WO2022250517A1 - 전립선암 진단용 바이오마커 및 이의 용도 - Google Patents

전립선암 진단용 바이오마커 및 이의 용도 Download PDF

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Publication number
WO2022250517A1
WO2022250517A1 PCT/KR2022/007695 KR2022007695W WO2022250517A1 WO 2022250517 A1 WO2022250517 A1 WO 2022250517A1 KR 2022007695 W KR2022007695 W KR 2022007695W WO 2022250517 A1 WO2022250517 A1 WO 2022250517A1
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WIPO (PCT)
Prior art keywords
mir
hsa
mirna
prostate cancer
mrna
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Ceased
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PCT/KR2022/007695
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English (en)
French (fr)
Korean (ko)
Inventor
최관용
김종민
이상일
허태양
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Sysbiogen Co Ltd
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Sysbiogen Co Ltd
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Priority to US18/565,020 priority Critical patent/US20250283176A1/en
Priority to EP22811708.1A priority patent/EP4365310A4/en
Priority to JP2023573568A priority patent/JP2024521869A/ja
Publication of WO2022250517A1 publication Critical patent/WO2022250517A1/ko
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the composition is a composition for diagnosing prostate cancer comprising an agent capable of measuring the expression level of the combination of: (a) lncRNA and miRNA, (b) mRNA and miRNA of a fusion gene, or ( c) mRNA of lncRNA, miRNA, and fusion gene, wherein the lncRNA is at least one selected from PCA3 and MALAT1, and the mRNA of the fusion gene is TMPRSS2:ERG.
  • Figures 2 and 3 show the results for single marker analysis:
  • Figure 2 is a box plot for trend analysis of target gene expression;
  • 3 is a Receiver Operator Characteristic (ROC) curve and Area Under the Curve (AUC) for biomarker performance analysis.
  • ROC Receiver Operator Characteristic
  • AUC Area Under the Curve
  • prostate cancer used in the present invention is a malignant tumor arising in the prostate, including 'localized prostate cancer' and 'advanced prostate cancer', but is not limited thereto.
  • diagnosis means confirming the presence or characteristics of a pathological condition.
  • diagnosis can be interpreted as confirming whether prostate cancer has developed or progressed.
  • the term "marker or diagnostic marker” refers to a substance that can be diagnosed by distinguishing a subject with prostate cancer from normal cells or normal subjects, and refers to cells or cells in which prostate cancer has developed or progressed compared to normal cells.
  • organic biomolecules such as polypeptides, proteins or nucleic acids (eg, lncRNA, miRNA, or mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in a subject; Not limited to this.
  • Prostate cancer diagnostic markers can be indicators for the onset and progression of prostate cancer, and thus can be used for diagnosis of prostate cancer onset, disease progression, and disease.
  • the lncRNA prostate cancer diagnostic marker of the present invention is at least one selected from PCA3 and MALAT1, the mRNA prostate cancer diagnostic marker of the fusion gene is TMPRSS2:ERG, and the miRNA prostate cancer diagnostic markers are hsa_miR_375, hsa_miR_27b_3p, hsa_miR_31_5p, hsa_miR_125b_5p, hsa_miR_146a_3p, hsa_miR_146a_5p, hsa_miR_17_3p, hsa_miR_200b_3p, hsa_miR_21_5p, hsa_miR_1185_2_3p, hsa_miR_141_5p, hsa_miR_222_3p, hsa_miR_24_3p, hsa_
  • each marker is known, and for example, the sequence of each marker may be a human-derived sequence.
  • probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundreds of bases as short as possible to form a specific binding with a gene or mRNA, oligonucleotide It can be manufactured in the form of a probe, single stranded DNA probe, double stranded DNA probe, RNA probe, etc., and can be labeled for easier detection.
  • the primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoramidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • the six markers of the present invention namely lncRNA (PCA3)
  • TMPRSS2:ERG mRNA of the fusion gene
  • miRNA hsa_miR_125b_5p, hsa_
  • the method for providing information for diagnosis of prostate cancer of the present invention may additionally use non-protein clinical information of the patient, that is, clinical information other than the marker, in addition to the analysis result of the biomarker.
  • non-protein clinical information includes, for example, the patient's age, sex, weight, eating habits, body mass, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), angiography, endoscopic retrograde cholangiopancreatography, ultrasound endoscopy, tumor markers, and/or laparoscopy.
  • Substances that specifically recognize the biomarkers of the present invention may be individually dispensed and present in a partitioned container, and therefore, the present invention includes molecules that can specifically recognize the markers of the present invention. It provides a device and / or mechanism that does.
  • the present invention provides a method for diagnosing prostate cancer using the composition or kit for diagnosing prostate cancer.
  • QIAGEN product (cat no. 55114) was used to extract miRNA from urine samples. Add 3 ml of urine sample to a 50 ml centrifuge tube into which 400 ⁇ l Proteinase K was pipetted, add 3.2 ml of Buffer ACL (without carrier RNA) and 1.0 ml of Buffer ATL and pulse-vortex for 30 seconds. mixed. After incubation at 60° C. for 30 minutes, 9.0 ml of buffer ACB and 7.0 ml of isopropanol were added to the lysate in the tube and mixed thoroughly by pulse-vortexing for 15-30 seconds.
  • the QIAamp Mini column was placed in a clean 2 ml collection tube, the lid was removed and the assembly was incubated at 56°C for 10 minutes to completely dry the membrane. Place the QIAamp Mini column in a clean 1.5 ml elution tube, discard the 2 ml collection tube, and carefully apply 20-150 ⁇ l of Buffer AVE to the center of the QIAamp Mini membrane, close the lid and incubate at room temperature for 3 minutes. Finally, nucleic acids were eluted by centrifugation for 1 minute at maximum speed (20,000 ⁇ g, 14,000 rpm) in a microcentrifuge.
  • PCa/BPH prostate cancer/benign prostatic hyperplasia
  • PCa/BPH prostate cancer/benign prostatic hyperplasia
  • RFECV Recursive Feature Elimination with Cross-Validation
  • LOO Leave-One-Out
  • RF Random Forest
  • the PCA3_4 and TMPRSS2:ERG also showed high feature importance.
  • 4 miRNA combinations (4 feature combinations) plus 1 lncRNA and 1 mRNA of the fusion gene (6 feature combinations) were selected for machine learning (ML). It became.
  • the method is not limited thereto as an example of a method for selecting a biomarker.
  • each machine learning algorithm showed significant differences in AUC and LOOCV scores.
  • the average AUC was 0.71 ⁇ 0.08 (Standard Deviation), 0.66 ⁇ 0.12, and 0.80 ⁇ 0.10, and the average LOOCV scores were 0.727 ⁇ 0.066, 0.658 ⁇ 0.080, and 0.711 ⁇ 0.071.
  • SVM and LR are more suitable than RF for single-biomarker-based ML classification.
  • the overall trends of AUC and LOOCV scores appear to be more influenced by biomarkers than ML algorithms. Since the above results are based on a single biomarker, when the same analysis is applied to multiple biomarkers, better results are expected due to synergy between biomarkers.
  • PCa/BPH prostate cancer/benign prostatic hyperplasia
  • Logistic Regression can calculate a score to distinguish between a prostate cancer patient and a BPH patient, and the method for calculating the score is as follows.
  • the final score is evaluated based on a critical value of 0.5, and a score of 0.5 or more is judged as prostate cancer, and a score of less than 0.5 is judged as benign prostatic hyperplasia.
  • PCa/BPH prostate cancer/benign prostatic hyperplasia
  • custom machine learning software using Python 3.8.5 was run on Linux.
  • the software includes open libraries such as Scikit Learn, Scikit Plot and Panda.
  • Scikit Learn's C-Support Vector Classification was used.
  • the Radial Basis Function was used as a kernel, and a Balanced Class Weight was used in consideration of the number difference between classes.
  • RF Random Forest
  • Scikit Learn's Random Forest Classifier was used, and the model consists of 100 decision trees with balanced class weights.
  • Logistic Regression was also imported from Scikit Learn, and all parameters followed the default settings except for the balanced class weights.
  • the combination of four miRNAs (hsa_miR_125b_5p, hsa_miR_141_5p, hsa_miR_17_3p, and hsa_miR_30c_5p) derived through random forest analysis through the RFECV algorithm proved to be particularly useful as a detection marker, and one lncRNA (PCA3) and The combination with the addition of the mRNA of one fusion gene (TMPRSS2-ERG) also proved useful as a detection marker.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
PCT/KR2022/007695 2021-05-28 2022-05-30 전립선암 진단용 바이오마커 및 이의 용도 Ceased WO2022250517A1 (ko)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US18/565,020 US20250283176A1 (en) 2021-05-28 2022-05-30 Biomarker for diagnosing prostate cancer and use thereof
EP22811708.1A EP4365310A4 (en) 2021-05-28 2022-05-30 Biomarkers for the diagnosis of prostate cancer and their use
JP2023573568A JP2024521869A (ja) 2021-05-28 2022-05-30 前立腺癌診断用バイオマーカー及びその使用

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KR10-2021-0069428 2021-05-28
KR1020210069428A KR102550113B1 (ko) 2021-05-28 2021-05-28 전립선암 진단용 바이오마커 및 이의 용도

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EP (1) EP4365310A4 (https=)
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130043104A (ko) * 2010-04-06 2013-04-29 카리스 라이프 사이언스 룩셈부르크 홀딩스 질병용 순환 생물학적 지표들
US10822661B2 (en) * 2014-09-04 2020-11-03 Miodx Method of multivariate molecule analysis

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ES2749651T3 (es) * 2015-02-11 2020-03-23 Region Midtjylland Método basado en microARN para la detección temprana de cáncer de próstata en muestras de orina
KR102414106B1 (ko) * 2020-03-12 2022-06-29 (주) 바이오인프라생명과학 유방암 진단용 다중 바이오마커 및 이의 용도

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130043104A (ko) * 2010-04-06 2013-04-29 카리스 라이프 사이언스 룩셈부르크 홀딩스 질병용 순환 생물학적 지표들
US10822661B2 (en) * 2014-09-04 2020-11-03 Miodx Method of multivariate molecule analysis

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BUSSEMAKERS ET AL., CANCER RESEARCH, vol. 59, no. 23, 1999, pages 5975 - 5979
FREDSØE JACOB, RASMUSSEN ANNE K.I., THOMSEN ANNI R., MOURITZEN PETER, HØYER SØREN, BORRE MICHAEL, ØRNTOFT TORBEN F., SØRENSEN KARI: "Diagnostic and Prognostic MicroRNA Biomarkers for Prostate Cancer in Cell-free Urine", EUROPEAN UROLOGY FOCUS, ELSEVIER, NL, vol. 4, no. 6, 1 December 2018 (2018-12-01), NL , pages 825 - 833, XP093007039, ISSN: 2405-4569, DOI: 10.1016/j.euf.2017.02.018 *
FREDSPJ ET AL., EUR. UROL. FOCUS, vol. 4, 2018, pages 825 - 833
FUMIHIKO URABE, JUNTARO MATSUZAKI, YUSUKE YAMAMOTO, TAKAHIRO KIMURA, TOMOHIKO HARA, MAKIKO ICHIKAWA, SATOKO TAKIZAWA, YOSHIAKI AOK: "Large-scale Circulating microRNA Profiling for the Liquid Biopsy of Prostate Cancer", CLINICAL CANCER RESEARCH, ASSOCIATION FOR CANCER RESEARCH, US, vol. 25, no. 10, 15 May 2019 (2019-05-15), US, pages 3016 - 3025, XP055684095, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-18-2849 *
HAN, H.BEARSS, D. J.BROWNE, L. W.CALALUCE, R.NAGLE, R. B.VON HOFF, D. D.: "Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray", CANCER RES, vol. 62, no. 10, 2002, pages 2890 - 6
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See also references of EP4365310A4
URABE, F ET AL., CLIN. CANCER RES., vol. 25, 2019, pages 3016 - 3025
URBINATI, GIORGIA ET AL., MOLECULAR THERAPY-NUCLEIC ACIDS, 2016, pages 5
WANG, F. ET AL., ONCOTARGET, vol. 5, no. 22, 2014, pages 11091 - 11102
XUE, D ET AL., JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, vol. 22, no. 6, 2018, pages 3223 - 3237
XUEPING ZHANG ET AL., CLIN EXP METASTASIS, vol. 26, no. 8, 2009, pages 965 - 79

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Publication number Publication date
EP4365310A4 (en) 2025-12-17
EP4365310A1 (en) 2024-05-08
KR102550113B1 (ko) 2023-07-03
KR20230106542A (ko) 2023-07-13
US20250283176A1 (en) 2025-09-11
JP2024521869A (ja) 2024-06-04
KR20220161039A (ko) 2022-12-06

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