JP2005211023A - Method for estimating possibility of metastasis or relapse of renal cell carcinoma - Google Patents
Method for estimating possibility of metastasis or relapse of renal cell carcinoma Download PDFInfo
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Abstract
Description
本発明は、腎細胞癌の転移若しくは再発の可能性を予測する方法並びにそれを行うためのキットに関するものである。 The present invention relates to a method for predicting the possibility of metastasis or recurrence of renal cell carcinoma and a kit for performing the method.
腎細胞癌の治療としては、腫瘍部分の外科的な手術による切除が一般的な方法であるが、患者によっては、切除後数年で、腎臓での再発、又は、肺などの他の臓器への転移が認められる場合が多い。通常、浸潤やリンパ節への転移の程度が激しく悪性度が高いと診断された腎細胞癌は、癌の転移や再発が起こりやすく、摘出手術後に、インターフェロンαなどのサイトカインや抗癌剤を投与して、手術により除去できなかった癌細胞を死滅させる治療が行われている。しかしながら、サイトカインや抗癌剤の投与は、患者への副作用が大きいことから、投与の決定に際して、癌転移や再発の可能性を予測することができる診断方法は有用である。 For the treatment of renal cell carcinoma, surgical removal of the tumor is a common method, but depending on the patient, recurrence in the kidney or other organs such as the lung may occur several years after the resection. Metastasis is often observed. Usually, renal cell carcinoma diagnosed with a high degree of malignancy and invasion and metastasis to lymph nodes is likely to cause metastasis and recurrence of the cancer. There is a treatment for killing cancer cells that could not be removed by surgery. However, since administration of cytokines and anticancer agents has great side effects on patients, a diagnostic method that can predict the possibility of cancer metastasis or recurrence is useful when determining the administration.
従来、癌の診断は、腫瘍の大きさや形状、組織への浸潤、リンパ節への転移の程度などで評価してランク付けして悪性度を示すことによって行われてきた。しかしながら、上記のようにランク付けする評価方法は、手術時に医師の目視により行われるため、曖昧な評価となり、また、手術後の癌の転移若しくは再発の予測が正確に行うことができるとはいえない。そこで、近年、癌患者のインターフェロンα受容体タイプ１遺伝子の発現量に着目して癌の悪性度を診断することが検討されており、その診断方法としては、例えば、腎細胞癌患者の血清中の遊離受容体を免疫学的な手法により測定する方法（非特許文献１）、肝臓癌をＲＴ−ＰＣＲによりインターフェロンα受容体タイプ１遺伝子の発現量を調べる方法（非特許文献２）がある。しかしながら、これらの方法は、腎細胞癌の手術後の転移若しくは再発の可能性を予測するものではない。 Conventionally, cancer diagnosis has been performed by evaluating and ranking the tumor according to the size and shape of the tumor, the invasion of tissue, the degree of metastasis to lymph nodes, and the like, and showing the grade of malignancy. However, since the evaluation method for ranking as described above is performed by visual observation by a doctor at the time of surgery, it is an ambiguous evaluation, and it can be said that cancer metastasis or recurrence after surgery can be accurately predicted. Absent. Therefore, in recent years, it has been studied to diagnose the malignancy of cancer by paying attention to the expression level of interferon α receptor type 1 gene in cancer patients. As a diagnostic method, for example, in the serum of patients with renal cell carcinoma There is a method (Non-patent document 1) for measuring the free receptor of ceramide, and a method (Non-patent document 2) for examining the expression level of interferon α receptor type 1 gene by RT-PCR in liver cancer. However, these methods do not predict the possibility of metastasis or recurrence after surgery for renal cell carcinoma.
本発明は、腎細胞癌の手術摘出後の患者の転移若しくは再発の可能性を予測する方法を提供することを第一の課題とする。 The first object of the present invention is to provide a method for predicting the possibility of metastasis or recurrence in a patient after surgical removal of renal cell carcinoma.
さらに本発明は、腎細胞癌の手術摘出後の患者の転移若しくは再発の可能性の予測を容易に実施することを可能とするキットを提供することを第ニの課題とする。 Furthermore, it is a second object of the present invention to provide a kit that makes it possible to easily predict the possibility of metastasis or recurrence of a patient after surgical removal of renal cell carcinoma.
上記課題を解決すべく、本発明者等は、腎細胞癌患者から摘出された腎細胞癌腫瘍組織におけるインターフェロンα受容体遺伝子の発現量をＲＴ−ＰＣＲ法により算出し、それを当該患者の正常腎組織におけるそれと比較した。さらに、手術後数年に渡り、肺転移若しくは再発の有無を調べた。その結果、インターフェロンα受容体、とりわけ、インターフェロンα受容体タイプ２遺伝子の発現量が正常腎組織に比べて高くなるほど、腎細胞癌の手術後の転移若しくは再発が起こる可能性が高いことを見出し、本発明を完成するに至った。
In order to solve the above-mentioned problems, the present inventors calculated the expression level of interferon α receptor gene in renal cell carcinoma tumor tissue extracted from renal cell carcinoma patients by RT-PCR method, Compared with that in renal tissue. In addition, we examined the presence or absence of lung metastasis or recurrence for several years after surgery. As a result, it has been found that the higher the expression level of interferon α receptor, especially interferon
すなわち、本発明は、腎細胞癌腫瘍組織におけるインターフェロンα受容体遺伝子、とりわけ、インターフェロンα受容体タイプ２（ＩＦＮＡＲ２）遺伝子の発現量と正常腎組織のそれとを比較することによって、腎細胞癌の転移若しくは再発の可能性を予測することにより第一の課題を解決するものである。 That is, the present invention relates to metastasis of renal cell carcinoma by comparing the expression level of interferon α receptor gene, particularly interferon α receptor type 2 (IFNAR2) gene in renal cell carcinoma tumor tissue with that of normal kidney tissue. Alternatively, the first problem is solved by predicting the possibility of recurrence.
さらに、本発明は、腎細胞癌の転移若しくは再発の可能性の予測を、インターフェロンα受容体遺伝子のＤＮＡ配列を有するオリゴヌクレオチドプライマーと、耐熱性ＤＮＡポリメラーゼを含んでなるキットを提供することにより、第二の課題を解決するものである。 Furthermore, the present invention provides a kit comprising an oligonucleotide primer having a DNA sequence of an interferon α receptor gene and a thermostable DNA polymerase for predicting the possibility of metastasis or recurrence of renal cell carcinoma. It solves the second problem.
この発明によれば、腎細胞癌の転移若しくは再発の可能性を予測することができる。したがって、本発明は、腎細胞癌の手術後のサイトカインや抗癌剤投与の判断基準になる。 According to this invention, the possibility of metastasis or recurrence of renal cell carcinoma can be predicted. Therefore, the present invention is a criterion for the administration of cytokines and anticancer agents after surgery for renal cell carcinoma.
本発明でいうインターフェロンα受容体とは、インターフェロンα又はインターフェロンβなどに特異的な受容体であり、タイプ１（ＧＥＮＢＡＮＫ ＭＮ＿０００６２９）（以下、「ＩＦＮＡＲ１」と記載することがある。）及びタイプ２（ＧＥＮＢＡＮＫ ＭＮ＿０００８７４）（以下、「ＩＦＮＡＲ２」と記載することがある。）の２種類の受容体が知られている。本発明においては、それら両方を用いることがができるが、ＩＦＮＡＲ２遺伝子の方が正確に予測可能なので有利に選択される。 The interferon α receptor referred to in the present invention is a receptor specific for interferon α or interferon β and the like, and is type 1 (GENBANK MN — 000629) (hereinafter sometimes referred to as “IFNAR1”) and type 2 ( GENBANK MN — 000874) (hereinafter sometimes referred to as “IFNAR2”) is known. In the present invention, both of them can be used, but the IFNAR2 gene is advantageously selected because it can be accurately predicted.
インターフェロンα受容体遺伝子の発現量を調べる方法としては、通常、組織又は細胞内で発現しているインターフェロンα受容体のｍＲＮＡを抽出し、必要に応じて精製、増幅した後、適宜の公知の方法により定量することによって行われる。例えば、逆転写酵素法、ノーザンブロッティング法、サザンブロッテイング法、ハイブリダイゼーション法、マグネットビーズ法、電気泳動法、ＰＣＲ法、ＤＮＡシークエンシング法などを単独で、若しくは適宜組み合わせて行うことができる。前記定量方法としては、ＰＣＲ法、とりわけ後述するリアルタイムＰＣＲ法は、処理速度、定量精度が共に優れていることから本発明においては最も有利に用いられる。 As a method for examining the expression level of the interferon α receptor gene, usually, mRNA of interferon α receptor expressed in tissues or cells is extracted, purified and amplified as necessary, and then an appropriate known method. By quantifying with For example, the reverse transcriptase method, the Northern blotting method, the Southern blotting method, the hybridization method, the magnet bead method, the electrophoresis method, the PCR method, the DNA sequencing method and the like can be carried out alone or in combination as appropriate. As the quantification method, the PCR method, particularly the real-time PCR method described later, is most advantageously used in the present invention because of its excellent processing speed and quantification accuracy.
インターフェロンα遺伝子の発現量を比較する方法として、通常、腎細胞癌患者から摘出された腫瘍部分を含む腎臓組織から、腎細胞癌腫瘍組織と正常腎組織を分取した後、それぞれの組織のインターフェロンα受容体遺伝子の発現量を測定し、下記の数式により、腎細胞癌腫瘍組織におけるインターフェロンα受容体遺伝子の発現量と正常腎組織におけるそれとを比較して、インターフェロンα受容体遺伝子の発現比率（以下、「Ｔ／Ｓ値」と略記することがある）を算出する方法が挙げられる。なお、発現量の比較において、ＲＮＡ量、ｍＲＮＡ量、ｃＤＮＡ量、細胞数などを単位量に設定して調整する必要がある。また、正常腎組織部分の入手が困難な場合、腎細胞癌腫瘍組織におけるグリセロアルデヒドリン酸脱水素酵素などの解糖系の酵素、又は、βアクチンなどの細胞骨格タンパク質などの発現量を内部標準にして、インターフェロンα受容体遺伝子の相対発現量を算出する方法を採用することもできる。 As a method for comparing the expression level of the interferon α gene, after separating the renal cell carcinoma tumor tissue and the normal kidney tissue from the kidney tissue including the tumor part usually removed from the renal cell carcinoma patient, the interferon of each tissue The expression level of the α-receptor gene was measured and compared with the expression level of the interferon α-receptor gene in renal cell carcinoma tumor tissue and that in normal renal tissue using the following formula. Hereinafter, a method of calculating “T / S value” may be mentioned. In comparison of expression levels, it is necessary to adjust the RNA quantity, mRNA quantity, cDNA quantity, cell number, etc. by setting them as unit quantities. In addition, when it is difficult to obtain normal kidney tissue, the expression level of glycolytic enzymes such as glyceraldehyde phosphate dehydrogenase or cytoskeletal proteins such as β-actin in renal cell carcinoma tumor tissues is used as an internal standard. Thus, a method of calculating the relative expression level of the interferon α receptor gene can also be employed.
得られたＴ／Ｓ値と、手術による腎細胞癌の摘出後、２乃至４年の転移の有無を比較し、インターフェロンα受容体遺伝子の発現量と転移若しくは再発の有無を、適宜の統計的手法、例えば、マン・ホイットニーのＵ検定によって、所望の有意差水準において有意差が存在するか否かに基づいて検証した結果、ＩＦＮＡＲ２遺伝子のＴ／Ｓ値と転移との間には相関性があることが判明した。
The obtained T / S value is compared with the presence or absence of
本発明でいう腎細胞癌とは、腎細胞組織に発生した悪性腫瘍を意味し、腎臓組織を原発巣とするもの及び他の組織から腎臓に転移した癌であってもよい。 The renal cell cancer referred to in the present invention means a malignant tumor that has occurred in the renal cell tissue, and may be a cancer that has the renal tissue as the primary focus and a cancer that has metastasized to the kidney from other tissues.
本発明の腎細胞癌の予測方法の利用方法について一例を述べると、まず、腎細胞癌患者から腎細胞癌腫瘍組織を含む腎臓組織を摘出手術により摘出するか、あるいは少量を診断用サンプルとして摘出し、摘出した組織から腎細胞癌腫瘍組織と正常腎組織を分離し、それぞれからＲＮＡを採取する。これを鋳型にして逆転写酵素によりｃＤＮＡを調製した後、インターフェロンα受容体遺伝子の一部又は全部を特異的に増幅可能なオリゴヌクレオチドプライマー（配列表における配列番号１乃至４で表される塩基配列）や耐熱性ＤＮＡポリメラーゼなどを含有するＰＣＲ反応液中でインターフェロンα受容体遺伝子の一部分を増幅し、増幅ＤＮＡ量を定量してインターフェロンα受容体遺伝子の発現量を算出する。このとき、正常腎組織から調製したＲＮＡを鋳型にして逆転写酵素によりｃＤＮＡを調製し、腎細胞癌腫瘍組織の場合と等量のｃＤＮＡ量を用いて、インターフェロンα受容体遺伝子の発現量を測定し、それらの測定値を前記数式に当てはめることにより、インターフェロンα受容体遺伝子の発現比率（Ｔ／Ｓ値）を求める。Ｔ／Ｓ値が高い患者ほど腎癌の転移若しくは再発する確率が高いので、腎細胞癌の転移若しくは再発を防止するためにインターフェロンαなどのサイトカインや抗癌剤を投与することが効果的であると予測できる。腎細胞癌患者の症状、手術後の状況などにより異なるが、腎細胞癌摘出手術後でのサイトカインや抗癌剤の投与を決定する目安としては、通常ＩＦＮＡＲ２遺伝子のＴ／Ｓ値が３以上の場合、転移若しくは再発の危険領域であると判断してサイトカインや抗癌剤の投与を決定することができる。また、Ｔ／Ｓ値が０．８乃至３の範囲内は、中間領域であり、患者の状況、他の予測結果と合わせてサイトカインや抗癌剤の投与を検討すればよい。Ｔ／Ｓ値が０．８以下、より厳密には０．４以下の場合は、ほぼ転移若しくは再発の危険性がない安全領域であると判断することができる。 An example of a method for using the method for predicting renal cell carcinoma of the present invention will be described. First, renal tissue containing renal cell carcinoma tumor tissue is removed from a renal cell carcinoma patient by surgery, or a small amount is removed as a diagnostic sample. Then, renal cell carcinoma tumor tissue and normal kidney tissue are separated from the excised tissue, and RNA is collected from each. After preparing cDNA with reverse transcriptase using this as a template, an oligonucleotide primer capable of specifically amplifying part or all of the interferon α receptor gene (base sequence represented by SEQ ID NO: 1 to 4 in the sequence listing) ) Or a thermostable DNA polymerase or the like, a part of the interferon α receptor gene is amplified, and the amount of the amplified DNA is quantified to calculate the expression level of the interferon α receptor gene. At this time, cDNA was prepared with reverse transcriptase using RNA prepared from normal kidney tissue as a template, and the amount of interferon α receptor gene expressed was measured using the same amount of cDNA as in renal cell carcinoma tumor tissue. Then, by applying these measured values to the above formula, the expression ratio (T / S value) of the interferon α receptor gene is determined. Patients with higher T / S values have a higher probability of metastasis or recurrence of renal cancer, so it is predicted that administration of cytokines such as interferon α and anticancer agents is effective in preventing metastasis or recurrence of renal cell carcinoma. it can. Although it depends on the symptoms of patients with renal cell carcinoma, the situation after surgery, etc., as a guideline for determining the administration of cytokines and anticancer agents after surgery for renal cell carcinoma, if the T / S value of IFNAR2 gene is usually 3 or more, The administration of cytokines or anticancer agents can be determined based on the risk of metastasis or recurrence. Further, the T / S value in the range of 0.8 to 3 is an intermediate region, and administration of cytokines and anticancer agents may be considered in combination with the patient's situation and other prediction results. When the T / S value is 0.8 or less, more strictly 0.4 or less, it can be determined that the safety region is almost free from the risk of metastasis or recurrence.
上記のＰＣＲによる検出を簡便におこなうべく、鋳型ＤＮＡ以外の成分、すなわち、インターフェロンα受容体遺伝子を増幅するプライマー、耐熱性ＤＮＡポリメラーゼ、基質のデオキシヌクレオチド三リン酸などをあらかじめ混合したＰＣＲ反応用プレミックス液を作製しておくこともできる。 In order to easily perform the above-described detection by PCR, a PCR reaction premix containing components other than the template DNA, that is, a primer for amplifying the interferon α receptor gene, a heat-resistant DNA polymerase, a substrate deoxynucleotide triphosphate, etc. in advance. A mixed solution can also be prepared.
以下、実験例をもとに、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail based on experimental examples.
＜実験：腎細胞癌患者のインターフェロンα受容体遺伝子のＴ／Ｓ値と手術後の経過についての相関関係＞
腎細胞癌患者から摘出した組織から、腎細胞癌腫瘍組織と正常腎組織を各約５０ｍｇ分取し、市販のＲＮＡ調製キット（キアゲン社販売、商品名『ＲＮｅａｓｙ Ｍｉｎｉ ｋｉｔ』）を使用して、常法にしたがってＲＮＡを調製した。このＲＮＡ１μｇを、別途調製した１００ｎｇのランダムヘキサマー、１００単位のマウス乳癌ウイルス逆転写酵素を含む１００μｌの反応溶液と、２５℃１０分間、４２℃３０分間反応させたのち、７０℃で１０分間処理して、反応を停止させ、ｃＤＮＡを得た。次に、ＤＮＡデータベースＧＥＮＢＡＮＫに登録されているＩＦＮＡＲ１遺伝子（ＧＥＮＢＡＮＫ ＭＮ＿０００６２９）又はＩＦＮＡＲ２遺伝子（ＧＥＮＢＡＮＫ ＭＮ＿０００８７４）に開示される塩基配列を基に、ＰＣＲで特異的に増幅可能なオリゴヌクレオチドプライマー（配列表における配列番号１乃至４で表される塩基配列を有する）を常法により作製した。さらに、常法にしたがって、リアルタイムＰＣＲ法を市販の装置（アプライドバイオシステムズ社販売、商品名『ＡＢＩＰＲＩＳＭ ７７００ シークエンス・ディテクション・システム』）を用いて行った。すなわち、２０ｎｇのｃＤＮＡ、各１００ｎＭのプライマー（ＩＦＮＡＲ１遺伝子用に配列表における配列番号１及び２の塩基配列からなるプライマー、あるいは、ＩＦＮＡＲ２遺伝子用に配列表における配列番号３及び４の塩基配列からなるプライマー）を水に溶解し１２．５μｌとし、それに１２．５μｌの『ＳＹＢＲグリーンＰＣＲマスターミックス』（アプライドバイオシステムズ社販売）を添加して反応溶液を調製した。これを、９０℃１５秒、６０℃１分を１サイクルとして、４５サイクルのＰＣＲを行った。腎細胞癌腫瘍組織と正常腎組織におけるインターフェロンα遺伝子の増幅断片の量を定量し、前記数式を適用してＴ／Ｓ値を算出した。次に、各患者の手術後２４乃至４８ヶ月までにおける、胸部Ｘ線撮影による肺への癌転移及び生存を調べた。これらの結果を表１に示す。さらに、転移の有無によりグループ分けした後、平均値及び標準偏差を算出した後、マン・ホイットニーのＵ検定を行い、有意水準ｐ＜０．０５及びｐ＜０．０１で有意差があるかどうか調べた。この結果を図１及び図２に示す。図中、棒状で示された値が平均値であり、線状で示された値が標準偏差である。
<Experiment: Correlation between T / S value of interferon alpha receptor gene in renal cell carcinoma patients and progress after surgery>
About 50 mg each of renal cell carcinoma tumor tissue and normal kidney tissue is extracted from the tissue removed from a renal cell carcinoma patient, and using a commercially available RNA preparation kit (trade name “RNeasy Mini kit” sold by Qiagen), RNA was prepared according to a conventional method. 1 μg of this RNA was reacted with 100 μl of a reaction solution containing 100 ng of a random hexamer separately prepared and 100 units of mouse mammary tumor virus reverse transcriptase at 25 ° C. for 10 minutes, 42 ° C. for 30 minutes, and then treated at 70 ° C. for 10 minutes. Then, the reaction was stopped and cDNA was obtained. Next, based on the nucleotide sequence disclosed in the IFNAR1 gene (GENBANK MN_000629) or IFNAR2 gene (GENBANK MN_000874) registered in the DNA database GENBANK, oligonucleotide primers (sequences in the sequence listing) that can be specifically amplified by PCR Having the base sequences represented by Nos. 1 to 4) were prepared by a conventional method. Furthermore, according to a conventional method, real-time PCR was performed using a commercially available apparatus (available from Applied Biosystems, Inc., trade name “ABIPRISM 7700 Sequence Detection System”). That is, 20 ng cDNA, each 100 nM primer (primer consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2 in the sequence listing for IFNAR1 gene, or primer consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 in the sequence listing for IFNAR2 gene ) Was dissolved in water to make 12.5 μl, and 12.5 μl of “SYBR Green PCR Master Mix” (available from Applied Biosystems) was added thereto to prepare a reaction solution. This was carried out for 45 cycles of PCR at 90 ° C. for 15 seconds and 60 ° C. for 1 minute. The amount of the amplified fragment of the interferon α gene in renal cell carcinoma tumor tissue and normal kidney tissue was quantified, and the T / S value was calculated by applying the above formula. Next, each patient was examined for cancer metastasis to the lung and survival by chest X-ray film from 24 to 48 months after the operation. These results are shown in Table 1. Furthermore, after grouping according to the presence or absence of metastasis, after calculating the mean value and standard deviation, Mann-Whitney U test is performed, and whether there is a significant difference at significance levels p <0.05 and p <0.01 Examined. The results are shown in FIGS. In the figure, the value indicated by a bar is an average value, and the value indicated by a line is a standard deviation.
図１及び図２の結果が示すとおり、インターフェロンα受容体遺伝子のＴ／Ｓ値は、転移が認められた群において高い値を示した。特にＩＦＮＡＲ２遺伝子において、統計的に有為に高い（ｐ＜０．０１）Ｔ／Ｓ値を示した。したがって、インターフェロンα受容体遺伝子は、腎細胞癌の手術摘出後の転移を予測する指標とすることが可能であることが判明した。 As shown in the results of FIGS. 1 and 2, the T / S value of the interferon α receptor gene was high in the group in which metastasis was observed. In particular, the IFNAR2 gene showed a statistically significant (p <0.01) T / S value. Therefore, it was found that the interferon α receptor gene can be used as an index for predicting metastasis after surgical removal of renal cell carcinoma.
また、表１において、ＩＦＮＡＲ２遺伝子のＴ／Ｓ値が３以上の場合、転移の起こる確率は９１％（１０／１１）であり、一方、０．８以下の場合、転移が起こる確率は０％（０／２２）であった。したがって、ＩＦＮＡＲ２遺伝子のＴ／Ｓ値が３以上の際は、インターフェロンαなどのサイトカインや抗癌剤などの投与が必要であると判断される危険領域であり、０．８以下、より厳密には０．４以下の場合は摘出手術後の処置が不要であると判断される安全領域であると考えられた。また、０．８乃至３の範囲内は、転移の起こる確率は約５０％程度であり、摘出手術後の処理は医師の判断に委ねられる中間領域であると考えられた。結果を表２に示す。 In Table 1, when the T / S value of the IFNAR2 gene is 3 or more, the probability of occurrence of metastasis is 91% (10/11), whereas when it is 0.8 or less, the probability of occurrence of metastasis is 0%. (0/22). Therefore, when the T / S value of the IFNAR2 gene is 3 or more, it is a risk area where it is determined that administration of cytokines such as interferon α and anticancer agents is necessary, and is 0.8 or less, more strictly 0. In the case of 4 or less, it was considered that it was a safe area where it was judged that treatment after the excision operation was unnecessary. Further, within the range of 0.8 to 3, the probability of metastasis was about 50%, and it was considered that the processing after the excision operation is an intermediate region that is left to the judgment of the doctor. The results are shown in Table 2.
さらに、表１において、Ｔ／Ｓ値が平均値（ＩＦＮＡＲ１遺伝子では３．７１、ＩＦＮＡＲ２遺伝子では２．９０）よりも高いグループと低いグループに分けて、カプラン−マイヤー（Ｋａｐｌａｎ−Ｍｅｉｅｒ）法により累積生存率を求めた。結果を図３及び図４に示す。なお、図中、「▲」はＴ／Ｓ値が低いグループ、「●」はＴ／Ｓ値が高いグループを示す。この結果、図４に示すとおり、ＩＦＮＡＲ２遺伝子のＴ／Ｓ値が高い場合、患者の生存率は極めて低下することが判明した。なお、生存率の低下は、腎細胞癌の転移若しくは再発によるものである。 Further, in Table 1, the T / S value is divided into a higher group and a lower group than the average value (3.71 for IFNAR1 gene and 2.90 for IFNAR2 gene), and cumulative by Kaplan-Meier method. Survival was determined. The results are shown in FIGS. In the figure, “▲” indicates a group having a low T / S value, and “●” indicates a group having a high T / S value. As a result, as shown in FIG. 4, it was found that when the T / S value of the IFNAR2 gene is high, the survival rate of the patient is extremely reduced. The decrease in survival rate is due to metastasis or recurrence of renal cell carcinoma.
以下、実施例により、本発明をさらに詳細に説明する。なお、本発明は実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited to an Example.
腎細胞癌患者５名（患者Ａ乃至Ｅ）から摘出した腎細胞癌腫瘍組織を含む腎臓組織から、腎細胞癌腫瘍組織と正常腎組織を５０ｍｇずつ分取し、常法にしたがって、それぞれＲＮＡを採取し、これらを鋳型にしてマウス乳癌ウイルス逆転写酵素によりｃＤＮＡを調製し、２０ｎｇ／１２．５μｌのｃＤＮＡ溶液とした。一方、ＩＦＮＡＲ２ｍＲＮＡ検出用のプライマー（配列表における配列番号３及び４）各１００ｎＭと、耐熱性ＤＮＡポリメラーゼなどを含有する『ＳＹＢＲグリーンＰＣＲマスターミックス』（アプライドバイオシステムズ社販売）とを混合したプレミックス反応液１２．５μｌを、上記ｃＤＮＡ溶液に添加して合計２５μｌのＰＣＲ反応溶液を調製した。これらを、リアルタイムＰＣＲ装置（アプライドバイオシステムズ社販売、商品名『ＡＢＩＰＲＩＳＭ ７７００ シークエンス・ディテクション・システム』）により、９０℃１５秒、６０℃１分を１サイクルとして４５サイクルのＰＣＲを行い、蛍光強度を測定し、前記数式を適用して、正常細胞に対する腫瘍細胞のＴ／Ｓ値を算出した。その結果、それぞれの腎細胞癌患者のＴ／Ｓ値については、患者Ａが０．３、患者Ｂが０．７、患者Ｃが１．５、患者Ｄが４．７、患者Ｅが９．３であった。そこで、患者Ｄ及びＥに対しては、転移の可能性が高い危険領域であると認められたので、常法に従い、３００万ＩＵ／日のインターフェロンαを１週間投与した。一方、患者Ａ及びＢに対しては転移の可能性が低い安全領域であると認められたのでインターフェロンαの投与は行わなかった。患者Ｃに対しては、中間領域と認められたが、副作用による患者への影響の方を重くみて、インターフェロンαの投与を行わなかった。 50 mg of renal cell carcinoma tumor tissue and normal kidney tissue are sampled from kidney tissue including renal cell carcinoma tumor tissue extracted from 5 renal cell carcinoma patients (patients A to E), and RNA is extracted according to a conventional method. Using these as templates, cDNA was prepared using mouse mammary tumor virus reverse transcriptase to prepare a 20 ng / 12.5 μl cDNA solution. On the other hand, a premix reaction in which 100 nM each of primers for detecting IFNAR2 mRNA (SEQ ID NOs: 3 and 4 in the sequence listing) and “SYBR Green PCR Master Mix” (sold by Applied Biosystems) containing heat-resistant DNA polymerase and the like are mixed. 12.5 μl of the solution was added to the cDNA solution to prepare a total of 25 μl of PCR reaction solution. These were subjected to 45 cycles of PCR using a real-time PCR device (Applied Biosystems, trade name “ABIPRISM 7700 Sequence Detection System”) at 90 ° C. for 15 seconds and 60 ° C. for 1 minute to obtain fluorescence intensity. And the above formula was applied to calculate the T / S value of tumor cells relative to normal cells. As a result, the T / S values of the respective renal cell carcinoma patients were 0.3 for patient A, 0.7 for patient B, 1.5 for patient C, 4.7 for patient D, and 9. for patient E. 3. Therefore, since it was recognized that patients D and E were in a dangerous area with a high possibility of metastasis, 3 million IU / day of interferon α was administered for 1 week according to a conventional method. On the other hand, since it was recognized that patients A and B were safe areas with a low possibility of metastasis, administration of interferon α was not performed. Although it was recognized as an intermediate region for patient C, administration of interferon α was not performed in view of the effect of side effects on the patient.
叙述のとおり、本発明によれば、腎細胞癌におけるインターフェロンα受容体遺伝子の発現量を調べることで、手術により腎細胞癌腫瘍組織を摘出した後の、腎細胞癌の転移若しくは再発を予測することができる。したがって、本発明を手術後における転移若しくは再発を阻止するために、インターフェロンαなどのサイトカインや抗癌剤による治療を無駄なく行うことができる。 As described above, according to the present invention, metastasis or recurrence of renal cell carcinoma after surgical removal of renal cell carcinoma tumor tissue is examined by examining the expression level of interferon α receptor gene in renal cell carcinoma. be able to. Therefore, in order to prevent metastasis or recurrence after surgery according to the present invention, treatment with cytokines such as interferon α and anticancer agents can be performed without waste.
Claims (4)
The prediction kit according to claim 3, wherein the interferon α receptor gene is an interferon α receptor type 2 gene.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007139742A (en) * | 2005-11-18 | 2007-06-07 | Chi Mei Foundation Medical Center | Biomarker for determining predisposition and/or prognosis of hepatocellular carcinoma |
| EP3029155A3 (en) * | 2010-01-11 | 2016-08-31 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| US10181008B2 (en) | 2013-05-30 | 2019-01-15 | Genomic Health, Inc. | Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer |
| US10260104B2 (en) | 2010-07-27 | 2019-04-16 | Genomic Health, Inc. | Method for using gene expression to determine prognosis of prostate cancer |
| US11011252B1 (en) | 2012-01-31 | 2021-05-18 | Genomic Health, Inc. | Gene expression profile algorithm and test for determining prognosis of prostate cancer |
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| CA2344995A1 (en) * | 1998-10-05 | 2000-04-13 | Ludwig Institute For Cancer Research | Cancer associated antigens and uses therefor |
| DE60230785D1 (en) * | 2001-10-16 | 2009-02-26 | Toshiba Kk | POLYMORPHE MARKER THAT CAN BE USED TO DETERMINE THE EFFECTIVENESS OF INTERFERON THERAPY |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007139742A (en) * | 2005-11-18 | 2007-06-07 | Chi Mei Foundation Medical Center | Biomarker for determining predisposition and/or prognosis of hepatocellular carcinoma |
| EP3029155A3 (en) * | 2010-01-11 | 2016-08-31 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| US9551034B2 (en) | 2010-01-11 | 2017-01-24 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| EP3178944A3 (en) * | 2010-01-11 | 2017-09-13 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| EP3561078A1 (en) * | 2010-01-11 | 2019-10-30 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| US10892038B2 (en) | 2010-01-11 | 2021-01-12 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| EP3831964A3 (en) * | 2010-01-11 | 2021-10-20 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| US11776664B2 (en) | 2010-01-11 | 2023-10-03 | Genomic Health, Inc. | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| US10260104B2 (en) | 2010-07-27 | 2019-04-16 | Genomic Health, Inc. | Method for using gene expression to determine prognosis of prostate cancer |
| US11011252B1 (en) | 2012-01-31 | 2021-05-18 | Genomic Health, Inc. | Gene expression profile algorithm and test for determining prognosis of prostate cancer |
| US10181008B2 (en) | 2013-05-30 | 2019-01-15 | Genomic Health, Inc. | Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer |
| US11551782B2 (en) | 2013-05-30 | 2023-01-10 | Genomic Health, Inc. | Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer |
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