisting of
本発明の腫瘍マーカーによれば、甲状腺分化癌の中でも最も頻度の多い乳頭癌を診断することができ、転移や予後を予測することで診断や治療に役立て、各個人に応じたオーダーメード医療を行うことができる。 According to the tumor marker of the present invention, papillary cancer, which is the most frequent of differentiated thyroid cancer, can be diagnosed, and it can be used for diagnosis and treatment by predicting metastasis and prognosis. It can be carried out.
本発明を以下の実施例において更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described in more detail in the following examples, but the present invention is not limited to the following examples.
（１）一次スクリーニング：
（サンプル）
一次スクリーニングにおいて、以下のサンプルを使用した：
・高リスク群（Ａ群：50歳以上の老年者であって、病理所見で甲状腺外に癌の転移・浸潤が見られた患者）２例
・低リスク群（Ｃ群：30歳以下の若年者であって、病理所見で癌が甲状腺内限局していた患者）２例。
(1) Primary screening:
(sample)
The following samples were used in the primary screen:
・ High-risk group (Group A: elderly patients over 50 years old with pathological findings of cancer metastasis and invasion outside the thyroid) ・ Low-risk group (Group C: young people under 30 years old) 2 patients whose pathological findings had localized cancer within the thyroid.
（組織サンプルおよびRNAの調製）
甲状腺乳頭癌（thyroid papillary carcinoma ; TPC）の組織を、患者のインフォームドコンセントを得た後、手術により得た。全ての組織を液体窒素中で急冷し、使用するまで−８０℃で保存した。腫瘍は、熟練した病理学者により甲状腺腫瘍のＷＨＯ組織学的分類に従って分類され、組織は高い純度を有した（腫瘍中９０％以上の新生物性細胞）。
(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 had a high purity (> 90% neoplastic cells in the tumor).
トータルRNAを、ChmczynskiおよびSacchiの方法（Single-Step Methods of RNA Isolation by Acid Guanidinium Thyocyanate- Phenol -Chloroform Extraction. Chomczynski P and Sacchi N. Anal.Biochem. 162:156-159, 1987.）に従って抽出し、そしてRNeasy Mini kit （Qiagen, Valencia, CA）を使用して精製した。 Total RNA was extracted according to the Chmczynski and Sacchi method (Single-Step Methods of RNA Isolation by Acid Guanidinium Thyocyanate-Phenol-Chloroform Extraction. Chomczynski P and Sacchi N. Anal. Biochem. 162: 156-159, 1987.) And it refine | purified using RNeasy Mini kit (Qiagen, Valencia, CA).
（マイクロアレイ分析）
マイクロアレイ分析をhttp://www.affymetrix.comで詳細に記載されているように行った。cRNAを１０μｇのトータルRNAから調製し、HG-U133A Gene chip, Affimetrix oligonucleotide arrays （２万以上のヒト遺伝子を含有）へハイブリダイズさせ、スキャンし、そしてAffymetrix (Santa Clara, CA) プロトコルに従って分析した。スキャンした画像ファイルを、artifactsのために視覚的に検査し、そしてGCOSソフトウエア（Affimetrix）を使用して標準化した。若年TPC腫瘍と老年TPC腫瘍との間で、遺伝子発現レベルの相対変化を示すthe fold change valuesを比較して、GCOSソフトウエア（Affimetrix）を使用することによってこれら２つの条件の間で異なって発現される遺伝子を同定した。
(Microarray analysis)
Microarray analysis was performed as described in detail at http://www.affymetrix.com. cRNA was 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 Affymetrix (Santa Clara, CA) protocol. Scanned image files were visually inspected for artifacts and standardized using GCOS software (Affimetrix). Compare the fold change values, which show relative changes in gene expression levels, between young and old TPC tumors and express differently between these two conditions by using GCOS software (Affimetrix) Identified genes.
（データ分析）
これらのｃＤＮＡをサンプルとしてAffinmetrix社製のGene chipを用いて予後の良いと考えられる乳頭癌と予後が悪いと考えられる乳頭癌の2群間で発現の異なる遺伝子群を既知の2万遺伝子の中から選出した。βアクチン遺伝子をEndgenous controlとして使用した。その結果、2群間で遺伝子発現相対比が2.0以上である有意に発現量が異なる遺伝子の中から上位95遺伝子を選択した。結果を下記の表２に示す。表２に記載の95遺伝子は、2群間での遺伝子発現相対比が3.6以上であり、甲状腺乳頭癌を診断するための遺伝子マーカーとして有用であり得る。次いで、これらの95遺伝子を用いて二次スクリーニングを行った。
(Data analysis)
Using these Affinmetrix Gene chips as a sample of these cDNAs, a group of genes whose expression is different between the two groups of papillary cancer that has a good prognosis and a papillary cancer that has a poor prognosis are known 20,000 genes. Elected from. The β-actin gene was used as an endgenous control. As a result, the top 95 genes were selected from genes whose expression levels differed significantly between the two groups with a gene expression relative ratio of 2.0 or more. The results are shown in Table 2 below. The 95 genes listed in Table 2 have a gene expression relative ratio between the two groups of 3.6 or more, and may be useful as genetic markers for diagnosing papillary thyroid cancer. Subsequently, secondary screening was performed using these 95 genes.
（２）二次スクリーニング
（サンプル）
二次スクリーニングにおいて、一次スクリーニングとは異なる以下のサンプルを使用した：
・高リスク群（Ａ群：50歳以上の老年者であって、病理所見で甲状腺外に癌の転移・浸潤が見られた患者）４例
・低リスク群（Ｂ群：50歳以上の老年者であって、病理所見で癌が甲状腺内限局していた患者）４例。
・低リスク群（Ｃ群：30歳以下の若年者であって、病理所見で癌が甲状腺内限局していた患者）５例。
(2) Secondary screening (sample)
The following samples were used in the secondary screen, which are different from the primary screen:
・ High-risk group (Group A: elderly patients over 50 years old, with pathological findings showing cancer metastasis / invasion outside the thyroid) ・ Low-risk group (Group B: elderly people over 50 years old) 4 patients with pathological findings and localized cancer within the thyroid).
-5 cases of low-risk group (Group C: young patients under 30 years old whose pathological findings had localized cancer within the thyroid).
（組織サンプルおよびRNAの調製）
一次スクリーニングと同様にして組織サンプルおよびRNAの調製とcDNA合成を行った。一次スクリーニングとは異なる甲状腺乳頭癌のサンプル（Ｃ群：若年者（30歳以下）で癌が甲状腺内に留まっている症例4例。Ａ群：老年者（50歳以上）で癌が遠隔転移あるいは甲状腺外に浸潤をもつ症例4例）をサンプルとしてこれらの遺伝子発現量をABI社のABI 7900HTとMicrofludic cardを用いてTaqMan PCR法により定量し（Distinctive gene expression of human lung adenocarcinoma carrying LKB1 mutations. Oncogene 23, 5084-5091, 2004）、再検討を行った。
(Tissue sample and RNA preparation)
Tissue samples and RNA were prepared and cDNA was synthesized as in the primary screening. A sample of papillary thyroid cancer different from the primary screening (Group C: young people (under 30 years old) with 4 cases of cancer remaining in the thyroid gland. Group A: elderly people (over 50 years old) with distant metastasis or cancer In 4 cases with extrathyroidal invasion, 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 LKB1 mutations. Oncogene 23 , 5084-5091, 2004).
（Micro Fluidic Cards）
Applied Biosystems fluorogenic 5’ nuclease assays を含むThe Micro Fluidic Card (Applied Biosystems, Foster City, CA )を、遺伝子チップによって選択された９５標的間での遺伝子発現の差異を検出するために使用した。遺伝子発現の相対レベルを、the ABI PRISM 7900 HT Sequence Detection (Applied Biosystems)を使用して、ＰＣＲリアルタイム定量ＲＴ−ＰＣＲ（TaqMan PCR）の間に発せられる蛍光データより測定した。外部の内部対照（external endogenous control）（FAM-GAPDH）を、相対定量計算（relative quantitation calculations）における標準物質として使用した。１００ｎｇのcDNAおよびTaqMan Universal Master Mix (Applied Biosystems)をアッセイのために使用した。
(Micro Fluidic Cards)
The Micro Fluidic Card (Applied Biosystems, Foster City, Calif.) Containing Applied Biosystems fluorogenic 5 ′ nuclease assays was used to detect differences in gene expression 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 internal control (FAM-GAPDH) was used as a standard in relative quantitation calculations. 100 ng cDNA and TaqMan Universal Master Mix (Applied Biosystems) were used for the assay.
その結果、Ａ群とＣ群との間で9.0倍以上発現の異なる11遺伝子(SUV39H2 24.9倍, CRLF1 16.3倍, TMPRSS2 12.96倍, FXYD3 12.7倍, MYCN 11.5倍, NMU 11.1倍, TREX1 10.4倍, KCNV1 9.9倍, CAPN6 9.9倍, PAPPA 9.0倍, SLC7A5 (hLAT1) 13.7倍)が得られた(＋GAPDH遺伝子をEndgenous controlとして使用)。結果を表３に示す。 As a result, 11 genes (SUV39H2 24.9 times, CRLF1 16.3 times, TMPRSS2 12.96 times, FXYD3 12.7 times, MYCN 11.5 times, NMU 11.1 times, TREX1 10.4 times, KCNV1 are expressed 9.0 times or more different between the A group and the C group. 9.9 times, CAPN6 9.9 times, PAPPA 9.0 times, SLC7A5 (hLAT1) 13.7 times) were obtained (+ GAPDH gene was used as Endgenous control). The results are shown in Table 3.
年齢による遺伝子発現への影響を除外するため、これらの11遺伝子の中から同年齢（老年者この場合は50歳以上）で癌が甲状腺内に留まっている症例及び癌が遠隔転移あるいは甲状腺外に浸潤をもつ症例間（即ち、Ａ群とＢ群との間）でも有意に遺伝子発現量の異なる遺伝子を検討したところ、これらのうちの3遺伝子（SLC7A5 (hLAT1) 137.57倍, PAPPA 70.57倍, KCNV1 23.6倍）が予後の差によって著しく発現が異なることが明らかとなった。結果を表３に示す。 To exclude the effect of age on gene expression, out of these 11 genes, cases where the cancer remained in the thyroid at the same age (older than 50 years in this case), and the cancer was distant from metastasis or extrathyroid When genes with significantly different gene expression levels were examined between cases with invasion (ie between group A and group B), three of these genes (SLC7A5 (hLAT1) 137.57 times, PAPPA 70.57 times, KCNV1 23.6 times), however, the expression was significantly different depending on the prognosis. The results are shown in Table 3.
即ち、これらの3遺伝子（hLAT1とSLC7A5とは同一遺伝子である）は、その遺伝子発現量について、Ａ群（老年者で予後の悪い群）とＣ群（若年者で予後の良い群）との間で差異（9.0倍以上）が認められ、かつＡ群（老年者で予後の悪い群）とＢ群（老年者で予後の良い群）との間でも顕著な差異が認められた（23.6倍以上）。即ち、これらの3遺伝子は、年齢よりもむしろ予後（癌の悪性度）でより大きな差異が認められた遺伝子であり、甲状腺乳頭癌を診断するマーカーとして非常に有用と考えられる。 That is, these three genes (hLAT1 and SLC7A5 are the same gene) are expressed in terms of the gene expression level between group A (the elderly and poor prognosis group) and group C (the young and good prognosis group). A difference (9.0 times or more) was observed between the two groups, and a significant difference was also observed between Group A (the elderly and poor prognosis group) and Group B (the elderly and good prognosis) (23.6 times). that's all). 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 diagnosing papillary thyroid cancer.
また、表３に記載の23遺伝子（RASGRF1、SFTPB、CYP４B１、C8orf4、EHF、CYP１B1、HS3ST1、PCDHA2、SCEL、CXCL2、KIAA1579、CH13L1、PRO2533、CD2AP、PROS1、CMG1、ERO1-L、OSF-2、AGR2、FOSB、ZNF407、SMC2L1、FLNB）についてもその遺伝子発現量を測定したところ、Ａ群（老年者で予後の悪い群）とＣ群（若年者で予後の良い群）との間では上記11遺伝子の場合ほど差異が認められられなかったが（6.8倍以下）、Ａ群（老年者で予後の悪い群）とＢ群（老年者で予後の良い群）との間ではより大きな差異が認められた（1.92〜51.8倍）。即ち、これらの23遺伝子も、年齢よりもむしろ予後（癌の悪性度）で差異が認められた遺伝子であり、甲状腺乳頭癌を診断するマーカーとして有用と考えられる。特に、これら23遺伝子の中でも6遺伝子（RASGRF1、SFTPB、CYP４B１、C8orf4、EHF、CYP１B１）は、Ａ群とＢ群との間でその発現量の差異が大きく（5.9〜51.8倍）、甲状腺乳頭癌を診断する遺伝子マーカーとして有用であり得る。 In addition, 23 genes listed in Table 3 (RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, CYP1B1, HS3ST1, PCDHA2, SCEL, CXCL2, KIAA1579, CH13L1, PRO2533, CD2AP, PROS1, CMG1, ERO1-L, OSF-2, The gene expression levels of AGR2, FOSB, ZNF407, SMC2L1, and FLNB) were also measured. As a result, the above-mentioned 11 was observed between group A (the elderly and poor prognosis group) and group C (the young and good prognosis) The difference was not found as much as in the case of genes (less than 6.8 times), but there was a greater difference between Group A (the elderly and poor prognosis group) and Group B (the elderly and good prognosis group) (1.92-51.8 times). That is, these 23 genes are genes that are different in prognosis (cancer malignancy) rather than age, and are considered useful as markers for diagnosing papillary thyroid cancer. In particular, among these 23 genes, 6 genes (RASGRF1, SFTPB, CYP4B1, C8orf4, EHF, CYP1B1) have a large difference in expression level 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 diagnosing
また、二次スクリーニングで用いたＡ、ＢおよびＣ群についての手術前の血中サイログロブリン値を測定した。結果を病理所見と共に表４に示す。 Moreover, the blood thyroglobulin level before operation about the A, B, and C groups used in the secondary screening was measured. The results are shown in Table 4 together with the pathological findings.
（３）三次スクリーニング：
これらの11遺伝子について結果の再現性を異なるより多くの症例のサンプルを用いて（若年者10例、老年者12例）同じくTaqMan PCR法にて定量し、確認した。
(3) Tertiary screening:
The reproducibility of the results for these 11 genes was also confirmed by quantification using the TaqMan PCR method using samples from more cases (10 young and 12 elderly).
（リアルタイム定量RT-PCR；TaqMan PCR）
上記11遺伝子転写物について、PCR増幅されたDNA鎖を蛍光色素を用いてリアルタイムに定量的検出を行った。50ｎｇのcDNAをTaq Man Universal PCR Master Mix を用いて、ABI PRISM7900HT Sequence Detection system上で遺伝子発現を定量的に測定した。標的遺伝子のcDNAは以下の条件でPCR法により増幅された［95℃で10分間、次いで（95℃で30秒続いて60℃で30秒）を40サイクルのPCR条件］。インプットcDNAにおける変動を補正するために内在性コントロール遺伝子としてGAPDHを使用した。
(Real-time quantitative RT-PCR; TaqMan PCR)
For the above 11 gene transcripts, PCR amplified DNA strands were quantitatively detected in real time using a fluorescent dye. 50 ng of cDNA was quantitatively measured on a ABI PRISM7900HT Sequence Detection system using Taq Man Universal PCR Master Mix. The cDNA of the target gene was amplified by PCR under the following conditions [95 ° C for 10 minutes, then (95 ° C for 30 seconds followed by 60 ° C for 30 seconds) for 40 cycles of PCR conditions]. GAPDH was used as an endogenous control gene to correct for variations in the input cDNA.
閾値サイクル（Ct）を測定し、そして相対的遺伝子発現を以下のように算出した：fold change ＝2−△(△Ct)（式中、△Ct＝Cttarget−CtGAPDH）（サイクル差異）。 The threshold cycle (Ct) was measured and the relative gene expression was calculated as follows: fold change = 2 −Δ (ΔCt) where ΔCt = Ct target −Ct GAPDH (cycle difference).
以上の結果から、これらの11遺伝子（中でも3遺伝子は特に）は甲状腺乳頭癌を診断するマーカーとして臨床検査上、有用と考えられる。また、これらの遺伝子の組み合わせあるいはサイログロブリンとの組み合わせが新しい甲状腺癌の腫瘍マーカーとして診断率を上げ、有用な可能性がある。手術組織のみならず、これらの遺伝子の発現量を血中の白血球を用いて血液レベルで定量が可能であり、あるいは蛋白レベルで抗原抗体法を用いて血清レベルで簡便な測定が可能であり、患者の予後を予測し、術後の再発や転移の検索の参考にすることができ、外来診療上で非常に有用である。 From the above results, these 11 genes (especially 3 genes) are considered to be useful in clinical examination as markers for diagnosing papillary thyroid cancer. In addition, a combination of these genes or a combination with thyroglobulin may be useful as a new tumor marker for thyroid cancer, raising the diagnostic rate. The expression level of these genes can be quantified at the blood level using leukocytes in the blood as well as the surgical tissue, or simple measurement at the serum level using the antigen-antibody method at the protein level, It can predict the prognosis of patients and can be useful for searching for recurrence and metastasis after surgery, which is very useful in outpatient practice.
Claims (9)
KCNV1
PAPPA
SLC7A5（ｈLAT1）
RASGRF1
SFTPB
CYP4B1
C8orf4 A prognostic marker for papillary thyroid cancer comprising at least one gene selected from the gene group described below:
KCNV1
PAPPA
SLC7A5 (hLAT1)
RASGRF1
SFTPB
CYP4B1
C8orf4
KCNV1
PAPPA
SLC7A5（ｈLAT1）。 The prognostic marker for papillary thyroid cancer according to claim 1, wherein the prognostic marker for papillary thyroid cancer comprises at least one gene of the gene group described below:
KCNV1
PAPPA
SLC7A5 (hLAT1).
KCNV1
PAPPA
SLC7A5（ｈLAT1）
RASGRF1
SFTPB
CYP4B1
C8orf4。 An oligonucleotide comprising a base sequence of at least one mRNA expressed by at least one gene selected from the gene group described below or a part of a complementary base sequence of the mRNA, wherein the site is located in the mRNA A method for predicting the prognosis of papillary thyroid cancer, characterized by quantifying the mRNA using a primer or probe containing an oligonucleotide that specifically binds:
KCNV1
PAPPA
SLC7A5 (hLAT1)
RASGRF1
SFTPB
CYP4B1
C8orf4.
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| US20070037186A1 (en) * | 2005-05-20 | 2007-02-15 | Yuqiu Jiang | Thyroid fine needle aspiration molecular assay |
| US20100330561A1 (en) | 2007-06-28 | 2010-12-30 | Nissin Foods Holdings Co., Ltd. | Marker Gene For Detection of Tumor Promoter, and Method For Detection of Tumor Promoter |
| AU2009314502B2 (en) * | 2008-11-17 | 2015-01-22 | Veracyte, Inc. | Methods and compositions of molecular profiling for disease diagnostics |
| US10236078B2 (en) | 2008-11-17 | 2019-03-19 | Veracyte, Inc. | Methods for processing or analyzing a sample of thyroid tissue |
| US9495515B1 (en) | 2009-12-09 | 2016-11-15 | Veracyte, Inc. | Algorithms for disease diagnostics |
| US9074258B2 (en) | 2009-03-04 | 2015-07-07 | Genomedx Biosciences Inc. | Compositions and methods for classifying thyroid nodule disease |
| EP3360978A3 (en) | 2009-05-07 | 2018-09-26 | Veracyte, Inc. | Methods for diagnosis of thyroid conditions |
| KR101049583B1 (en) | 2009-06-30 | 2011-07-14 | 한국과학기술연구원 | A marker for diagnosing papillary thyroid cancer containing 3-indole acetonitrile as an active ingredient |
| PT2456889E (en) * | 2009-07-24 | 2014-11-21 | Geadic Biotec Aie | Markers for endometrial cancer |
| US10446272B2 (en) | 2009-12-09 | 2019-10-15 | Veracyte, Inc. | Methods and compositions for classification of samples |
| EP2714903B1 (en) * | 2011-06-03 | 2018-03-21 | OncoTherapy Science, Inc. | Suv39h2 as a target gene for cancer therapy |
| GB201121924D0 (en) * | 2011-12-20 | 2012-02-01 | Fahy Gurteen Labs Ltd | Detection of breast cancer |
| US11976329B2 (en) | 2013-03-15 | 2024-05-07 | Veracyte, Inc. | Methods and systems for detecting usual interstitial pneumonia |
| US12297505B2 (en) | 2014-07-14 | 2025-05-13 | Veracyte, Inc. | Algorithms for disease diagnostics |
| JP7356788B2 (en) | 2014-11-05 | 2023-10-05 | ベラサイト インコーポレイテッド | Systems and methods for diagnosing idiopathic pulmonary fibrosis in transbronchial biopsies using machine learning and high-dimensional transcriptional data |
| US11217329B1 (en) | 2017-06-23 | 2022-01-04 | Veracyte, Inc. | Methods and systems for determining biological sample integrity |
| JP2019158777A (en) * | 2018-03-16 | 2019-09-19 | 東ソー株式会社 | Tumor marker and method for collecting and detecting tumor cell by distinguishing it from contaminant cell |
| CN115792247B (en) * | 2023-02-09 | 2023-09-15 | 杭州市第一人民医院 | Application of protein combination in preparation of thyroid papillary carcinoma risk auxiliary layering system |
| CN120254289B (en) * | 2025-06-03 | 2025-08-22 | 杭州广科安德生物科技有限公司 | Biomarker for predicting recurrence risk of papillary thyroid carcinoma and application thereof |
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| DK1052292T3 (en) * | 1997-12-22 | 2003-07-28 | Genset Sa | Prostate cancer gene |
| US6703202B2 (en) * | 1999-11-30 | 2004-03-09 | Oxo Chemie Ag | Evaluating and predicting clinical outcomes by gene expression analysis |
| JP2006081416A (en) | 2004-09-14 | 2006-03-30 | Japan Industrial Technology Association | Method for determining dna |
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