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WO2025237289A1 - Kit de marqueur de méthylation pour carcinome urothélial - Google Patents

Kit de marqueur de méthylation pour carcinome urothélial

Info

Publication number
WO2025237289A1
WO2025237289A1 PCT/CN2025/094580 CN2025094580W WO2025237289A1 WO 2025237289 A1 WO2025237289 A1 WO 2025237289A1 CN 2025094580 W CN2025094580 W CN 2025094580W WO 2025237289 A1 WO2025237289 A1 WO 2025237289A1
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WO
WIPO (PCT)
Prior art keywords
region
gene
human
dna
application
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/094580
Other languages
English (en)
Chinese (zh)
Inventor
楼峰
陈利斌
王慧娜
张雅风
刘磊
曹善柏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acornmed Biotechnology Co Ltd Beijing
Acornmed Medical Instrument Co Ltd Tianjin
Tianjin Acornmed Medical Laboratory Co Ltd
Original Assignee
Acornmed Biotechnology Co Ltd Beijing
Acornmed Medical Instrument Co Ltd Tianjin
Tianjin Acornmed Medical Laboratory Co Ltd
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Filing date
Publication date
Application filed by Acornmed Biotechnology Co Ltd Beijing, Acornmed Medical Instrument Co Ltd Tianjin, Tianjin Acornmed Medical Laboratory Co Ltd filed Critical Acornmed Biotechnology Co Ltd Beijing
Publication of WO2025237289A1 publication Critical patent/WO2025237289A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • This application relates to the field of biomedicine, specifically to a biomarker, method, and kit for detecting urothelial carcinoma.
  • Urothelial carcinoma includes upper urinary tract urothelial carcinoma (UTUC) and lower urinary tract urothelial carcinoma (mainly bladder cancer, BC), characterized by high incidence, multiple primary sites, and a high recurrence rate after surgery.
  • bladder cancer examination include cystoscopy (which allows for biopsy of suspected lesions), urine cytology, imaging examinations, and fluorescence in situ hybridization.
  • UC urothelial carcinoma
  • Traditional imaging examinations are difficult to use for small or atypical lesions, leading to challenges in localization and qualitative diagnosis.
  • Urine tests are widely used as an important diagnostic tool in clinical practice, but they all have certain limitations. For example, urine cytology and FISH tests have relatively low sensitivity and specificity and cannot replace endoscopy and biopsy.
  • Endoscopy (ureteroscopy/cystoscopy) combined with biopsy is the gold standard for the diagnosis and follow-up of urothelial tumors, but due to its invasiveness and relatively high cost, patient compliance is poor, making it difficult to detect early tumors and tumor recurrence in some patients, thus missing the opportunity for treatment.
  • UC ulcerative bladder cancer
  • MIBC muscle-invasive bladder cancer
  • FISH fluorescence in situ hybridization
  • cystoscopy is the gold standard for diagnosing bladder cancer, but its invasive nature leads to patient resistance, making adherence to this method difficult to guarantee, and carcinoma in situ is not easily detected under cystoscopy and is easily confused with inflammatory changes in the bladder, requiring biopsy for definitive diagnosis.
  • ureteroscopy allows direct visualization of the tumor and enables biopsy.
  • the location and angle of the tumor can affect the endoscopic examination, often preventing the use of a rigid ureteroscope to reach the tumor site and thus hindering diagnosis.
  • Ureteral obstruction can also affect endoscopic results, making it difficult to determine the cause of obstruction.
  • Causes of obstruction include ureteral stones, ureteropelvic junction stenosis, and ureteral malignancies.
  • ureteroscopy itself presents certain challenges. Besides the inherent risks of trauma and infection associated with ureteroscopy, the likelihood of postoperative exudation and adhesions in surrounding tissues significantly increases, complicating radical surgery and making it unsuitable for routine procedures.
  • this application provides a method for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma occurrence, and/or assessing the prognosis/progress of urothelial carcinoma, as well as a corresponding detection kit and/or detection model.
  • This application is the first to discover the correlation between the methylation of the target gene and urothelial carcinoma, and applies it to the detection of urothelial carcinoma.
  • the target gene of this application can be used alone or in combination with other urothelial carcinoma detection biomarkers for the development of detection products.
  • the detection method uses urinary gDNA as a template for initial sample testing, making it a non-invasive method that effectively avoids complications such as urogenital infections, urethral and bladder bleeding, urethral injury, and urethral stricture. It is also easy to perform and can be done at home.
  • conventional urinary cytology testing has insufficient sensitivity and a risk of false negatives, especially for G1 grade (well-differentiated papillary carcinoma) and low-grade tumors, where the sensitivity is only 16%.
  • the detection method in this application detects biomarkers including HOXA9, VIM, SIM2, ZIC4, ZNF154, KCNMA1, and FOXF2, or HOXA9, TRPS1, RAB37, SEPT9, LHX8, NKX6-2, ZNF571-AS1, NKX1-1, and MARC.
  • Methylation status of genes including H11, PENK, TBX15, NKX2-8, NRN1, BCL11B, ALX1, and KCNQ1DN, or LRRC9, BNC1, IRX4, SLC26A4, and PAX1 effectively utilizes multiplex information within a limited sample to effectively differentiate tumors, especially early-stage tumors, exhibiting high sensitivity and specificity.
  • the detection method of this application provides rapid data results, for example, using quantitative real-time PCR, which facilitates timely subsequent treatment for patients.
  • the method, kit, and/or detection model of this application which includes specific gene combinations, have higher sensitivity and specificity.
  • this application provides the use of a reagent capable of detecting the methylation status of a DNA region containing a target gene in a sample in the preparation of a urothelial carcinoma detection kit, wherein the target gene comprises HOXA9.
  • this application provides a method for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma occurrence, and/or assessing the prognosis/progression of urothelial carcinoma, the method comprising detecting the methylation status of a DNA region containing a target gene in a sample, the target gene comprising HOXA9.
  • the DNA region of the HOXA9 gene is derived from the region defined in human chr7:27203916-27207001. In some embodiments, the DNA region of the HOXA9 gene includes the region defined in human chr7:27203916-27207001. In some embodiments, the DNA region of the HOXA9 gene is derived from a region selected from the group consisting of: the region defined in human chr7:27206851-27207001, the region defined in human chr7:27203926-27204076, the region defined in human chr7:27205754-27205904, and the region defined in human chr7:27206632-27206782.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27206851-27207001. In some embodiments, the DNA region of the HOXA9 gene includes the region defined by human chr7:27203926-27204076. In some embodiments, the DNA region of the HOXA9 gene includes the region defined by human chr7:27205754-27205904. In some embodiments, the DNA region of the HOXA9 gene includes the region defined by human chr7:27206632-27206782.
  • the samples are from cancer patients, suspected cancer patients, cancer-susceptible populations, high-risk cancer populations, or healthy individuals.
  • the cancer patients include patients who have not received anticancer therapy and/or patients who have received anticancer therapy.
  • the sample comprises tissue, cells, and/or body fluids.
  • the sample is selected from blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, and tissue samples.
  • the sample comprises blood.
  • the sample comprises urine.
  • the target gene comprises HOXA9, and also comprises any one of the following genes selected from the group consisting of: VIM, KCNQ1DN, SIM2, ZNF571-AS1, NKX1-1, MARCH11, ZIC4, BNC1, IRX4, SLC26A4, TBX15, NKX2-8, NRN1, ZNF154, TRPS1, RAB37, SEPT9, LRRC9, PAX1, KCNMA1, FOXF2, LHX8, NKX6-2, PENK, BCL11B, and ALX1.
  • the target gene comprises HOXA9 and any two genes selected from the group consisting of: VIM, KCNQ1DN, SIM2, ZNF571-AS1, NKX1-1, MARCH11, ZIC4, BNC1, IRX4, SLC26A4, TBX15, NKX2-8, NRN1, ZNF154, TRPS1, RAB37, SEPT9, LRRC9, PAX1, KCNMA1, FOXF2, LHX8, NKX6-2, PENK, BCL11B, and ALX1.
  • the target gene comprises three genes and is selected from any combination of those in Table 5. It should be noted that each combination in Table 5 may be a DNA region used in the embodiments or other DNA regions listed in this application.
  • the target gene may include HOXA9, VIM and SIM2, but this does not mean that the target gene contains HOXA9, VIM and SIM2 and can only contain the DNA regions corresponding to HOXA9_1, VIM_1 and SIM2_1 or their primer and probe sequences.
  • the method includes providing reagents capable of recognizing and/or binding to the DNA region containing the target gene, its base-transformed region, or its complementary region.
  • the reagent comprises a nucleic acid molecule capable of recognizing and/or binding to the DNA region containing the target gene, its base-transformed region, or its complementary region.
  • the reagent comprises primers and/or probes capable of recognizing and/or binding to the DNA region containing the target gene, its base-transformed region, or its complementary region.
  • the DNA region comprises one, two, three, four or more DNA regions.
  • the method includes obtaining nucleic acids from the sample.
  • the method includes transforming the nucleic acid such that methylated bases and unmethylated bases form different substances after transformation.
  • the transformation includes transformation by a deamination reagent and/or a methylation-sensitive restriction endonuclease.
  • the method includes contacting the sample with a nucleic acid molecule or fragment thereof capable of recognizing, amplifying, and/or binding to the DNA region containing the target gene, its base-transformed region, its complementary region, or a region thereof.
  • the DNA region fragment is a fragment of 50-300 bp in length.
  • the DNA region fragment is a fragment of 100-200 bp in length.
  • the method for determining the methylation state includes determining the presence and/or content of a substance formed after the conversion by a base having a methylation state.
  • the method for determining methylation status includes determining the presence and/or content of DNA regions or fragments thereof that have a methylation status.
  • the presence and/or quantity of the DNA region or fragment thereof having the modified state is determined by the fluorescence Ct value detected by the fluorescence PCR method.
  • the presence of urothelial carcinoma, or the risk of urothelial carcinoma formation is determined by confirming the presence of a modification state of the DNA region or fragment thereof and/or the amount of the DNA region or fragment thereof having a higher level of modification state relative to a reference level.
  • the method further includes amplifying the DNA region or fragment thereof in the sample to be tested before determining the presence and/or content of modifications to the DNA region or fragment thereof.
  • the amplification includes PCR amplification.
  • the method further includes: if one or more methylation states of the target gene in the sample are detected to be positive, the test result is considered positive; if all methylation states of the target gene in the sample are detected to be negative, the test result is considered negative.
  • the product further comprises a reagent that selectively modifies unmethylated cytosine residues in the DNA of the sample to produce detectable modified residues, but the reagent does not modify methylated cytosine residues.
  • the product further comprises a deamination agent and/or a methylation-sensitive restriction endonuclease.
  • the deamination agent comprises a bisulfite.
  • the product is a reagent kit.
  • this application provides upstream primers and corresponding downstream primers selected from SEQ ID NO:1-216 in Table 11.
  • this application provides probes selected from any one of the sequences in SEQ ID NO:217-324 for use in the methods, products and/or kits of this application.
  • Figures 1 to 15 show the NGS sequencing results of methylation levels of the HOXA9, KCNQ1DN, SIM2, VIM, ZIC4, ZNF154, KCNMA1, FOXF2, PAX1, BCL11B, LHX8, MARCH11, RAB37, TBX15, and ZNF571-AS1 genes in newly diagnosed and follow-up samples of urothelial carcinoma, respectively.
  • TD1, TD2...TD10 are newly diagnosed positive samples
  • ND1, ND2...ND10 are newly diagnosed negative samples
  • TR1, TR2...TR10 are follow-up positive samples
  • NR1, NR2...NR10 are follow-up negative samples.
  • HOXA9 generally refers to the gene encoding the homeobox protein Hox-A9, also known as HOX1 or HOX1G.
  • the human HOXA9 gene is located in Chromosome 7:27,202,054-27,210,117 on the reverse strand (GRCh37:CM000669.1). More information about the human HOXA9 gene can be found in ensemble number ENSG00000078399.
  • VIM generally refers to the gene encoding vimentin, also known as CTRCT30 or HEL113.
  • the human VIM gene is located in Chromosome 10:17,270,258-17,279,592 on the forward strand (GRCh37:CM000672.1). More information about the human VIM gene can be found in ensemble number ENSG00000026025.
  • SIM2 generally refers to the gene encoding SIM bHLH transcription factor 2, also known as BHLHE15, MGC119447, or SIM.
  • the human SIM2 gene is located in Chromosome 21:38,071,433-38,122,218 on the forward strand (GRCh37:CM000683.1). More information about the human SIM2 gene can be found in ensemble number NSG00000159263.
  • ZIC4 generally refers to the gene encoding member 4 of the Zic family.
  • the human ZIC4 gene is located in the reverse strand of Chromosome 3:147,103,833-147,124,647 (GRCh37:CM000665.1). More information about the human ZIC4 gene can be found in ensemble number NSG00000174963.
  • ZNF154 generally refers to the gene encoding zinc finger protein 154, also known as PHZ-92.
  • the human ZNF154 gene is located in Chromosome 19:58,208,735-58,220,579 on the reverse strand (GRCh37:CM000681.1). More information about the human ZNF154 gene can be found in ensemble number ENSG00000179909.
  • KCNMA1 generally refers to the potassium-conducting calcium-activated channel, subfamily M, ⁇ member 1, also known as KCNMA.
  • the human KCNMA1 gene is located in Chromosome 10:78,629,359-79,398,353 on the reverse strand (GRCh37:CM000672.1). More information about the human KCNMA1 gene can be found in ensemble number ENSG00000156113.
  • FOXF2 generally refers to the gene encoding the forkhead box protein F2, also known as FKHL6, FREAC-2, or FREAC2.
  • the human FOXF2 gene is located on the forward strand of Chromosome 6:1,390,069-1,395,832 (GRCh37:CM000668.1). More information about the human FOXF2 gene can be found in ensemble number ENSG00000137273. Additionally, the FOXF2 gene contains a branched transcript, FOXF2-DT.
  • the human FOXF2-DT transcript is located on the reverse strand of Chromosome 6:1,384,025-1,385,301. More information about the human FOXF2-DT gene can be found in ensemble number ENSG00000261730.
  • TRPS1 generally refers to the gene encoding the transcriptional repressor GATA-binding 1, also known as GC79 or LGCR.
  • the human TRPS1 gene is located in Chromosome 8:116,420,724-116,821,899 on the reverse strand (GRCh37:CM000670.1). More information about the human TRPS1 gene can be found in ensemble number ENSG00000104447.
  • RAB37 generally refers to RAB37, a member of the RAS oncogene family.
  • the human RAB37 gene is located in Chromosome 17:72,666,717-72,743,474 on the forward strand (GRCh37:CM000679.1). More information about the human RAB37 gene can be found in ensemble number ENSG00000172794.
  • SEPT9 generally refers to the gene encoding the Septin9 protein, and may also be referred to as AF17Q25, KIAA0991, MSF, MSF1, PNUTL4, SEPTIN9, or SEPTD1.
  • the human SEPT9 gene is located in Chromosome 17:75,276,651-75,496,678 on the forward strand (GRCh37:CM000679.1). More information about the human SEPT9 gene can be found in ensemble number ENSG00000184640.
  • LHX8 generally refers to the gene encoding LIM homeobox protein 8, also known as LHX7.
  • the human LHX8 gene is located in Chromosome 1:75,594,119-75,627,218 on the forward strand (GRCh37:CM000663.1). More information about the human LHX8 gene can be found in ensemble number ENSG00000162624.
  • NKX6-2 generally refers to the gene encoding the homeobox protein Nkx-6.2, and may also be referred to as GTX, NKX6.1, or NKX6B.
  • the human NKX6-2 gene is located in Chromosome 10:134,598,297-134,599,556 on the reverse strand (GRCh37:CM000672.1). More information about the human NKX6-2 gene can be found in ensemble number ENSG00000148826.
  • ZNF571-AS1 generally refers to antisense RNA 1 of the ZNF571 gene.
  • the human ZNF571-AS1 gene is located in Chromosome 19:38,039,816-38,078,249 on the forward strand (GRCh37:CM000681.1). More information about the human ZNF571-AS1 gene can be found in ensemble number ENSG00000267470.
  • NKX1-1 generally refers to the gene encoding NK1 homeobox protein 1, also known as HSPX153 or SAX2.
  • the human NKX1-1 gene is located in Chromosome 4:1,396,720-1,400,119 inverted strand (GRCh37:CM000666.1). More information about the human NKX1-1 gene can be found in ensemble number ENSG00000235608.
  • MARCH11 generally refers to the gene encoding membrane-associated ring finger protein antibody 11, also known as MARCH-XI.
  • the human MARCH11 gene is located in Chromosome 5:16,067,248-16,180,871 on the reverse strand (GRCh37:CM000667.1). More information about the human MARCH11 gene can be found in ensemble number ENSG00000183654.
  • PENK generally refers to the gene encoding the endogenous hormone proenkephalin.
  • the human PENK gene is located in Chromosome 8:57,349,233-57,359,293 on the reverse strand (GRCh37:CM000670.1). More information about the human PENK gene can be found in ensemble number ENSG00000181195.
  • TBX15 generally refers to a transcription factor in the T-box that plays a crucial role in embryonic development.
  • the human TBX15 gene is located in Chromosome 1:119,425,669-119,532,179 on the reverse strand (GRCh37:CM000663.1). More information about the human TBX15 gene can be found in ensemble number ENSG00000092607.
  • NKX2-8 generally refers to the gene encoding NK2 homeobox protein 8, and may also be referred to as NKX2-9, NKX2.8, and NKX2H.
  • the human NKX2-8 gene is located in Chromosome 14:37,049,784-37,051,812 on the reverse strand (GRCh37:CM000676.1). More information about the human NKX2-8 gene can be found in ensemble number ENSG00000136327.
  • NRN1 generally refers to the gene encoding the neurotogenic factor neuroitin, also known as NRN.
  • the human NRN1 gene is located in Chromosome 6:5,998,232-6,007,200 on the reverse strand (GRCh37:CM000668.1). More information about the human NRN1 gene can be found in ensemble number ENSG000000124785.
  • BCL11B generally refers to a gene encoding a zinc finger protein, also known as ZNF856B or ATL1.
  • the human BCL11B gene is located in the reverse strand of Chromosome 14:99,635,624-99,737,861 (GRCh37:CM000676.1). More information about the human BCL11B gene can be found in ensemble number ENSG00000127152.
  • ALX1 generally refers to the gene encoding ALX homeobox protein 1, also known as CART1, FND3, or HEL23.
  • the human ALX1 gene is located in Chromosome 12:85,673,885-85,695,562 on the forward strand (GRCh37:CM000674.1). More information about the human ALX1 gene can be found in ensemble number ENSG00000180318.
  • KCNQ1DN generally refers to the downstream neighbor gene of KCNQ1, also known as BWRT or HSA404617.
  • the human KCNQ1DN gene is located in Chromosome 11:2,891,263-2,893,335 on the forward strand (GRCh37:CM000673.1). More information about the human KCNQ1DN gene can be found in ensemble number NSG00000237941.
  • LRRC9 generally refers to the gene rich in leucine repeat sequence 9, also known as FLJ46156.
  • the human LRRC9 gene is located in Chromosome 14:60,386,431-60,530,277 on the forward strand (GRCh37:CM000676.1). More information about the human LRRC9 gene can be found in ensemble number ENSG00000131951.
  • BNC1 generally refers to the gene encoding basic nucleoprotein 1, also known as BNC, BSN1, or HsT19447.
  • the human BNC1 gene is located in Chromosome 15:83,924,655-83,953,466 on the reverse strand (GRCh37:CM000677.1). More information about the human BNC1 gene can be found in ensemble number ENSG00000169594.
  • IRX4 generally refers to the gene encoding iroquois homeobox protein 4, also known as IRXA3.
  • the human IRX4 gene is located in Chromosome 5:1,877,541-1,887,350 in the reverse strand (GRCh37:CM000667.1). More information about the human IRX4 gene can be found in ensemble number ENSG00000113430.
  • SLC26A4 generally refers to member 4 of solute carrier family 26 (anion exchanger), also known as DFNB4, EVA, PDS, or TDH2B.
  • the human SLC26A4 gene is located in Chromosome 7:107,301,080-107,358,254 (GRCh37:CM000669.1). More information about the human SLC26A4 gene can be found in ensemble number ENSG00000091137.
  • PAX1 generally refers to PAX1 in the PAX family gene, also known as HUP48, OFC2.
  • the human PAX1 gene is located in Chromosome 20:21,686,297-21,696,620 in the forward strand (GRCh37:CM000682.1). More information about the human PAX1 gene can be found in ensemble number ENSG00000125813.
  • genes in this application can be described by their names and their chromosomal coordinates.
  • sample or “sample to be tested” generally refers to a sample that needs to be tested. For example, it can be used to detect whether one or more gene regions on the sample to be tested are modified.
  • the term "healthy population” generally refers to a population whose tumors are currently undetectable by tumor detection methods.
  • a healthy population can develop into a cancer patient, a high-risk group for cancer, a suspected cancer patient, or a cancer-susceptible population.
  • “Healthy population” is a concept relative to "cancer patient,” which typically refers to a population confirmed to have a tumor through pathological diagnosis or other testing methods.
  • high-risk population for cancer generally refers to a population with a higher risk of developing cancer.
  • people with a family history of cancer people who carry cancer susceptibility genes, people with unhealthy lifestyles (such as smokers, alcoholics, and those who frequently stay up late), and people who are frequently exposed to toxic or harmful substances in their living or working environment.
  • the term "suspected cancer patient” generally refers to a group of people who have cancer symptoms but have not been confirmed to have cancer by pathological diagnosis or other testing methods.
  • tumor-susceptible population generally refers to a population that is prone to developing tumors.
  • people with a family history of cancer people who carry tumor susceptibility genes, people with unhealthy lifestyles (such as smokers, alcoholics, and those who frequently stay up late), and people who are frequently exposed to toxic or harmful substances in their living or working environment.
  • the susceptible population or high-risk population may include, but is not limited to, the following groups: patients with unclear clinical diagnosis, high-risk populations for urothelial carcinoma (age > 50 years; long-term smokers; people with long-term exposure to carcinogens such as industrial chemicals; people with a family history of bladder cancer; and people who ingest aristolochic acid-containing traditional Chinese medicine).
  • DNA region generally refers to a sequence of two or more covalently bonded naturally occurring or modified deoxyribonucleotides.
  • a DNA region of a gene can refer to the location of a specific deoxyribonucleotide sequence in which the gene is located, such as a sequence that encodes the gene.
  • the DNA region of this application includes the full length of the DNA region, its complementary region, or fragments thereof. For example, a sequence of at least about 20 kb upstream and downstream of the detection region provided in this application can be used as the detection region.
  • a sequence of at least about 20 kb, at least about 15 kb, at least about 10 kb, at least about 5 kb, at least about 3 kb, at least about 2 kb, at least about 1 kb, or at least about 0.5 kb upstream and downstream of the detection region provided in this application can be used as the detection region.
  • suitable primers and probes can be designed based on the region for the detection of sample methylation.
  • a DNA region when referring to a DNA region, unless otherwise specified, it generally refers to the Human Reference Genome Version GRCh37, UCSC version number hg19.
  • methylation generally refers to the methylation state of a gene fragment, nucleotide, or base thereof in this application.
  • the DNA fragment containing the gene in this application may be methylated on one or more strands.
  • the DNA fragment containing the gene in this application may be methylated at one or more sites.
  • methylation may refer to the conversion of the 5′ cytosine of a CpG island dinucleotide on a DNA sequence to 5′ methylcytosine (5mC), and may also include the formation of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG).
  • methylation state generally refers to the methylation state of one or more nucleotides or bases in the DNA region containing the gene.
  • the methylation state of the DNA region containing the gene may be the methylation state of one or more CpG dinucleotides within that DNA region.
  • methylation state can refer to the presence or absence of 5-methylcytosine ("5-mC” or “5-mCyt”) at one or more CpG dinucleotides within a DNA sequence.
  • the methylation state at one or more specific CpG methylation sites (each with two CpG dinucleotide sequences) within a DNA sequence includes “unmethylated,” “fully methylated,” and “hemimethylated.”
  • hemimethylated can refer to the methylation state of double-stranded DNA where only one strand is methylated.
  • hypomethylation can refer to an average methylation state corresponding to an increased presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence of the test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample.
  • hypomethylation can refer to an average methylation state corresponding to a decreased presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence of the test DNA sample compared to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample.
  • the methylation state in this application can refer to the presence, absence, and/or content of methylation at one or more specific nucleotides within a DNA region.
  • the methylation state in this application can refer to the methylation state of each base or each specific base (e.g., cytosine) in a specific DNA sequence.
  • the methylation state in this application can refer to the methylation state of base pair combinations and/or base combinations in a specific DNA sequence.
  • the methylation state in this application can refer to information about the regional methylation density in a specific DNA sequence (including the DNA region containing the gene or a specific regional segment thereof), without providing precise location information of where methylation occurs within the sequence.
  • having or having a high methylation state can be associated with transcriptional silencing of a specific region.
  • having or having a high methylation state can be associated with the ability to be transformed by methylation-specific transforming agents (e.g., deaminating agents and/or methylation-sensitive restriction enzymes).
  • transformation can refer to being converted into other substances and/or being cleaved or digested.
  • transformation generally refers to the conversion of one or more structures into another.
  • the transformations in this application can be specific.
  • cytosine without methylation modification can be transformed into other structures (e.g., uracil), while cytosine with methylation modification can remain substantially unchanged after transformation.
  • cytosine without methylation modification can be cleaved after transformation, while cytosine with methylation modification can remain substantially unchanged after transformation.
  • a deamination agent generally refers to a substance capable of removing an amino group.
  • a deamination agent can remove the amino group from unmodified cytosine.
  • bisulfite generally refers to a reagent capable of distinguishing between modified and unmodified DNA regions.
  • a bisulfite may include bisulfite, its analogues, or combinations thereof.
  • a bisulfite can deamination of the amino group of unmodified cytosine to distinguish it from modified cytosine.
  • analyte generally refers to a substance having a similar structure and/or function.
  • an analogue of a bisulfite may have a similar structure to a bisulfite.
  • an analogue of a bisulfite may refer to a reagent that can also distinguish between modified and unmodified DNA regions.
  • methylation-sensitive restriction endonuclease generally refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. For example, for a restriction endonuclease that specifically cleaves when the recognition site is unmethylated, cleavage may not occur or may occur with significantly reduced efficiency when the recognition site is methylated. For instance, a methylation-specific restriction endonuclease may recognize sequences containing CG dinucleotides (e.g., cgcg or cccggg).
  • the term "primer” can be a natural or synthetic oligonucleotide that, upon forming a double helix with a polynucleotide template, can act as a starting point for nucleic acid synthesis and extend from its 3' end along the template to form an extended double helix.
  • the sequence of the nucleotides added during the extension process is determined by the sequence of the template polynucleotide.
  • Primers can typically be extended using polymerases such as nucleic acid polymerases.
  • complementary can include hybridization or base pairing or duplex formation between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between primer binding sites on oligonucleotide primers and single-stranded nucleic acids.
  • Complementary nucleotides can typically be A and T (or A and U) or C and G.
  • they can be considered substantially complementary when the nucleotides of one strand pair with at least about 80% (typically at least about 90% to about 95%, or even about 98% to about 100%) of the other strand in optimal alignment and comparison and with appropriate nucleotide insertions or deletions.
  • two complementary nucleotide sequences are capable of hybridization and can have less than 25% mismatch between the reversed nucleotides, more likely less than 15% mismatch, less than 5% mismatch, or no mismatch.
  • the two molecules can hybridize under highly stringent conditions.
  • Urothelial carcinoma generally refers to cancer associated with the malignant proliferation of urothelial cells.
  • Urothelial carcinoma can include upper urinary tract urothelial carcinoma (UTUC, e.g., renal pelvis cancer and ureteral cancer) and lower urinary tract urothelial carcinoma (e.g., bladder cancer).
  • Urothelial carcinoma can occur in the renal pelvis, ureter, or bladder.
  • progression generally refers to a change in a disease from a less severe state to a more severe state.
  • tumor progression can include an increase in the number or severity of the tumor, the extent of cancer cell metastasis, or the rate of cancer growth or spread.
  • tumor progression can include the stages of this cancer from a less severe state to a more severe state, such as progression from stage I to stage II, from stage II to stage III, etc.
  • occurrence generally refers to the presence of a lesion in an individual's body.
  • the individual can be diagnosed as a cancer patient.
  • fluorescent PCR generally refers to a quantitative or semi-quantitative PCR technique.
  • it may be a real-time quantitative polymerase chain reaction (PCR), a quantitative polymerase chain reaction (PCR), or a kinetic polymerase chain reaction (PCR).
  • PCR can be used to amplify and quantitatively detect the amount of the initial target nucleic acid using an intercalating fluorescent dye or a sequence-specific probe, which may contain a fluorescent reporter molecule that is detectable only by hybridization with the target nucleic acid.
  • PCR amplification generally refers to a polymerase chain amplification reaction.
  • PCR amplification in this application may include any polymerase chain amplification reaction currently known for DNA amplification.
  • fluorescence Ct value generally refers to a measurement for quantitative or semi-quantitative evaluation of target nucleic acids. For example, it may refer to the number of amplification reaction cycles required for the fluorescence signal to reach a set threshold.
  • the term “comprising” generally indicates that a scope or element includes other elements or features. This is used to define the scope of a particular technology, making it more flexible and inclusive. When the word “comprising” is used, it means that the scheme includes not only the explicitly listed elements but also other related elements or features to achieve similar functionality.
  • the term "and/or” is generally used to indicate a range of choices, representing an inclusive relationship between two or more elements or conditions. When “and/or” is used, it allows one or both of these elements or conditions to be present to achieve the same technical effect. For example, if the description contains an expression such as "A and/or B,” it means that it can include either A or B, or both A and B.
  • This application provides a combination of gene markers that can be used for the diagnostic detection of tumors, confirming the presence of a tumor, assessing the risk of tumor development, and/or evaluating the prognosis/progress of a tumor. Therefore, this application provides a method for the diagnostic detection of tumors, confirming the presence of a tumor, assessing the risk of tumor development, and/or evaluating the prognosis/progress of a tumor. This method can be used for the early diagnosis and/or recurrence monitoring of tumors.
  • This application also provides a reagent combination for detecting the combination of gene markers, and a kit containing the reagent combination. The reagent combination contains reagents that can determine the methylation status of the DNA region containing the gene marker in a sample, its base-transformed region, or its complementary region.
  • the tumor may be a tumor of the urinary system.
  • the tumor may be urothelial carcinoma.
  • the tumor may be bladder cancer.
  • the inventors of this application have discovered that by detecting the methylation level or status of key genes in a test sample, the presence of urinary system malignancies can be confirmed with high sensitivity and specificity, the risk of developing urinary system malignancies can be assessed, and/or the prognosis/progression of urinary system malignancies can be evaluated.
  • This application has also identified several target genes that can serve as biomarkers for the detection of urinary system tumors (e.g., urothelial carcinoma).
  • the target gene described in this application may include HOXA9.
  • the DNA region of the HOXA9 gene is derived from the region defined in human chr7:27203916-27207001.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27203916-27207001.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27203916-27204200.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27203916-27204800.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27205500-27207001.
  • the DNA region of the HOXA9 gene includes the region defined in human chr7:27205754-27206782. In some embodiments, the HOXA9 gene DNA region includes the region defined by human chr7:27205754-27207001. In some embodiments, the HOXA9 gene DNA region includes the region defined by human chr7:27206500-27207001. For example, the HOXA9 gene DNA region may include the region defined by human chr7:27206851-27207001. For example, the HOXA9 gene DNA region may include the region defined by human chr7:27203926-27204076.
  • the HOXA9 gene DNA region may include the region defined by human chr7:27205754-27205904.
  • the HOXA9 gene DNA region may include the region defined by human chr7:27206632-27206782.
  • the target gene described in this application may include VIM.
  • the DNA region of the VIM gene is derived from any one of the DNA regions corresponding to the VIM gene in Table 1 or Table 2.
  • the DNA region of the VIM gene includes any one of the DNA regions corresponding to the VIM gene in Table 1 or Table 2.
  • the target gene described in this application may include SIM2.
  • the DNA region of the SIM2 gene is derived from any one of the DNA regions corresponding to the SIM2 gene in Table 1 or Table 2.
  • the DNA region of the SIM2 gene includes any one of the DNA regions corresponding to the SIM2 gene in Table 1 or Table 2.
  • the target gene described in this application may include ZIC4.
  • the DNA region of the ZIC4 gene originates from any one of the DNA regions corresponding to the ZIC4 gene in Table 1 or Table 2.
  • the DNA region of the ZIC4 gene includes any one of the DNA regions corresponding to the ZIC4 gene in Table 1 or Table 2.
  • the target gene described in this application may include ZNF154.
  • the DNA region of the ZNF154 gene is derived from any one of the DNA regions corresponding to the ZNF154 gene in Table 1 or Table 2.
  • the DNA region of the ZNF154 gene includes any one of the DNA regions corresponding to the ZNF154 gene in Table 1 or Table 2.
  • the target gene described in this application may include KCNMA1.
  • the DNA region of the KCNMA1 gene is derived from any one of the DNA regions corresponding to the KCNMA1 gene in Table 1 or Table 2.
  • the DNA region of the KCNMA1 gene includes any one of the DNA regions corresponding to the KCNMA1 gene in Table 1 or Table 2.
  • the target gene described in this application may include FOXF2.
  • the DNA region of the FOXF2 gene is derived from any one of the DNA regions corresponding to the FOXF2 gene in Table 1 or Table 2.
  • the DNA region of the FOXF2 gene includes any one of the DNA regions corresponding to the FOXF2 gene in Table 1 or Table 2.
  • the target gene described in this application may include TRPS1.
  • the DNA region of the TRPS1 gene is derived from any one of the DNA regions corresponding to the TRPS1 gene in Table 1 or Table 2.
  • the DNA region of the TRPS1 gene includes any one of the DNA regions corresponding to the TRPS1 gene in Table 1 or Table 2.
  • the target gene described in this application may include RAB37.
  • the DNA region of the RAB37 gene is derived from any one of the DNA regions corresponding to the RAB37 gene in Table 1 or Table 2.
  • the DNA region of the RAB37 gene includes any one of the DNA regions corresponding to the RAB37 gene in Table 1 or Table 2.
  • the target gene described in this application may include SEPT9.
  • the DNA region of the SEPT9 gene is derived from any one of the DNA regions corresponding to the SEPT9 gene in Table 1 or Table 2.
  • the DNA region of the SEPT9 gene includes any one of the DNA regions corresponding to the SEPT9 gene in Table 1 or Table 2.
  • the target gene described in this application may include LHX8.
  • the DNA region of the LHX8 gene is derived from any one of the DNA regions corresponding to the LHX8 gene in Table 1 or Table 2.
  • the DNA region of the LHX8 gene includes any one of the DNA regions corresponding to the LHX8 gene in Table 1 or Table 2.
  • the target gene described in this application may include NKX6-2.
  • the DNA region of the NKX6-2 gene is derived from any one of the DNA regions corresponding to the NKX6-2 gene in Table 1 or Table 2.
  • the DNA region of the NKX6-2 gene includes any one of the DNA regions corresponding to the NKX6-2 gene in Table 1 or Table 2.
  • the target gene described in this application may include ZNF571-AS1.
  • the DNA region of the ZNF571-AS1 gene is derived from any one of the DNA regions corresponding to the ZNF571-AS1 gene in Table 1 or Table 2.
  • the DNA region of the ZNF571-AS1 gene includes any one of the DNA regions corresponding to the ZNF571-AS1 gene in Table 1 or Table 2.
  • the target gene described in this application may include NKX1-1.
  • the DNA region of the NKX1-1 gene is derived from any one of the DNA regions corresponding to the NKX1-1 gene in Table 1 or Table 2.
  • the DNA region of the NKX1-1 gene includes any one of the DNA regions corresponding to the NKX1-1 gene in Table 1 or Table 2.
  • the target gene described in this application may include MARCH11.
  • the DNA region of the MARCH11 gene is derived from any one of the DNA regions corresponding to the MARCH11 gene in Table 1 or Table 2.
  • the DNA region of the MARCH11 gene includes any one of the DNA regions corresponding to the MARCH11 gene in Table 1 or Table 2.
  • the target gene described in this application may include PENK.
  • the DNA region of the PENK gene is derived from any one of the DNA regions corresponding to the PENK genes in Table 1 or Table 2.
  • the DNA region of the PENK gene includes any one of the DNA regions corresponding to the PENK genes in Table 1 or Table 2.
  • the target gene described in this application may include TBX15.
  • the DNA region of the TBX15 gene is derived from any one of the DNA regions corresponding to the TBX15 gene in Table 1 or Table 2.
  • the DNA region of the TBX15 gene includes any one of the DNA regions corresponding to the TBX15 gene in Table 1 or Table 2.
  • the target gene described in this application may include NKX2-8.
  • the DNA region of the NKX2-8 gene is derived from any one of the DNA regions corresponding to the NKX2-8 gene in Table 1 or Table 2.
  • the DNA region of the NKX2-8 gene includes any one of the DNA regions corresponding to the NKX2-8 gene in Table 1 or Table 2.
  • the target gene described in this application may include NRN1.
  • the DNA region of the NRN1 gene is derived from any one of the DNA regions corresponding to the NRN1 gene in Table 1 or Table 2.
  • the DNA region of the NRN1 gene includes any one of the DNA regions corresponding to the NRN1 gene in Table 1 or Table 2.
  • the target gene described in this application may include BCL11B.
  • the DNA region of the BCL11B gene is derived from any one of the DNA regions corresponding to the BCL11B gene in Table 1 or Table 2.
  • the DNA region of the BCL11B gene includes any one of the DNA regions corresponding to the BCL11B gene in Table 1 or Table 2.
  • the target gene described in this application may include ALX1.
  • the DNA region of the ALX1 gene is derived from any one of the DNA regions corresponding to the ALX1 gene in Table 1 or Table 2.
  • the DNA region of the ALX1 gene includes any one of the DNA regions corresponding to the ALX1 gene in Table 1 or Table 2.
  • the target gene described in this application may include KCNQ1DN.
  • the DNA region of the KCNQ1DN gene is derived from any one of the DNA regions corresponding to the KCNQ1DN gene in Table 1 or Table 2.
  • the DNA region of the KCNQ1DN gene includes any one of the DNA regions corresponding to the KCNQ1DN gene in Table 1 or Table 2.
  • the target gene described in this application may include LRRC9.
  • the DNA region of the LRRC9 gene originates from any one of the DNA regions corresponding to the LRRC9 gene in Table 1 or Table 2.
  • the DNA region of the LRRC9 gene includes any one of the DNA regions corresponding to the LRRC9 gene in Table 1 or Table 2.
  • the target gene described in this application may include BNC1.
  • the DNA region of the BNC1 gene originates from any one of the DNA regions corresponding to the BNC1 gene in Table 1 or Table 2.
  • the DNA region of the BNC1 gene includes any one of the DNA regions corresponding to the BNC1 gene in Table 1 or Table 2.
  • the target gene described in this application may include IRX4.
  • the DNA region of the IRX4 gene is derived from any one of the DNA regions corresponding to the IRX4 gene in Table 1 or Table 2.
  • the DNA region of the IRX4 gene includes any one of the DNA regions corresponding to the IRX4 gene in Table 1 or Table 2.
  • the target gene described in this application may include SLC26A4.
  • the DNA region of the SLC26A4 gene is derived from any one of the DNA regions corresponding to the SLC26A4 gene in Table 1 or Table 2.
  • the DNA region of the SLC26A4 gene includes any one of the DNA regions corresponding to the SLC26A4 gene in Table 1 or Table 2.
  • the target gene described in this application may include PAX1.
  • the DNA region of the PAX1 gene is derived from any one of the DNA regions corresponding to the PAX1 gene in Table 1 or Table 2.
  • the DNA region of the PAX1 gene includes any one of the DNA regions corresponding to the PAX1 gene in Table 1 or Table 2.
  • the target gene of this application may include HOXA9, and one gene selected from the following group: VIM, KCNQ1DN, SIM2, ZNF571-AS1, NKX1-1, MARCH11, ZIC4, BNC1, IRX4, SLC26A4, TBX15, NKX2-8, NRN1, ZNF154, TRPS1, RAB37, SEPT9, LRRC9, PAX1, KCNMA1, FOXF2, LHX8, NKX6-2, PENK, BCL11B, and ALX1.
  • the target gene of this application may include HOXA9 and VIM.
  • the target gene of this application may include HOXA9 and SIM2.
  • the target gene of this application may include HOXA9 and ZIC4.
  • the target gene of this application may include HOXA9 and ZNF154.
  • the target gene of this application may include HOXA9 and KCNMA1.
  • the target gene of this application may include HOXA9 and FOXF2.
  • the target gene of this application may include HOXA9 and TRPS1.
  • the target gene of this application may include HOXA9 and RAB37.
  • the target gene of this application may include HOXA9 and MARCH11.
  • the target gene of this application may include HOXA9 and LHX8.
  • the target gene of this application may include HOXA9 and NKX6-2.
  • the target gene of this application may include HOXA9 and ZNF571-AS1.
  • the target gene of this application may include HOXA9 and NKX1-1.
  • the target gene of this application may include HOXA9 and SEPT9.
  • the target gene of this application may include HOXA9 and PENK.
  • the target gene of this application may include HOXA9 and TBX15.
  • the target gene of this application may include HOXA9 and NKX2-8.
  • the target gene of this application may include HOXA9 and NRN1.
  • the target gene of this application may include HOXA9 and BCL11B.
  • the target gene of this application may include HOXA9 and ALX1.
  • the target gene of this application may include HOXA9 and KCNQ1DN.
  • the target gene of this application may include HOXA9 and LRRC9.
  • the target gene of this application may include HOXA9 and BNC1.
  • the target gene of this application may include HOXA9 and IRX4.
  • the target gene of this application may include HOXA9 and SLC26A4.
  • the target gene of this application may include HOXA9 and PAX1.
  • the DNA region of the target gene of this application may be selected from those mentioned above.
  • the target gene of this application may include HOXA9, and two genes selected from the following group: VIM, KCNQ1DN, SIM2, ZNF571-AS1, NKX1-1, MARCH11, ZIC4, BNC1, IRX4, SLC26A4, TBX15, NKX2-8, NRN1, ZNF154, TRPS1, RAB37, SEPT9, LRRC9, PAX1, KCNMA1, FOXF2, LHX8, NKX6-2, PENK, BCL11B, and ALX1.
  • the confirmed presence of methylation in a target gene's DNA region, or an increased number of methylations relative to a reference level can be associated with the occurrence of disease.
  • the DNA region in this application can refer to a specific segment of genomic DNA.
  • the DNA region in this application can be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location determined by referring to its name, or by referring to its chromosomal coordinates.
  • This application uses the methylation status of these specific DNA regions as a series of analytical indicators, which can provide significant improvements in sensitivity and/or specificity, and can simplify the screening process.
  • sensitivity can refer to the proportion of correctly identified positive results, i.e., the percentage of individuals correctly identified as having the disease in question;
  • specificity can refer to the proportion of correctly identified negative results, i.e., the percentage of individuals correctly identified as not having the disease in question.
  • the detection method of this application may include determining the presence of a disease based on the determination of the presence and/or content of the modification state of the DNA region, or its complementary region, or the aforementioned fragment in the sample to be tested.
  • the method of this application may include assessing whether a disease has been diagnosed based on the determination of the presence and/or content of the modification state of the DNA region, or its complementary region, or the aforementioned fragment in the sample to be tested.
  • the method of this application may include assessing the risk of a confirmed disease and/or the level of that risk based on the determination of the presence and/or content of the modification state of the DNA region, or its complementary region, or the aforementioned fragment in the sample to be tested.
  • the method of this application may include assessing the progression of the disease based on the determination of the presence and/or content of the modification state of the DNA region, or its complementary region, or the aforementioned fragment in the sample to be tested.
  • the DNA region of this application may contain all forms of these molecules, as well as fragments or variants thereof.
  • variants may contain at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the DNA region described in this application, and variants may contain one or more deletions, additions, substitutions, inversions, etc.
  • the modification state of the variants described in this application can achieve the same evaluation results.
  • the DNA region of this application may contain all other forms of mutations, polymorphic variations, or allelic variations.
  • the method of this application includes providing a reagent capable of detecting the methylation status of any DNA region selected from Table 1 or Table 2, or a nucleic acid capable of recognizing, binding to, and/or amplifying the following DNA regions, their complementary regions, their base-transformed regions, or fragments thereof.
  • the method of this application may include obtaining nucleic acids from a sample.
  • the nucleic acids may comprise cell-free, free nucleic acids.
  • the sample to be tested may comprise tissues, cells, and/or body fluids.
  • the sample to be tested may comprise plasma.
  • the sample to be tested may comprise urine.
  • the detection method of this application can be performed on any suitable biological sample.
  • the sample to be tested can be any sample of biological material, such as that derived from an animal, but not limited to cellular material, biological fluids (e.g., blood), excrement, tissue biopsy specimens, surgical specimens, or fluids that have been introduced into an animal and subsequently removed.
  • the sample to be tested in this application may comprise a sample that has undergone any form of processing after sample separation.
  • the method of this application may include transforming the DNA region or fragment thereof.
  • methylated bases and unmethylated bases may form different substances after transformation.
  • methylated bases remain substantially unchanged after transformation, while unmethylated bases may be transformed into other bases different from the methylated bases (e.g., the other bases may include uracil) or cleaved after transformation.
  • the bases may include cytosine.
  • the transformation may include transformation by a deamination agent and/or a methylation-sensitive restriction enzyme.
  • the deamination agent may include bisulfite or its analogues, such as sodium bisulfite or potassium bisulfite.
  • the method of this application may optionally include amplifying the DNA region or fragment thereof in the sample to be tested before determining the presence and/or content of modifications to the DNA region or fragment thereof.
  • the amplification may include PCR amplification.
  • the amplification of this application may include any known amplification system.
  • the amplification step of this application may be optional.
  • "amplification” may refer to the process of producing multiple copies of the desired sequence.
  • Multiple copies may refer to at least two copies.
  • “Copy” may not mean perfect sequence complementarity or identity with the template sequence.
  • a copy may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (e.g., sequence alterations introduced by primers containing sequences that hybridize to but are not complementary to the template), and/or sequence errors that may occur during the amplification process.
  • nucleotide analogs such as deoxyinosine, intentional sequence alterations (e.g., sequence alterations introduced by primers containing sequences that hybridize to but are not complementary to the template), and/or sequence errors that may occur during the amplification process.
  • the method for determining the presence and/or content of methylation, or the method for detecting methylation may include confirming the presence and/or content of substances formed after the transformation by bases having methylation.
  • the method for determining the presence and/or content of methylation may include determining the presence and/or content of DNA regions or fragments having methylation.
  • the presence and/or content of DNA regions or fragments having methylation can be detected directly.
  • it can be detected that DNA regions or fragments having methylation may have different characteristics from DNA regions or fragments not having methylation during a reaction (e.g., an amplification reaction).
  • DNA regions or fragments having methylation may be specifically amplified and emit fluorescence; DNA regions or fragments not having methylation may be substantially not amplified and substantially not emit fluorescence.
  • alternative methods for determining the presence and/or content of substances formed after the transformation by bases having methylation may be included within the scope of this application.
  • the presence and/or content of methylated DNA regions or fragments can be determined by the fluorescence Ct value detected by fluorescence PCR.
  • the presence of urothelial carcinoma, or the risk of urothelial carcinoma formation can be determined by the presence of the modification state of the DNA region or fragment and/or by the higher content of the modification state of the DNA region or fragment relative to a reference level.
  • the fluorescence Ct value of the test sample is lower than the reference fluorescence Ct value
  • the presence of methylation of the DNA region or fragment can be determined, and/or the content of the modification state of the DNA region or fragment can be determined to be higher than the content of methylation in the reference sample.
  • the reference fluorescence Ct value can be determined by detecting the reference sample. For example, when the fluorescence Ct value of the test sample is higher or substantially equivalent to the reference fluorescence Ct value, the presence of modification of the DNA region or fragment cannot be ruled out; when the fluorescence Ct value of the test sample is higher or substantially equivalent to the reference fluorescence Ct value, the content of modification of the DNA region or fragment can be confirmed to be lower than or substantially equal to the content of modification in the reference sample.
  • this application can use a cycle threshold (i.e., Ct value) to represent the presence and/or content of methylation status of a specific DNA region or fragment, such as including the methylation level of the sample to be tested and a reference level.
  • Ct value can refer to the cycle number at which the fluorescence of the PCR product can be detected above the background signal.
  • the Ct value can be negatively correlated with the initial amount of the target marker in the sample, that is, the lower the Ct value, the greater the number of modified states of the DNA region or fragment in the sample to be tested.
  • the Ct value of a test sample when the Ct value of a test sample is the same as or lower than its corresponding reference Ct value, it can confirm the presence of a specific disease, diagnose the formation of a specific disease, or assess a risk of formation of a specific disease, or evaluate a certain progression of a specific disease.
  • the Ct value of a test sample when the Ct value of a test sample is lower than its corresponding reference Ct value by at least one cycle, at least two cycles, at least five cycles, at least ten cycles, at least twenty cycles, or at least fifty cycles, it can confirm the presence of a specific disease, diagnose the formation of a specific disease, or assess a certain progression of a specific disease.
  • the Ct value of a cell sample, tissue sample, body fluid sample, or sample derived from a subject is the same as or higher than its corresponding reference Ct value, it can be confirmed that a specific disease is not present, the formation of a specific disease is diagnosed, the risk of formation of a specific disease is present, or a certain progression of a specific disease is assessed.
  • the Ct value of a cell sample, tissue sample, body fluid sample, or sample derived from a subject is higher than its corresponding reference Ct value by at least one cycle, at least two cycles, at least five cycles, at least ten cycles, at least twenty cycles, or at least fifty cycles, it can be confirmed that a specific disease is not present, the formation of a specific disease is diagnosed, the risk of formation of a specific disease is present, or a certain progression of a specific disease is assessed.
  • the Ct value of a cell sample, tissue sample, body fluid sample, or sample derived from a subject is the same as its corresponding reference Ct value, it can be confirmed that a specific disease is present or not present, the formation or absence of a specific disease is diagnosed, the risk of formation of a specific disease is present or not present, or a certain progression of a specific disease is assessed, and a recommendation for further testing can be given.
  • the reference level or control level in this application can refer to a normal level or a healthy level.
  • the normal level can be the modification status level of a sample DNA region derived from cells, tissues, or individuals without the disease.
  • the normal level when used for tumor assessment, can be the modification status level of a sample DNA region derived from tumor-free cells, tissues, or individuals.
  • the normal level when used for urothelial carcinoma assessment, can be the modification status level of a sample DNA region derived from cells, tissues, or individuals without urothelial carcinoma.
  • a reference level can refer to a threshold level at which a subject or sample is identified as having or not having a specific disease.
  • a reference level can refer to a threshold level at which a subject is diagnosed as having developed or at risk of developing a specific disease.
  • a reference level can refer to a threshold level at which a subject is assessed as having a certain progression of a specific disease.
  • the reference level could refer to the modification status of a DNA region in a patient without a specific disease—it can be confirmed as having a specific disease, diagnosed as having developed or at risk of developing a specific disease, or assessed as having a certain progression of a specific disease.
  • substantially equal to A and B can mean that the difference between A and B is 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, or 0.0001% or less.
  • the modification status of DNA regions in cell samples, tissue samples, body fluid samples, or samples derived from the subject is higher than the corresponding reference level by at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 1, at least 2, at least 5, at least 10, or at least 20 times, it can be confirmed as the presence of a specific disease, diagnosed as the formation of a specific disease, or at risk of forming a specific disease, or assessed as a certain progression of a specific disease.
  • the modification status of DNA regions in cell samples, tissue samples, body fluid samples, or samples derived from the subject is higher than the corresponding reference level by at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 1, at least 2, at least 5, at least 10, or at least 20 times, it can be confirmed as the presence of a specific disease, diagnosed as the formation of a specific disease, or at risk of forming a specific disease, or assessed as a certain progression of a specific disease.
  • the modification status of DNA regions in cell samples, tissue samples, body fluid samples, or samples derived from a subject is below or substantially equal to a corresponding reference level—where the reference level could refer to the modification status of DNA regions in a patient with a specific disease—it can be confirmed that the specific disease is absent, the formation of the specific disease is diagnosed, the risk of its formation is identified, or a certain progression of the specific disease is assessed.
  • the modification status of DNA regions in cell samples, tissue samples, body fluid samples, or samples derived from a subject is below the corresponding reference level by at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, or at least 100%, it can be confirmed that the specific disease is absent, the formation of the specific disease is diagnosed, the risk of its formation is identified, or a certain progression of the specific disease is assessed.
  • the reference level in various situations within this application can be readily identified by those skilled in the art, such as by confirming a suitable reference level through a limited number of attempts and/or by suitable means of obtaining the reference level.
  • the reference level can be derived from one or more reference samples, wherein the reference level is obtained from experiments conducted in parallel with the experiment targeting the sample.
  • the reference level can also be obtained from a database comprising a collection of data, standards, or levels from one or more reference samples or disease reference samples.
  • the collection of data, standards, or levels can be standardized or normalized so that it can be used for comparison with data from one or more samples, thereby reducing errors arising under different detection conditions.
  • reference levels can be derived from a database, which may be a reference database, such as including modification status levels of the target marker and/or other laboratory and clinical data from one or more reference samples.
  • a reference database can be established by aggregating reference level data from reference samples obtained from healthy individuals and/or individuals without the corresponding disease (i.e., individuals known not to have the disease).
  • a reference database can be established by aggregating reference level data from reference samples obtained from individuals receiving treatment for the corresponding disease.
  • a reference database can be established by aggregating data from reference samples obtained from individuals at different stages of the disease. For example, different stages can be demonstrated by different modification status levels of the target marker of this application.
  • Those skilled in the art can also determine whether an individual has the corresponding disease or is at risk of having the corresponding disease based on various factors such as age, sex, medical history, family history, symptoms, etc.
  • the method of this application may include the following steps: obtaining nucleic acids from a sample to be tested; transforming the DNA region or a fragment thereof; and confirming the presence and/or content of a substance formed after the transformation by bases in a methylated state.
  • the method of this application may include the following steps: obtaining nucleic acids from a sample to be tested; transforming the DNA region or a fragment thereof; amplifying the DNA region or a fragment thereof in the sample to be tested; and confirming the presence and/or content of the substance formed after the transformation by bases in a methylated state.
  • the method of this application may include the following steps: obtaining nucleic acids from a sample to be tested; treating DNA obtained from the sample to be tested with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA; optionally amplifying the DNA region or fragment thereof in the sample to be tested; quantitatively, semi-quantitatively, or qualitatively analyzing the presence and/or content of the methylation state of the treated DNA in the sample to be tested; comparing the methylation level of the treated DNA in the sample to a corresponding reference level, wherein when the methylation state of the DNA region in the sample to be tested is higher than or substantially equal to the corresponding reference level, it can be confirmed as the presence of a specific disease, diagnosed as the formation of a specific disease or at risk of formation, or assessed as a certain progression of a specific disease.
  • one or more of the target regions mentioned above can be used as amplification regions and/or detection regions.
  • the target region described above can be detected using upstream primers and corresponding downstream primers selected from SEQ ID NO:1-216 in Table 11.
  • the target region described above can be detected using a probe selected from any one of the sequences in SEQ ID NO: 217-324.
  • the probe may have end modifications, and suitable modifications include, but are not limited to, the following: FAM, BHQ1, BHQ3, 5'6-FAM, 3'BHQ1, MGB, CY5, and VIC.
  • this application provides a nucleic acid, which may contain a sequence capable of binding to the DNA region containing the target gene of this application, or its complementary region, or the region derived from the above-mentioned transformation, or the fragment described above.
  • the nucleic acid may be any probe of this application.
  • this application provides a method for preparing nucleic acid, which may involve designing a nucleic acid capable of binding to the DNA region containing the target gene of this application, or its complementary region, or the region derived from the above-mentioned transformation, or the fragment described above, based on the modification state of the DNA region containing the target gene of this application, or its complementary region, or the region derived from the above-mentioned transformation, or the fragment described above.
  • the method for preparing nucleic acid may be any suitable method known in the art.
  • a single probe or primer configured to hybridize with the target polynucleotide can be used to assess the methylation status of the target polynucleotide.
  • multiple probes or primers configured to hybridize with the target polynucleotide can be used.
  • this application provides a kit that may contain nucleic acids and/or nucleic acid groups as described in this application.
  • the kit of this application may optionally contain a reference sample for a specific purpose or provide a reference level for a specific purpose.
  • the methods of this application can be used for the diagnosis, prognosis, stratification, risk assessment, or treatment monitoring of cancer or tumor formation in subjects.
  • subject can refer to an organism, or a part or component of such an organism, to which the methods, nucleic acids, nucleomes, kits, devices, and systems provided in this application can be administered or applied.
  • the subject can be a mammal or a cell, tissue, organ, or part of such a mammal.
  • mammal means any kind of mammal, preferably a human (including a person, a human subject, or a human patient).
  • Subjects and mammals include, but are not limited to, farm animals, livestock, pets, primates, horses, dogs, cats, and rodents such as mice and rats.
  • the method described in this application can be used to assess cancer or tumor formation in any suitable subject.
  • the method described in this application can be used to assess cancer or tumor formation in mammals.
  • the mammal can be a non-human mammal, such as a pet, farm animal, companion animal, or laboratory animal.
  • the mammal is a human.
  • the subject can be someone who needs to undergo cancer or tumor formation risk screening, someone in a high-risk group, someone diagnosed with cancer or tumor formation but requiring further stratification or grading, someone diagnosed with cancer or tumor formation and receiving active treatment, or someone with cancer or tumor formation and in remission.
  • this application provides the use of reagents and/or kits as described in this application in the preparation of substances that determine the modification state of DNA regions or fragments thereof.
  • this application provides a method for determining the modification state of the DNA region or fragment thereof, which may include providing the reagents and/or kits of this application.
  • this application provides reagents and/or kits as described in this application, which can be used to determine the modification status of the DNA region or fragment thereof.
  • this application provides the use of reagents and/or kits as described in this application in the preparation of disease detection products.
  • this application provides a disease detection method, which may include providing the reagents and/or kits of this application.
  • this application provides a method for confirming the existence of a disease, assessing the formation or risk of disease formation, and/or assessing the progression of a disease, which may include providing the reagents and/or kits of this application.
  • this application provides reagents and/or kits as described in this application, which can be used for disease detection.
  • this application provides reagents and/or kits as described in this application, which can be used to confirm the presence of a disease, assess the formation or risk of disease formation, and/or assess the progression of a disease.
  • this application provides the use of reagents and/or kits as described in this application in the preparation of substances for confirming the presence of a disease, assessing the formation or risk of disease formation, and/or assessing the progression of a disease.
  • the reagent may contain primers and/or probes.
  • the disease may include tumors, such as solid tumors.
  • the disease may include urothelial carcinoma.
  • the disease may include bladder cancer.
  • the kit may contain primers and/or probes for detecting the combination of said gene markers, as well as optionally pharmaceutically acceptable solvents, carriers, and/or instructions for use.
  • the primers and/or probes for detecting each of the said gene markers, along with other necessary reagents, may be placed in the same container or in different containers.
  • the kit is an early cancer diagnostic kit.
  • the kit is a cancer recurrence detection kit.
  • this application provides an apparatus that may include the storage medium of this application.
  • this application provides a non-volatile computer-readable storage medium having a computer program stored thereon, which is executed by a processor to implement any one or more methods described in this application.
  • the method may include a method for classifying samples with different carcinogenic potentials using methylation indicators.
  • the device of this application may also include a processor coupled to the storage medium, the processor being configured to execute the method of this application based on a program stored in the storage medium.
  • Example 1 Genome sequencing for the selection of detection biomarkers and CpG regions
  • the gene biomarkers include TRPS1, RAB37, SEPT9, LHX8, NKX6-2, ZNF571-AS1, NKX1-1, MARCH11, PENK, TBX15, NKX2-8, NRN1, BCL11B, ALX1, KCNQ1DN, LRRC9, BNC1, IRX4, SLC26A4, PAX1, HOXA9, VIM, SIM2, ZIC4, ZNF154, KCNMA1, and FOXF2.
  • Table 1 exemplary methylation site regions for the 27 genes are shown in Table 1.
  • Exemplary sequencing results for HOXA9, VIM, KCNQ1DN, SIM2, ZIC4, ZNF154, KCNMA1, FOXF2, PAX1, BCL11B, LHX8, MARCH11, RAB37, TBX15, and ZNF571-AS1 are shown in Figures 1-15. Within these regions, the methylation levels of newly diagnosed positive samples of urothelial carcinoma were significantly higher than those of newly diagnosed negative samples, and the methylation levels of follow-up positive samples were significantly higher than those of follow-up negative samples. This indicates that the DNA regions in Table 1 can serve as target regions for methylation detection in urothelial carcinoma. As a specific example, the next-generation sequencing results for HOXA9 are shown in Example 7.
  • methylated regions of appropriate size e.g., about 150 bp
  • an appropriate number e.g., more than one
  • the obtained biomarkers all exhibit excellent detection performance compared to other DNA regions of these genes.
  • Example 2 Evaluation of the detection performance of biomarkers using PCR method
  • the methylation levels of the above biomarkers were determined by PCR. Specifically, a representative smaller region within the DNA region range of each gene in Table 1 was selected for PCR to evaluate the detection performance of each region of the gene for urothelial carcinoma.
  • the methylation level of positive samples was detected using quantitative PCR (qMSP) specifically targeting DNA methylation modification.
  • qMSP quantitative PCR
  • the instrument's software analyzed the data, adjusting the threshold to above the baseline fluorescence value to ensure that the negative control Ct value did not show any value.
  • the Ct value of the sample was recorded. A Ct value that was not detected was considered below the detection limit and was converted to 45.
  • the test sample can be considered positive, and the subject from whom the test sample originated can be considered to have urothelial carcinoma, a high risk of developing urothelial carcinoma, or a high risk of urothelial carcinoma recurrence.
  • a method for determining whether each gene marker is positive based on the Ct value can be that if the Ct value of the methylated fragment is less than a certain value, the test result is considered positive, indicating that the target gene is hypermethylated; otherwise, the target gene is hypomethylated. Values for comparison with Ct can be found in Table 2. Those skilled in the art can also choose appropriate judgment values according to actual needs.
  • the primer and probe sequences used are shown in Table 11 and can be used as exemplary primer and probe sequences.
  • Example 3 Detection performance of a single gene for urothelial carcinoma
  • biomarkers related to urothelial carcinoma reported in the literature were also used for detection, and their performance was compared.
  • the results are shown in Table 3.
  • the results show that the gene biomarkers of this application performed well in terms of sensitivity and specificity in single-gene detection, even outperforming the detection performance of biomarkers related to urothelial carcinoma reported in the literature.
  • the sensitivity was between 40% and 70%, and the specificity was above 90%.
  • Some biomarkers had a sensitivity between 50% and 70% and a specificity above 95%. While some genes of biomarkers related to urothelial carcinoma reported in the literature had slightly higher specificity, their sensitivity was lower.
  • Example 4 Detection performance of two genes for urothelial carcinoma
  • the HOXA9 gene was selected as the main gene from the biomarkers in Table 3, and other genes were combined with it to detect the samples in Example 3.
  • the results are shown in Table 4. The results show that the detection performance of the two-gene combination of the biomarkers in this application is improved compared with that of the single gene, especially in terms of sensitivity, the detection sensitivity of the two-gene combination reached more than 70%.
  • the performance is better when there are two gene combinations such as VIM and HOXA9, KCNMA1 and HOXA9, BCL11B and HOXA9, and BNC1 and HOXA9.
  • Example 5 Detection performance of three genes for urothelial carcinoma
  • a biomarker from this application was added to the two-gene combination, forming a three-gene combination, which was then used to detect the samples in Example 3.
  • the results are shown in Table 5.
  • the results show that adding any one of the biomarkers from this application to the two-gene combination can improve sensitivity and specificity.
  • the detection performance of the three-gene combination of this application for newly diagnosed samples and follow-up samples is significantly higher than that of three-gene combinations of genes other than those in this application.
  • the detection performance of three-gene combinations of genes other than those in this application is even lower than that of two-gene combinations of genes from this application.
  • Example 6 NGS evaluation of detection performance of each CpG site within the gene biomarker region
  • the methylation level of the biomarker regions was determined using NGS. Specifically, NGS sequencing was performed on the DNA regions of each gene within the regions listed in Table 1 to evaluate the detection performance of each CpG site within the gene regions for urothelial carcinoma.
  • Table 6 shows the detection performance of each CpG site within the chr7_27203916-27207001 region of the HOXA9 gene in initial and follow-up samples.
  • the sensitivity ranged from 0.7248 to 0.9194, and the specificity ranged from 0.8461 to 0.9230.
  • follow-up samples the sensitivity ranged from 0.7083 to 0.875, and the specificity ranged from 0.8425 to 0.9259.
  • the methylation levels of each CpG site are shown in Figure 16, demonstrating significant differentiation between positive and negative samples.
  • Table 7 shows the detection performance of each CpG site within the intervals chr21_38068194-38073891, chr21_38076763-38077685, chr21_38079942-38081833, and chr21_38119794-38120742 of the SIM2 gene in initial and follow-up samples.
  • the sensitivity ranged from 0.7046 to 0.9261, and the specificity ranged from 0.8076 to 0.9230.
  • follow-up samples the sensitivity ranged from 0.6944 to 0.8472, and the specificity ranged from 0.7962 to 0.8981.
  • the methylation levels of each CpG site are shown in Figure 17, demonstrating significant differentiation between positive and negative samples.
  • Example 7 NGS performance in evaluating the detection of average methylation levels at all CpG sites within a gene biomarker region.
  • Table 8 shows the detection performance of the average methylation level of all CpG sites within the chr7_27203916_27207001 region of the HOXA9 gene in initial and follow-up samples.
  • the specificity was 0.8566 and the sensitivity was 0.9022.
  • the specificity was 0.8513 and the sensitivity was 0.9028.
  • TBX3 which had the highest sensitivity (0.4022) and specificity (0.5955), HOXA9 showed significantly higher sensitivity and specificity.
  • Table 9 shows the detection performance of the average methylation level of all CpG sites within the chr21_38068194_38120742 region of the SIM2 gene in initial and follow-up samples.
  • the specificity was 0.8514 and the sensitivity was 0.9011.
  • the specificity was 0.8546 and the sensitivity was 0.9055.
  • TBX3 which had the highest sensitivity (0.4022) and specificity (0.5955), SIM2 showed significantly higher sensitivity and specificity.
  • Example 8 NGS evaluation of the detection performance of the combination of HOXA9 and SIM2 for urothelial carcinoma
  • the HOXA9 gene chr7_27203916_27207001 region and the SIM2 gene chr21_38068194_38120742 region were combined, and the combined performance for detecting urothelial carcinoma samples was calculated.
  • the sensitivity for initial diagnostic samples is 0.9124 and the specificity is 0.9531, while the sensitivity for follow-up samples is 0.9215 and the specificity is 0.9032.
  • the HOXA9 and TBX3 combination had a sensitivity of 0.6828 and a specificity of 0.6721 for initial diagnostic samples and a sensitivity of 0.6798 and a specificity of 0.6566 for follow-up samples, which were significantly lower than the HOXA9 and SIM2 combination.
  • Table 8 Average methylation detection performance of all CpG sites within the HOXA9 chr7_27203916_27207001 interval.
  • Table 9 Average methylation detection performance of all CpG sites within the SIM2 chr21_38068194_38120742 interval.

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Abstract

La présente demande concerne un kit de marqueur de méthylation pour carcinome urothélial, et concerne également un réactif pouvant détecter l'état de méthylation d'une région d'ADN dans laquelle un gène cible est situé dans un échantillon, ainsi qu'une utilisation du réactif dans la préparation d'un kit de détection d'un carcinome urothélial. Les gènes cibles comprennent HOXA9 et/ou un ou plusieurs gènes choisis parmi : VIM, SIM2, ZIC4, ZNF154, KCNMA1, FOXF2, TRPS1, RAB37, SEPT9, LHX8, NKX6-2, ZNF571-AS1, NKX1-1, MARCH11, PENK, TBX15, NKX2-8, NRN1, BCL11B, ALX1, KCNQ1DN, LRRC9, BNC1, IRX4, SLC26A4 et PAX1. La présente demande concerne également une méthode de détection de carcinome urothélial.
PCT/CN2025/094580 2024-05-13 2025-05-13 Kit de marqueur de méthylation pour carcinome urothélial Pending WO2025237289A1 (fr)

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