WO2020067498A1 - Procédé d'acquisition de données pour le diagnostic du cancer de la prostate - Google Patents

Procédé d'acquisition de données pour le diagnostic du cancer de la prostate Download PDF

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WO2020067498A1
WO2020067498A1 PCT/JP2019/038347 JP2019038347W WO2020067498A1 WO 2020067498 A1 WO2020067498 A1 WO 2020067498A1 JP 2019038347 W JP2019038347 W JP 2019038347W WO 2020067498 A1 WO2020067498 A1 WO 2020067498A1
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prostate cancer
sf3b2
expression level
gene
expression
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PCT/JP2019/038347
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English (en)
Japanese (ja)
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圭祐 二村
憲彦 川村
安史 金田
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株式会社ダイセル
国立大学法人大阪大学
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Priority to JP2020549474A priority Critical patent/JP7473132B2/ja
Publication of WO2020067498A1 publication Critical patent/WO2020067498A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to a data acquisition method for prostate cancer diagnosis.
  • prostate cancer is a cancer mainly occurring in the external gland (marginal region).
  • Prostate cancer is a cancer that accounts for the highest number of affected males in Japan and the United States by site, and there are still many patients. In Japan, the number of people with prostate cancer is estimated to exceed 80,000, and about 12,000 of them are expected to die (National Cancer Research Center Cancer Information Service "Cancer Registry and Statistics", 2017 cancer statistics prediction).
  • Treatment of prostate cancer includes surgical treatment, radiation treatment, chemotherapy, endocrine therapy (hormone therapy) and the like.
  • endocrine therapy is a treatment for suppressing the progression of prostate cancer by suppressing the secretion of androgen, which plays a role in promoting the progression of prostate cancer, or by inhibiting the function of androgen.
  • long-term treatment may result in resistance to drugs used in endocrine therapy, and the therapeutic effect of endocrine therapy may be reduced.
  • Prostate cancer whose prostate cancer symptoms have worsened as a result of becoming resistant to endocrine therapy in this way is called castration-resistant prostate cancer.
  • AR-V7 the mechanism of castration-resistant prostate cancer involves the androgen receptor (AR) (Non-patent Document 1). Furthermore, one of the AR variants, AR-V7, has become ligand-independent. It has been found that the activation of a transcription factor can promote the growth of prostate cancer even in a castrated state (Non-Patent Documents 2 and 3).
  • AR-FL the full-length AR gene
  • AR-FL its variant AR-V7 has only exons 1-3 and cryptic exon CE3, and has no ligand binding site. Therefore, it can have activity as a transcription factor in a ligand-independent manner. That is, it is thought that reducing the expression level of AR-V7 leads to treatment of castration-resistant prostate cancer.
  • the Gleason score is used as an index of the malignancy of prostate cancer, but the Gleason score is 8 or higher, and the metastasis is not treated with metastatic endocrine therapy. It is known that castration resistance is likely to be acquired early in risk prostate cancer and the prognosis is poor. Alternatively, if the Gleason score is high, it may be that castration-resistant prostate cancer has already developed. For the high-risk prostate cancer as described above, the LATITUDE trial (international phase III study) comparing the overall survival rate between the endocrine therapy (ADT) group and the two groups administered with ADT plus abiraterone and prednisone was conducted.
  • ADT endocrine therapy
  • abiraterone is an inhibitor of cytochrome P450 17A1, and is a drug that damages the AR signal by depleting androgens in kidney and tumor.
  • Enzalmid is an AR signal inhibitor, and inhibits an AR signal by binding to an AR ligand such as testosterone by binding to a ligand binding domain of AR. Therefore, enzalumid is used as an indication for castration-resistant prostate cancer.
  • Non-Patent Document 4 a factor for predicting the effect of endocrine treatment with abiraterone or enzalutamide in high-risk prostate cancer was required.
  • a method for determining whether or not AR-V7 is expressed has not been established in both image diagnosis and pathological tissue diagnosis, and a method for predicting the therapeutic effect of abiraterone or enzalutamide has not been established so far.
  • an object of the present invention is to provide a data acquisition method for diagnosing prostate cancer.
  • the present inventors have conducted intensive studies to solve the above-described problems, and as a result, identified the SF3B2 (splicing factor 3B subunit 2) gene as a gene that regulates the expression of AR-V7, and the expression of the SF3B2 gene indicates And correlated with prognosis.
  • SF3B2 splicing factor 3B subunit 2
  • SF3B2 was found to be highly expressed in castration-resistant prostate cancer. It was also found that high expression of SF3B2 correlates with AR-V7 expression even in localized prostate cancer. Further, in a mouse model, it has been found that a tumor formed by prostate cancer cells that express SF3B2 at a high level has enhanced proliferation.
  • SF3B2 increases expression of not only AR-V7 but also genes involved in tumor exacerbation such as NEAT1 and AKT1 in prostate cancer cells. Therefore, by examining the gene expression of SF3B2 in prostate cancer tissue, it becomes possible to determine the degree of malignancy of the tumor. Based on the above findings, the invention of a data acquisition method for diagnosing prostate cancer using the SF3B2 gene was completed.
  • a method for acquiring data for diagnosing prostate cancer comprising a step of measuring the expression level of SF3B2 gene.
  • the SF3B2 gene is represented by a DNA having a nucleotide sequence of SEQ ID NO: 1 or a DNA having a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and a DNA hybridizing under stringent conditions, [1] The method described in. [3] The method of [1] or [2], wherein the diagnosis of prostate cancer is a prognosis of prostate cancer. [4] The method according to [3], wherein the prognosis of prostate cancer is a castration resistance diagnosis. [5] A kit for diagnosing prostate cancer, comprising a reagent capable of measuring the expression level of SF3B2 gene.
  • data for diagnosing prostate cancer using the SF3B2 gene can be obtained, and a case progressing to castration-resistant prostate cancer can be accurately diagnosed.
  • patients at risk of recurrence can be screened, and by following such risk cases, recurrence of prostate cancer can be detected at an early stage, and treatment can be performed at an early stage. It can be expected to increase the survival rate.
  • it is possible to screen cases showing resistance to drugs such as abiraterone and enzalutamide it can be expected that it is possible to select cases in which the effects of these drugs can be expected.
  • FIG. 4 is a western blot diagram showing the change in the expression level of AR-V7 protein by expressing SF3B2 in prostate cancer cells (a photograph as a substitute for a drawing).
  • prostate cancer cell CWR22Rv1 22Rv1
  • prostate cancer cell LNCaP95 was used.
  • the present invention provides a method for obtaining data for diagnosing prostate cancer, which comprises a step of measuring the expression level of the SF3B2 gene.
  • Prostate cancer is classified into stages by, for example, judging the degree of malignancy of the cancer by Gleason score or judging the degree of progression of the cancer by TNM classification.
  • the Gleason score can be classified by observing the tissue structure of a cancer cell under a microscope using a pathological image to determine the degree of malignancy of the cancer.
  • TNM classification the disease is classified according to the presence and degree of metastasis (lymph node metastasis, distant metastasis).
  • the prostate cancer in the present invention includes any staging determined based on these indices.
  • the TNM classification is one of the indices used for staging malignant tumors, and includes T factor (the size and degree of invasion of the primary tumor), N factor (the presence and degree of lymph node metastasis), And M factors (presence or absence and extent of distant metastasis).
  • the prostate cancer includes castration-resistant prostate cancer. Castration-resistant prostate cancer is a condition in which prostate cancer, once suppressed by endocrine therapy for prostate cancer, becomes resistant to endocrine therapy drugs by long-term endocrine therapy. Is prostate cancer that has become worse again.
  • SF3B2 is a positive splicing regulator of AR-V7. That is, SF3B2 controls splicing of AR-V7 by removing the intron to which SF3B2 is bound while leaving the exon to which SF3B2 is bound.
  • the SF3B2 gene a gene having the nucleotide sequence shown in SEQ ID NO: 1 is exemplified. Further, as long as the SF3B2 gene encodes an SF3B2 protein having a function of enhancing the expression of AR-V7, a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the nucleotide sequence of SEQ ID NO: 1 Is included.
  • examples of stringent conditions include, for example, conditions for washing under conditions of 0.1 ⁇ SDS, 0.1 ⁇ SSC, and 68 ° C.
  • SF3B2 is a protein that more easily binds to the AR-V7 pre-mRNA sequence than the AR-FL pre-mRNA sequence, and further binds directly to the CE3 region, which is a potential exon of AR-V7.
  • measuring the expression level of the SF3B2 gene is not particularly limited, it means, for example, measuring the expression level of mRNA or protein.
  • the expression level of the SF3B2 gene can be measured by, for example, RT-PCR, quantitative PCR, microarray, Northern blot, spectrophotometry, or fluorometry, and the expression level of SF3B2 protein can be measured, for example, by flow cytometry. , Western blot, and ELISA.
  • the base sequence of the coding region of the SF3B2 gene is, for example, the sequence of SEQ ID NO: 1. Using this sequence and the like, primers and probes for gene expression analysis can be designed or obtained.
  • the nucleotide sequence may have 90% or more, preferably 95% or more, more preferably 98% or more identity with the nucleotide sequence of SEQ ID NO: 1.
  • the above-mentioned primers and probes have 90% or more, preferably 95% or more, more preferably 98% or more identity with the complementary sequence of the above-mentioned base sequence as long as they can specifically bind to the above-mentioned base sequence. May be.
  • As the antibody used to measure the protein expression level a commercially available antibody can be used, or an antibody prepared using a part of the amino acid sequence of the SF3B2 protein represented by SEQ ID NO: 2 as an antigen can be used. it can.
  • the amino acid sequence has 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of SEQ ID NO: 2 as long as it is an SF3B2 protein having a function of enhancing AR-V7 expression. May be something.
  • the antibody or the like has 90% or more, preferably 95% or more, more preferably 98% or more identity with a complementary sequence of the amino acid sequence as long as it can specifically bind to the amino acid sequence. Good.
  • the expression level of the SF3B2 gene in a biological sample obtained from a test subject is increased by, for example, 20% or more, preferably 50% or more, more preferably 100% or more, as compared with those obtained from a healthy subject. If so, it can be determined that the subject has prostate cancer.
  • the expression level of the SF3B2 gene in a biological sample obtained from a subject is equal to or greater than the value in a prostate cancer patient (control value), it is determined that the subject has prostate cancer.
  • diagnostic criteria can be formulated in combination with the expression levels of other genes and the like, and based on the results, it may be determined whether or not the subject has prostate cancer.
  • a healthy subject is generally defined as a subject who does not have a specific chronic disease. In the present invention, it may be used in the sense as defined above, but may also be used in the sense of a subject who does not have prostate cancer but has another disease.
  • the healthy subject is not particularly limited as long as it is a subject that develops prostate cancer, but includes a mammal, and is preferably a human.
  • the biological sample includes cells or tissues of the prostate, cells or tissues around the prostate, and cells or tissues at any site where metastasis of prostate cancer can be considered, which is collected from a test subject or a healthy subject.
  • the biological sample may be a body fluid such as blood or lymph that may contain prostate cancer cells, and preferably includes circulating cancer cells. Circulating cancer cells are not particularly limited, but can be collected by, for example, applying magnetic beads coated with an antibody recognizing prostate cancer to blood and performing positive selection by magnetism. .
  • the diagnosis may be a prognosis.
  • Prognosis generally refers to future prospects for the disease. In the prognosis, for example, if prostate cancer remission or improvement, or long-term survival, long-term progression-free period, if the prognosis is determined to be good, while, for example, prostate cancer recurrence, relapse, Alternatively, the prognosis is determined to be poor in the case of metastasis, short-term survival, or short-term progression-free period.
  • Diagnosis according to the present invention includes, before the onset of prostate cancer, evaluation of the possibility of becoming prostate cancer in the future, preferably of castration-resistant prostate cancer.
  • Treatment strategies can be determined based on the results of diagnosis or prognosis. For example, it is possible to determine whether to perform surgical treatment, radiation therapy, chemotherapy, or endocrine therapy (hormonal therapy), or to determine which other endocrine agent to use as endocrine therapy.
  • the present invention also includes a kit for use in prostate cancer diagnosis.
  • the contents of the kit are composed of a combination of devices or reagents. If the kit contains a substance that is essentially the same as each component described below, or a substance that is essentially the same as a part thereof, the configuration or form is Even if they are different, they are included in the kit of the present invention.
  • the reagent includes, for example, a primer capable of specifically amplifying the SF3B2 gene when the expression level of the SF3B2 gene is measured by the PCR method. If necessary, a reverse transcriptase, a polymerase, a buffer, a fluorescent reagent and the like may be included. As the device, for example, a fluorometer, a thermal cycler or the like can be used.
  • a support such as glass, plastic, silicon, or a membrane on which the SF3B2 gene fragment is spotted is included. If necessary, a labeling reagent, a hybridization buffer, a fragmentation buffer, and the like may be included.
  • an anti-SF3B2 antibody is included.
  • a diluent of a biological sample an antibody-immobilized solid phase, a buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a reagent for detecting a labeled body, a standard substance, and the like are also included.
  • the diluent for the biological sample include an aqueous solution containing a protein such as BSA or casein in a surfactant or a buffer.
  • the antibody-immobilized solid phase a material obtained by immobilizing an anti-molecular marker antibody or an antibody fragment thereof on a material obtained by shaping various polymer materials to suit the intended use is used. Tubes, beads, plates, latex and other fine particles, sticks, etc. are used as materials, and polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, etc. Polymer materials, glass, ceramics and metals.
  • known methods such as a physical method and a chemical method or a combination method thereof can be used. For example, there may be mentioned a polystyrene 96-well immunoassay microterplate having an antibody or antibody fragment immobilized on a hydrophobic solid phase.
  • the reaction buffer may be any as long as it provides a solvent environment for the binding reaction between the antibody in the antibody-immobilized solid phase and the antigen in the biological sample, but may be a surfactant, a buffer, a BSA, Reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators, and the like.
  • the labeled secondary antibody or its antibody fragment is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine intestinal alkaline phosphatase, ⁇ -galactosidase, and a buffer. , A mixture of proteins such as BSA and casein, preservatives and the like.
  • HRP horseradish peroxidase
  • bovine intestinal alkaline phosphatase bovine intestinal alkaline phosphatase
  • ⁇ -galactosidase a buffer
  • a mixture of proteins such as BSA and casein, preservatives and the like.
  • horseradish peroxidase may be used as a reagent for detecting a labeled substance, a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine, or a fluorescent substance such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid.
  • a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine
  • a fluorescent substance such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid.
  • the luminescent substrate such as a substrate or luminol is alkaline phosphatase
  • examples thereof include a substrate for measuring absorbance such as 4-nitrophenyl phosphate and a fluorescent substrate such as 4-methylumbelliferyl phosphate.
  • Example 1 Correlation between Gleason score and SF3B2 expression
  • the Gleason score and SF3B2 gene expression level in prostate cancer were examined by TCGA data (The Cancer Genome Atlas Research Network, 2015) (Fig. 1).
  • the relationship between SF3B2 gene expression level and non-relapse rate in prostate cancer patients was examined by TCGA data (Fig. 2).
  • the prostate cancer patient group was divided into two groups depending on whether the expression level of SF3B2 was high or low.
  • 3 shows a Kaplan-Meier curve.
  • SF3B2 Increase in expression level of AR-V7 due to SF3B2 overexpression
  • SF3B2 was overexpressed in two types of prostate cancer cells 22Rv1 and LNCaP95. By expression, increase / decrease of expression of AR-V7 was confirmed.
  • Overexpression of SF3B2 in prostate cancer cells was performed by transfecting a vector having a CAG promoter into which a SF3B2 gene was inserted downstream of the CAG promoter into prostate cancer cells. Further, puronycin resistance gene was simultaneously expressed in the cells by IRES. As shown in FIG.
  • SF3B2 overexpression increased the expression level of AR-V7 in any of the two types of prostate cancer cells.
  • overexpression of SF3B2 did not affect AR-FL (full length). That is, even if the presence or absence of the expression of AR-V7, which is an androgen receptor, cannot be directly measured by diagnostic imaging or the like, the expression of AR-V7 is indirectly measured by measuring the expression level of SF3B2 instead. It is considered that the presence or absence can be estimated.

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé d'acquisition de données pour le diagnostic du cancer de la prostate. Dans ce procédé, le niveau d'expression du gène SF3B2 est mesuré, et en fonction du niveau d'expression, le dépistage est réalisé pour des cas pour lesquels il existe un risque de récidive du cancer de la prostate, et le dépistage est réalisé pour des cas qui démontrent une résistance aux médicaments.
PCT/JP2019/038347 2018-09-28 2019-09-27 Procédé d'acquisition de données pour le diagnostic du cancer de la prostate WO2020067498A1 (fr)

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JP2013507987A (ja) * 2009-10-26 2013-03-07 アボット・ラボラトリーズ 非小細胞肺癌の予後を決定するための診断方法
US20130130241A1 (en) * 2010-03-08 2013-05-23 Regents Of The University Of Minnesota Androgen receptor isoforms and methods
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WO2017091706A1 (fr) * 2015-11-23 2017-06-01 Acceleron Pharma Inc. Méthode de traitement de troubles oculaires
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WO2002029099A1 (fr) * 2000-10-03 2002-04-11 Takara Bio Inc. Technique de criblage de substance physiologiquement active
JP2005080524A (ja) * 2003-09-05 2005-03-31 Japan Science & Technology Agency 前立腺癌マーカポリペプチド、該ポリペプチドに対する抗体、及び該ポリペプチドを利用した前立腺癌の診断方法
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JP2013507987A (ja) * 2009-10-26 2013-03-07 アボット・ラボラトリーズ 非小細胞肺癌の予後を決定するための診断方法
US20130130241A1 (en) * 2010-03-08 2013-05-23 Regents Of The University Of Minnesota Androgen receptor isoforms and methods
US20170268063A1 (en) * 2014-05-19 2017-09-21 The Johns Hopkins University Methods for identifying androgen receptor splice variants in subjects having castration resistant prostate cancer
WO2017091706A1 (fr) * 2015-11-23 2017-06-01 Acceleron Pharma Inc. Méthode de traitement de troubles oculaires
EP3214444A1 (fr) * 2016-03-04 2017-09-06 AIT Austrian Institute of Technology GmbH Procédé de diagnostic du cancer de la prostate et moyens associés

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DATABASE Genbank [online] 15 September 2018 (2018-09-15), Database accession no. NM_006842. 2 *

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