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WO2017067477A1 - Méthodes de diagnostic et/ou de pronostic d'un néoplasme gynécologique - Google Patents

Méthodes de diagnostic et/ou de pronostic d'un néoplasme gynécologique Download PDF

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WO2017067477A1
WO2017067477A1 PCT/CN2016/102718 CN2016102718W WO2017067477A1 WO 2017067477 A1 WO2017067477 A1 WO 2017067477A1 CN 2016102718 W CN2016102718 W CN 2016102718W WO 2017067477 A1 WO2017067477 A1 WO 2017067477A1
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seq
target gene
subject
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methylation
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Hung-Cheng Lai
Rui-lan HUANG
Yu-ping LIAO
Po-Hsuan SU
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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
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to cancer diagnosis and/or prognosis. More particularly, the disclosed invention relates to methods for cancer diagnosis and/or prognosis based on the methylation state of selected markers.
  • Gynecologic cancer is the uncontrolled growth of abnormal cells that originate from women’s reproductive organs.
  • Main types of gynecologic cancers include cervical, ovarian, endometrial/uterine (hereinafter, endometrial) , vaginal, and vulvar cancers, while some rare gynecologic cancers include gestational trophoblastic disease (GTD) and primary peritoneal cancer.
  • GTD gestational trophoblastic disease
  • CDC Centers for Disease Control and Prevention
  • Endometrial cancer is the leading cause of gynecologic cancer in the Sates, with approximately 35,000 women being diagnosed each year. Most endometrial cancers are found in women who are going through or have gone through menopause. However, for those who do not have any signs or symptoms, there are no simple and reliable ways to test for uterine cancer. For patients showing symptoms or at a higher risk for endometrial cancer, an endometrial biopsy or a transvaginal ultrasound may be performed to help diagnose or rule out uterine cancer.
  • Ovarian cancer is second to endometrial cancer as the most common cause of gynecologic cancer, whereas the mortality rate of ovarian cancer is the highest among gynecologic cancers.
  • Ovarian cancer symptoms are often vague during the early stage, and hence ovarian cancer is rarely diagnosed in its early stages. By the time the diagnosis is made, it is usually quite advanced and the tumor has often spread beyond the ovaries. To be specific, about 90 percent of ovarian cancer cases develop from epithelial cells, and up to 70 percent of epithelial ovarian cancer cases are diagnosed at stage III or IV.
  • ovarian cancer may be curable; for women diagnosed with stage I ovarian cancer, the 10-year survival rate is close to 90%. In contrast, the five-year survival rate for women in stage III or IV is only about 15%-20%. Accordingly, a suitable method for screening women for early stage ovarian cancer would be of great benefit.
  • Current tests for screening ovarian cancer include, BRCA testing, transvaginal ultrasonography and serum cancer antigen (CA) –125 testing.
  • BRCA testing is primarily used for prognosis.
  • transvaginal ultrasonography and CA-125 testing have a high false-positive rate (about 10%) , thereby leading to unneeded oophorectomy that may cause major complications.
  • the U.S. Preventive Service Task Force recommended against screening for ovarian cancer in asymptomatic, average-risk women using serum CA-125 testing and transvaginal ultrasonography for the harms caused by these screening procedures outweigh the benefits.
  • the present invention is directed to a method for assessing whether a subject has a gynecologic neoplasm, whether benign or malignant.
  • the method comprises the following steps:
  • determining the methylation state of at least one target gene in the sample wherein the at least one target gene is selected from the group consisting of, ADAMTS16 (SEQ ID NO. 1) , APBB1 IP (SEQ ID NO. 4) , CBLN4 (SEQ ID NO. 7) , CCDC140 (SEQ ID NO. 10) , CELF4 (SEQ ID NO. 11) , CNTNAP5 (SEQ ID NO. 14) , EFS (SEQ ID NO. 17) , EMILIN1 (SEQ ID NO. 18) , FERD3L (SEQ ID NO. 23) , FEZF2 (SEQ ID NO. 24) , GALNTL6 (SEQ ID NO.
  • GRIA4 SEQ ID NO. 30
  • NEFL SEQ ID NO. 44
  • PARP15 SEQ ID NO. 49
  • PAX6 SEQ ID NO. 50
  • PRLHR SEQ ID NO. 56
  • SSTR1 SEQ ID NO. 64
  • ST8SIA5 SEQ ID NO. 65
  • VWC2 SEQ ID NO. 73
  • ZNF300 SEQ ID NO. 77
  • ZNF334 SEQ ID NO. 78
  • ZNF662 SEQ ID NO. 79
  • CCDC105 SEQ ID NO. 9)
  • CYP26C1 SEQ ID NO.
  • OSBPL2 SEQ ID NO. 48
  • PCDH17 SEQ ID NO. 51
  • RYK SEQ ID NO. 58
  • SDPR SEQ ID NO. 59
  • SKIL SEQ ID NO. 60
  • TEAD1 SEQ ID NO. 69
  • ZNF669 SEQ ID NO. 80
  • step (d) assessing whether the subject has the gynecologic neoplasm based on the result of the step (c) , wherein the hypermethylation of the at least one target gene indicates that the subject has the gynecologic neoplasm.
  • the at least one target gene is selected from the group consisting of, ADAMTS16, APBB1 IP, CBLN4, CCDC140, CELF4, CNTNAP5, EMILIN1, FERD3L, FEZF2, GALNTL6, GRIA4, NEFL, PAX6, PRLHR, SSTR1, ST8SIA5, VWC2, ZNF300, ZNF334, ZNF662, CCDC105, CYP26C1, HORMAD2, IFFO1, LOC100130872, NKAPL, PCDHGA11, SLC25A2, TCTEX1 D1, TMEM101, TRIM61 and WDR69.
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, NRN1, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB, COLQ, FAM49B, NR5A2, OSBPL2, PCDH17, RYK, SDPR, SKIL, TEAD
  • the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage) , tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma) , serum, lymph, spinal fluid) .
  • the sample is derived from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.
  • the present disclosure is directed to a method for assessing whether a subject has a malignant endometrial cancer.
  • the method comprises the following steps:
  • determining the methylation state of at least one target gene in the sample wherein the at least one target gene is selected from the group consisting of, ADAMTS16, APBB1 IP, CBLN4, CCDC140, CELF4, CNTNAP5, EFS, EMILIN1, FERD3L, FEZF2, GALNTL6, GRIA4, NEFL, PARP15, PAX6, PRLHR, SSTR1, ST8SIA5, VWC2, ZNF300, ZNF334, ZNF662, CCDC105, CYP26C1, HORMAD2, IFFO1, LOC100130872, NKAPL, PCDHGA11, SLC25A2, TCTEX1 D1, TMEM101, TRIM61 and WDR69;
  • step (d) assessing whether the subject has the malignant endometrial cancer based on the result of the step (c) , wherein the hypermethylation of the at least one target gene indicates the subject has the malignant endometrial cancer.
  • the at least one target gene is selected from the group consisting of, APBB1 IP, CELF4, EFS, FEZF2, GRIA4, NEFL, PAX6, FERD3L, SSTR1, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, CELF4, FERD3L, FEZF2, EFS, PARP15, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, EFS, FEZF2 and ZNF300.
  • the methylation states of two or all of EFS, FEZF2 and ZNF300 are determined.
  • the at least one target gene is selected from the group consisting of, CELF4, EFS, ZNF334 and ZNF662.
  • the methylation states of two, three or all of CELF4, EFS, ZNF334 and ZNF662 are determined.
  • the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage) , tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma) , serum, lymph, spinal fluid) .
  • the sample is derived from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.
  • the present disclosure is directed to a method for assessing whether a subject has an ovarian neoplasm.
  • the method comprises the following steps:
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, COL6A3, CPEB1, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, NRN1, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX15, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB, COLQ, FAM49B,
  • step (d) assessing whether the subject has the ovarian neoplasm based on the result of the step (c) , wherein the hypermethylation of the at least one target gene indicates that subject has the ovarian neoplasm.
  • the hypermethylation of the at least one target gene indicates that the ovarian neoplasm is malignant
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, COL6A3, CPEB1, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB,
  • the at least one target gene is selected from the group consisting of, AOX1, FAM115A, NRN1, PCDHGA11, PHOX2A, PYGO1, TBX15, UCKL1 and WDR86.
  • the at least one target gene is NRN1, TBX15, or both.
  • the at least one target gene is selected from the group consisting of, FEZF2, PAX6 and ZNF662.
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, COL6A3, CPEB1, FAM115A, NRN1, TBX15, PCDHGA11, PHOX2A, UCKL1, and WDR86.
  • the at least one target gene is selected from the group consisting of, NRN1, AOX1, TBX15, and COL6A3.
  • the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage) , tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma) , serum, lymph, spinal fluid) .
  • the sample is derived from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.
  • the present invention is directed to a method for assessing whether a subject has an ovarian neoplasm and the malignancy thereof.
  • the method comprises the following steps:
  • step (d) assessing whether the subject has the ovarian neoplasm and the malignancy thereof based on the result of the step (c) , wherein the hypermethylation of the at least one target gene indicates that the subject has the ovarian neoplasm; and the hypermethylation of AOX1, WDR86 or both indicates that the ovarian neoplasm is malignant.
  • the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage) , tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma) , serum, lymph, spinal fluid) .
  • the sample is derived from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.
  • the step of determining the methylation state of a gene can be achieved by performing methylation-specific polymerase chain reaction (MSP) , quantitative methylation-specific polymerase chain reaction (qMSP) , bisulfite sequencing (BS) , bisulfite pyrosequencing, microarrays, mass spectrometry, denaturing high-performance liquid chromatography (DHPLC) , pyrosequencing, methylated DNA immunoprecipitation (MeDIP or mDIP) coupled with quantitative polymerase chain reaction, methylated DNA immunoprecipitation sequencing (MeDIP-seq) , or nanopore sequencing.
  • MSP methylation-specific polymerase chain reaction
  • qMSP quantitative methylation-specific polymerase chain reaction
  • BS bisulfite sequencing
  • pyrosequencing bisulfite pyrosequencing
  • microarrays microarrays
  • mass spectrometry denaturing high-performance liquid chromatography
  • Figure 1 summarizes the methylation levels of twelve candidates genes in the endometrial tissue specimens according to one embodiment of the present disclosure
  • Figure 2 summarizes the methylation levels of twelve candidates genes in the cervical scraping specimens according to one embodiment of the present disclosure
  • Figure 3 summarizes the DNA methylation using individual cervical scrapings for endometrial cancer detection according to one embodiment of the present disclosure
  • Figure 4 provides area under the curve (AUC) of three gene combinations for detection endometrial cancer detection using cervical scrapings according to one embodiment of the present disclosure
  • Figure 5 summarizes the methylation levels of nine candidates genes in the ovarian tissue specimens according to one embodiment of the present disclosure
  • Figure 6 summarizes the DNA methylation level in cervical scrapings from patient diagnosed with ovarian malignant tumor and normal ovary according to one embodiment of the present disclosure
  • Figure 7 summarizes the high methylation of NRN1 and TBX15 show in myoma, benign and malignant ovarian tumor according to one embodiment of the present disclosure
  • Figure 8 summarizes the results of cross-testing of candidate genes in other gynecological cancers according to one embodiment of the present disclosure.
  • Figures 9A to 11 D are drawings summarizing the specificity and sensitivity of some target genes for use in the diagnosis of ovarian neoplasm.
  • phrases “at least one of A, B, and C” , “at least one of A, B, or C” and “at least one of A, B and/or C, ” as use throughout this specification and the appended claims, are intended to cover A alone, B alone, C alone, A and B together, B and C together, A and C together, as well as A, B, and C together.
  • the term “assessing” refers to a process in which the health status of a subject is determined.
  • the health status of the subject may indicate a diagnosis, prognosis, or increased risk of a gynecologic neoplasm in said subject.
  • diagnosis refers to the identification of a pathological state, disease, or condition, such as neoplasms of various gynecologic tissue origins, including, cervix, ovary, endometrium/uterus, vagina, vulvar, uterus, and peritoneum lining the uterus. In some cases, the term diagnosis also refers to distinguishing between the malignant and benign neoplasms. In some other cases, the term diagnosis refers to distinguishing between the malignant neoplasm and normal tissues.
  • prognosis refers to the prediction of the likelihood of cancer-attributable death or progression, including any of, the recurrence rate and metastatic spread of a neoplastic disease, and the recurrence-free survival time, progression-free time, or the overall survival time of a subject diagnosed with a neoplastic disease.
  • prognosis concerns an estimation of the recurrence-free survival (RFS) , progression-free survival (PFS) , or overall survival (OS) .
  • the term "poor prognosis” as used herein means that a subject’s chance of having a given remaining expectancy of life is substantially decreased, as compared with another subject who has a normal methylation profile of one or more markers identified herein.
  • a subject with a poor RFS suggests that the subject’s RFS time may be less than 12 or 24 months.
  • a poor overall or progression-free survival indicates that the subject’s OS or PFS time may be less than 12 or 24 months.
  • a poor RFS or PFS may suggest that the subject has a higher probability of experiencing tumor recurrence or progression during a given time period, as compared with a reference subject group.
  • neoplasm refers to a new and abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal.
  • a neoplasm creates an unstructured mass (atumor) , which can be either benign or malignant.
  • benign refers to a neoplasm or tumor that is noncancerous, e.g. its cells do not invade surrounding tissues or metastasize to distant sites; whereas the term “malignant” refers to a neoplasm or tumor that is metastatic, invades contiguous tissue or no longer under normal cellular growth control.
  • cancer refers to all types of cancer, or malignant neoplasm or tumor found in animals.
  • the methods according to various embodiments of the present disclosure are directed to the diagnosis one or more carcinoma of gynecologic origin.
  • carcinoma refers to a malignant tumor originating from epithelial cells.
  • Exemplary gynecologic carcinomas of embodiments of the present disclosure include, but are not limited to, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, and primary peritoneal cancer.
  • methylation refers to the covalent attachment of a methyl group at the C5-position of cytosine within the CpG dinucleotides of the core promoter region of a gene.
  • methylation state refers to the presence or absence of 5-methyl-cytosine (5-mCyt) at one or a plurality of CpG dinucleotides within a gene or nucleic acid sequence of interest.
  • methylation level refers to the amount of methylation in one or more copies of a gene or nucleic acid sequence of interest. The methylation level may be calculated as an absolute measure of methylation within the gene or nucleic acid sequence of interest.
  • a “relative methylation level” may be determined as the amount of methylated DNA, relative to the total amount DNA present or as the number of methylated copies of a gene or nucleic acid sequence of interest, relative to the total number of copies of the gene or nucleic acid sequence. Additionally, the “methylation level” can be determined as the percentage of methylated CpG sites within the DNA stretch of interest.
  • methylation profile refers to a set of data representing the methylation level of one or more target genes in a sample of interest.
  • the methylation profile is compared to a reference methylation profile derived from a known type of sample (e.g., cancerous or noncancerous samples or samples from different stages of cancer) .
  • the term "differential methylation” refers to a difference in the methylation level of one or more target genes in one sample or group, as compared with the methylation level of said one or more target genes in another sample or group.
  • the differential methylation can be classified as an increased methylation ( “hypermethylation” ) or a decreased methylation ( “hypomethylation” ) .
  • the term “hypermethylation” of a target gene in a test sample refers to an increased methylation level of at least 10%, relative to the average methylation level of the target gene in a reference sample.
  • the increased methylation level may be at least 15, 20, 25, 30, 35, 40, 45, or 50%.
  • genes or polynucleotide sequences described herein respectively comprise their variants that have at least 75%nucleotide sequence identity to the named genes or polynucleotide sequences.
  • target gene sequences also encompass the bisulfite conversion nucleotide sequence thereof. Accordingly, unless otherwise expressly specified, all of the genes or polynucleotide sequences described herein should be understood as modified in all instances by the phrase “and a bisulfite conversion nucleotide sequence thereof, and a polynucleotide sequence having at least 75%nucleotide sequence identity to said gene or said bisulfite conversion nucleotide sequence. ”
  • Percentage (%) nucleotide sequence identity with respect to a gene or nucleotide sequence identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the referenced polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • the percentage nucleotide sequence identity of a given polynucleotide sequence A to a referenced polynucleotide sequence B (which can alternatively be phrased as a given polynucleotide sequence A that has a certain %nucleotide sequence identity to a referenced polynucleotide sequence B) is calculated by the formula as follows:
  • X is the number of nucleotide residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of nucleotide residues in A or B, whichever is shorter.
  • subject and patient are used interchangeably herein and are intended to mean an animal including the human species that can be subjected to the diagnosis methods of the present invention. Accordingly, the term “subject” or “patient” comprises any mammal, which may benefit from the method of the present disclosure.
  • mammal refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat.
  • non-human mammal refers to all members of the class Mammalis except human. In one exemplary embodiment, the patient is a human.
  • sample used herein comprises any samples obtained from a patient.
  • the sample contains DNA molecules and the methylation level thereof can be determined.
  • body samples include, but are not limited to, blood, smears, sputum, urine, stool, liquor, bile, gastrointestinal secretions, lymph fluid, osteosarcoma marrow, organ aspirates and organ or tissue biopsies.
  • body samples can be obtained from the patient by routine measures known to persons having ordinary skill in the art. Further, persons having ordinary skill in the art are also familiar with methods and reagents for the DNA isolation from the sample, e.g. extraction with phenol/chloroform or by means of commercial kits.
  • the present disclosure is based, at least in part, on the finding that differential methylation (in particular, hypermethylation) of one or more target genes, as identified hereinbelow, relates to the tumor progression, or the absence thereof, in a subject. Accordingly, these target genes, alone or in combination, can be used as biomarkers for the prediction of risk or susceptibility of a subject developing a neoplasm, the determination of the malignancy of the neoplasm, and/or making prognosis of the patient being diagnosed with said neoplasm. Further, the methylation profile of relevant genes of the patient can be used as a guide for tailoring suitable therapy regime individually. For example, for patients with one or more hypermethylated target genes listed herein, de-methylation agents or other epigenetic drugs can be administered to the patients to treat the neoplasm.
  • de-methylation agents or other epigenetic drugs can be administered to the patients to treat the neoplasm.
  • the present disclosure provides various diagnostic and/or prognostic methods, which will be separately addressed below.
  • all methods involve the determination of the methylation state or methylation level of at least one target gene.
  • steps common to most, if not all, claimed methods are first described in the following paragraph.
  • a sampling step (a) in which a biological sample is obtained from the subject; at least one methylation determination step (b) , in which the methylation state of at least one target gene in the sample is determined; and at least one determining step (c) , in which the presence or absence of hypermethylation of said at least one target gene is determined.
  • a methylation determination step (b) in which the methylation state of at least one target gene in the sample is determined
  • determining step (c) in which the presence or absence of hypermethylation of said at least one target gene is determined.
  • the sample is a sample obtained from a subject, preferably a human subject, or present within a subject, preferably a human subject, including a tissue, tissue sample, or cell sample (e.g., a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy an endoscopic biopsy, cervical scraping cells, uterus scraping cells or a vaginal lavage) , tumor, tumor sample, or biological fluid (e.g., peritoneal fluid, blood (including plasma) , serum, lymph, spinal fluid) .
  • the sample is derived from the ovarian tissue, cell samples (e.g., cervical scraping cells) and body fluid (e.g., serum and plasma) of the subject.
  • the methylation state is determined using qMSP or bisulfite pyrosequencing, according to working examples provided below.
  • the present method is not limited to the methods described above; rather, the scope of the claimed invention encompasses the use of other equivalent methods for quantitatively determining the methylation state or level of a particular gene.
  • the above-mentioned methods and equivalents thereof are also applicable to the embodiments described hereinbelow, hence, the method suitable for determining the methylation state or methylation level of the gene is not repeated in the following aspects/embodiments, for the sake of brevity.
  • the present disclosure provides a method for assessing whether a subject has a gynecologic neoplasm, which comprises the common steps (a) , (b) and (c) as described above, and an assessement step (d) , in which the presence or absence of the gynecologic neoplasm in the subject is determined based on the result of the step (c) .
  • the hypermethylation of the at least one target gene indicates that the subject has the gynecologic neoplasm.
  • the absence of the hypermethylation of the at least one target gene indicates that the subject does not have the gynecologic neoplasm or the gynecologic neoplasm is benign.
  • the at least one target gene is selected from the group consisting of, ADAMTS16, APBB1 IP, CBLN4, CCDC140, CELF4, CNTNAP5, EFS, EMILIN1, FERD3L, FEZF2, GALNTL6, GRIA4, NEFL, PARP15, PAX6, PRLHR, SSTR1, ST8SIA5, VWC2, ZNF300, ZNF334, ZNF662, CCDC105, CYP26C1, HORMAD2, IFFO1, LOC100130872, NKAPL, PCDHGA11, SLC25A2, TCTEX1 D1, TMEM101, TRIM61, WDR69, AOX1, CACNA2D4, COL6A3, CPEB1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210,
  • the at least one target gene is selected from the group consisting of, ADAMTS16, APBB1 IP, CBLN4, CCDC140, CELF4, CNTNAP5, EMILIN1, FERD3L, FEZF2, GALNTL6, GRIA4, NEFL, PAX6, PRLHR, SSTR1, ST8SIA5, VWC2, ZNF300, ZNF334, ZNF662, CCDC105, CYP26C1, HORMAD2, IFFO1, LOC100130872, NKAPL, PCDHGA11, SLC25A2, TCTEX1 D1, TMEM101, TRIM61 and WDR69.
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, NRN1, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB, COLQ, FAM49B, NR5A2, OSBPL2, PCDH17, RYK, SDPR, SKIL, TEAD1 and ZNF669.
  • the present disclosure provides a method for assessing whether the subject has a malignant endometrial cancer.
  • the method also comprises the common steps (a) , (b) and (c) as described above, and an assessement step (d) in which the presence of the malignant endometrial cancer in the subject is determined based on the result of the step (c) .
  • the hypermethylation of the at least one target gene indicates that the subject has the malignant endometrial cancer.
  • the absence of the hypermethylation of the at least one target gene indicates that the subject does not have an endometrial neoplasm or the endometrial neoplasm is benign.
  • the at least one target gene is selected from the group consisting of, ADAMTS16, APBB1 IP, CBLN4, CCDC140, CELF4, CNTNAP5, EFS, EMILIN1, FERD3L, FEZF2, GALNTL6, GRIA4, NEFL, PARP15, PAX6, PRLHR, SSTR1, ST8SIA5, VWC2, ZNF300, ZNF334, ZNF662, CCDC105, CYP26C1, HORMAD2, IFFO1, LOC100130872, NKAPL, PCDHGA11, SLC25A2, TCTEX1 D1, TMEM101, TRIM61 and WDR69.
  • the at least one target gene is selected from the group consisting of, APBB1 IP, CELF4, EFS, FEZF2, GRIA4, NEFL, PAX6, FERD3L, SSTR1, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, CELF4, FERD3L, FEZF2, EFS, PARP15, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, ZNF300, ZNF334 and ZNF662.
  • the at least one target gene is selected from the group consisting of, EFS, FEZF2 and ZNF300.
  • the methylation states of two or all of EFS, FEZF2 and ZNF300 are determined.
  • the at least one target gene is selected from the group consisting of, CELF4, EFS, ZNF334 and ZNF662.
  • the methylation states of two, three or all of CELF4, EFS, ZNF334 and ZNF662 are determined.
  • the present disclosure directs to a method for assessing whether a subject has an ovarian neoplasm.
  • the presence or absence of the ovarian neoplasm is determined based on the result of the step (c) .
  • the hypermethylation of the at least one target gene indicates that the subject has the ovarian neoplasm.
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, COL6A3, CPEB1, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, NRN1, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX15, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB, COLQ, FAM49B, NR5A2, OSBPL2, PCDH17, RYK
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, CBLN4, CCDC140, COL6A3, CPEB1, CYP26C1, EMILIN1, FAM101A, FAM115A, FBXL22, FIGLA, FLJ32255, FOXB2, GIGYF1, GSX1, KIAA1731 NL, LBX2, LINC00925, LINC01210, LINC01475, LOC283692, LOC440982, MIR124-2HG, MKX, PCDHGA11, PFN3, PHOX2A, PIP4K2A, PRLHR, PYGO1, SKOR1, SKOR2, TBX5-AS1, UCKL1, WDR86, ZDHHC19, APBA1, CACNA1A, CBS, CIDEB, CKB, COLQ, FAM49B, NR5A2, OSBPL2, PCDH17, RYK, SDPR, SKIL, T
  • the at least one target gene is selected from the group consisting of, AOX1, FAM115A, NRN1, PCDHGA11, PHOX2A, PYGO1, TBX15, UCKL1 and WDR86.
  • the at least one target gene is NRN1, TBX15, or both.
  • the at least one target gene is selected from the group consisting of, FEZF2, PAX6 and ZNF662.
  • the at least one target gene is selected from the group consisting of, AOX1, CACNA2D4, COL6A3, CPEB1, FAM115A, NRN1, TBX15, PCDHGA11, PHOX2A, UCKL1, and WDR86.
  • the at least one target gene is selected from the group consisting of, NRN1, AOX1, TBX15, and COL6A3.
  • the present invention is directed to a method for assessing whether a subject has an ovarian neoplasm and whether the ovarian neoplasm is malignant, in which the assessment is based on the hypermethylation of the at least one target gene as determined in the step (c) .
  • the at least one target gene is selected from the group consisting of, NRN1, AOX1, TBX15, COL6A3 and WDR86.
  • the method further comprises an assessment step (d) , in which the hypermethylation of the at least one target gene indicates that the subject has the ovarian neoplasm; and the hypermethylation of AOX1, WDR86 or both indicates that the ovarian neoplasm is malignant.
  • the ovarian or tumor specimens were obtained during surgery and were frozen immediately in liquid nitrogen and stored at -80°C until further use.
  • the pathological diagnosis of each sample was confirmed with histological examination by gynecologic pathologists.
  • Normal epithelial ovarian tissue was collected by scraping the ovarian epithelium from patients diagnosed with uterine leiomyoma.
  • cervical scraping cells were collected from patients diagnosed with benign ovarian tumor or malignant tumors, and female patients were free of ovarian and cervical diseases at the sampling time.
  • cervical scraping cells were collected from patients diagnosed with myoma or malignant endometrial tumors, and female patients were free of ovarian and cervical diseases at the sampling time.
  • the endocervical scraping cells were collected as follows. Inserting a new endocervical brush into the endocervical canal and gently rotating the brush three to five times to ensure appropriate sampling.
  • the brush was then plunged into a centrifuge tube (15 ml) containing 2 ml (Ambion, Life technologies, USA) , and closed the cap of the centrifuge tube.
  • the endocervical scraping cells specimens were stored at 4°C. After vortexing for 10 seconds, the swab cells-containing sample was aliquoted into three microcentrifuge tubes (1 ml per tube) , which were stored at -80°C until further analysis.
  • TCGA Cancer Genome Atlas
  • Em-MethylCap-seq including 80 malignant endometrial tumor tissues and 10 normal endometrial tissues
  • methylation biomarker of benign tumors including endometrial hyperplasia, benign ovarian tumors and myoma
  • 10 myoma tumor tissues, 10 benign ovarian tumors, 6 cervical scrapings collected from subjects with myoma, 11 cervical scrapings collected from subjects with benign ovarian tumor, and 5 cervical scrapings collected from subjects with endometrial hyperplasia were used.
  • Methylated DNA was enriched from 2 ⁇ g genomic DNA using the MethylMiner methylated DNA Enrichment Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Briefly, genomic DNA was sonicated to about 200-bp, captured by MBD proteins and precipitated using 1 M salt buffer. Enriched methylated DNA was used to generate libraries for sequencing following the standard protocols from Illumina (San Diego, CA) . MethylCap-seq libraries were sequenced using the Illumina Genome Analyzer IIx System. Image analysis and base calling were performed using the standard Illumina pipeline. Unique reads (up to 36-bp reads) were mapped to the human reference genome (hg18) using the ELAND algorithm, with up to two base-pair mismatches.
  • the uniquely mapped reads were used for additional linear normalization and differential methylation analysis.
  • the methylation level was calculated by accumulating the number of reads using the following Equations 1 and 2.
  • N_ (Read, i) is the number of normalized reads at the ith bin; U_ (Read, i) is the number of uniquely mapped reads at the ith bin, and N_U is the number of total uniquely mapped reads. “INT” rounds the element to the nearest integers towards minus infinity, “ ⁇ ” means the power operator.
  • AVG R, G means the average methylation level of group G at the R region.
  • M R, G is the methylation levels of each sample of group G at the R region.
  • the statistical significance of different methylation at the R region was identified using two-tailed and Mann–Whitney U test (P ⁇ 0.001) . Differentially methylated loci were analyzed between case and control samples.
  • TCGA data portal used 450K methylation Chip array to analyze the tissue samples, and the data portal contained datasets from 417 endometrial tissues. Among them, 371 cancer cases with ⁇ 40%neoplastic cellularity were chosen for this study. Non-parametric test was used to investigate the differential methylated CpG sites between case and control groups. Probes with P ⁇ 0.001 and the top 5%differential methylation of high ⁇ -values were initially selected. Then, significant probes located within the region spanning -1000 to +1000 relative to the transcription start site of coding genes were identified. Hypermethylated genes with at least five of the identified probes were selected per our selection criteria.
  • Genomic DNA was extracted from specimens using QIAmp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) .
  • the genomic DNA (1 ⁇ g) was bisulfite modified using the EZ DNA Methylation Kit (ZYMO RESEARCH, CA, USA) , according to the manufacturer's recommendations, and dissolved in 70 ⁇ L of nuclease-free water.
  • the bisulfite DNA was used as the temple for the DNA methylation analysis.
  • Quantitative methylation-specific PCR using specific probes and primers was performed to identify the relative DNA methylation level by making reference to the un-methylated (COL2A1) total input DNA.
  • Bisulfite pyrosequencing was used to quantitate the DNA methylation level, in which the methylation level is expressed as the percentage of methylation (e.g., 0%to 100%) for each CpG site of the pyrosequencing amplicons.
  • Non-parametric test and percentile rank of differential median between two groups were carried out. Mann-Whitney U test was used to test whether the means of two unpaired groups are equal or not. Wilcoxon signed-rank test was used to compare paired samples of two groups.
  • the true positive rate i.e., the sensitivity
  • the false positive rate that is, 1–specificity
  • ROC receiver operating characteristic
  • AUC area under the curve
  • DMRs Differential methylation regions
  • TSS transcription start site
  • the results ( Figure 1) indicate that twelve of the candidate genes exhibit significant hypermethylation in malignant endometrial cancer tissues. Said twelve genes are, APBB1 IP, CELF4, EFS, FEZF2, GRIA4, NEFL, PAX6, FERD3L, SSTR1, ZNF300, ZNF334 and ZNF662.
  • the results ( Figure 2) indicate that the above-mentioned twelve genes are also capable of distinguishing subjects with malignant tumor and normal endometrial tissue using cervical scrapings samples.
  • the threshold is the optimal cut-off calculated from the close. left method of pROC package.
  • the methylation level is higher than threshold is positive. vice versa is negative to compute the sensitivity, specificity and accuracy using data of TCGA 450k methylation array
  • CELF4 CELF4, EFS, ZNF334, and ZNF662 were used in the subsequent independent cohort to determine the best accuracy for the diagnostic and/or prognostic stratification power.
  • Sen sensitivity
  • Spe specificity
  • ACC accuracy
  • AUC area under the receiver operating characteristic (ROC) curve
  • CI confidence interval
  • the pROC package was used to identify the best threshold of each gene.
  • the methylation level higher than the threshold was determined to be positive, whereas the methylation level lower than the threshold was determined to be negative.
  • the results, as summarized in Figure 4 indicate that three types of gene combinations resulted in an area under the curve (AUC) value of at least 90%.
  • the three-gene signatures are better than any single gene.
  • novel hypermethylated genes in endometrial cancer were identified using data from Ov-MethyCap sequencing to identify DMRs that locate at ⁇ 1 kb spanning from the TSS of each gene.
  • 124 consistently high-methylation genes present in any two subtypes of the malignant ovarian tumor were identified.
  • 52 genes were selected.
  • a total of 130 genes were identified; among them, 51 genes in which the hypermethylation thereof had never been reported to be associated with malignant ovarian cancer were identified (see, Table 3) .
  • the accuracy of these 51 genes in distinguishing malignant and non-malignant ovarian tissues is about 71%to 95.12%.
  • the threshold is the optimal cut-off calculated from the close. left method of pROC package.
  • the methylation level is higher than threshold is positive, vice versa is negative to compute the sensitivity, specificity and accuracy using data of Ov
  • the results ( Figure 5) indicate that nine of the candidate genes distinguish between the malignant ovarian tumor and normal ovarian tissue. Said nine genes are, AXO1, FAM115A, NRN1, PCDHGA11, PHOX2A, PYGO1, TBX15, UCKL1 and WDR86.
  • the results ( Figure 6) indicate that the above-mentioned nine genes are also capable of distinguishing subjects with malignant ovarian tumor and normal ovarian tissue using cervical scrapings samples.
  • FEZF2, PAX6 and ZNF662 were further investigated for their ability in distinguishing abnormal gynecologic diseases, and the results are summarized in Figure 8 (each symbol represents the data from the pooled DNA of 5 people) .
  • FEZF2 and PAX6 respectively exhibited a higher methylation level in the malignant ovarian tumor, as compared with the methylation level in normal ovarian tissue.
  • FEZF2 and PAX6 also exhibited a higher methylation in patients with malignant ovarian tumor, cervical intraepithelial neoplasia 3 (CIN3) or squamous cell carcinoma of the cervix (SCC) , as compared with the methylation level of samples from normal subject or those with cervical intraepithelial neoplasia 1 (CIN1) .
  • CIN3 cervical intraepithelial neoplasia 3
  • SCC squamous cell carcinoma of the cervix
  • the close. left method of pROC package was used to calculate the optimal cut-off value.
  • the methylation level of a target gene is higher than the threshold, said target gene is hypermethylated, whereas when the methylation level of a gene is less than the threshold, said gene is not hypermethylated.
  • the sensitivity , specify and accuracy of some target genes were also determined using the data from Ov MethyCap-seq, and the results are summarized in Table 4 below.
  • the threshold values i.e., the optimal cut-off values of target genes or target gene combinations were determined using the M-index data from qMSP; the results summarized in Table 6 and Figures 10A to 10D.
  • the threshold values i.e., the optimal cut-off values of target genes or target gene combinations were determined using the M-index data from qMSP; the results summarized in Table 7 and Figures 11A to 11 D.
  • the sample is derived from cervical testing, and the diagnosis procedure was as follows. First, the samples were stratified as positive (i.e., with ovarian neoplasm) when of at least one of NRN1, AOX, TBX15, and COL6A3 was hypermethylated, and negative when none of NRN1, AOX, TBX15, and COL6A3 was hypermethylated (sensitivity 88.5%; specificity 95%) .
  • the positive samples were further stratified as malignant ovarian neoplasm when WDR86 or AOX1, or both was/were hypermethylated, whereas positive samples without hypermethylation of WDR86 and/or AOX1 were stratified as benign ovarian neoplasm (sensitivity 90%; specificity 81.8%) .

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Abstract

L'invention concerne des méthodes pour évaluer la présence de néoplasmes gynécologiques ou la malignité du néoplasme gynécologique, sur la base de l'hyperméthylation d'un ou de plusieurs gènes marqueurs.
PCT/CN2016/102718 2015-10-20 2016-10-20 Méthodes de diagnostic et/ou de pronostic d'un néoplasme gynécologique Ceased WO2017067477A1 (fr)

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CN110628910A (zh) * 2019-10-17 2019-12-31 湖南大地同年生物科技有限公司 一种膀胱癌驱动基因点突变甲基化联合辅助诊断方法、试剂盒、系统及应用
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CN114990213A (zh) * 2022-07-11 2022-09-02 重庆大学附属肿瘤医院 一种基于znf334基因的检测试剂盒及应用
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CN107326078A (zh) * 2017-07-19 2017-11-07 宁波大学 一种胱硫醚β‑合酶基因启动子区甲基化程度的检测试剂盒及测定方法
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CN109706242A (zh) * 2019-01-31 2019-05-03 佛山市顺德区辉锦创兴生物医学科技有限公司 心肌细胞受损检测试剂盒及其应用
CN110628910A (zh) * 2019-10-17 2019-12-31 湖南大地同年生物科技有限公司 一种膀胱癌驱动基因点突变甲基化联合辅助诊断方法、试剂盒、系统及应用
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CN110628910B (zh) * 2019-10-17 2023-06-20 湖南大地同年生物科技有限公司 一种膀胱癌驱动基因点突变甲基化联合辅助诊断方法、试剂盒、系统及应用
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US12018259B2 (en) 2020-12-23 2024-06-25 Regeneron Pharmaceuticals, Inc. Treatment of liver diseases with cell death inducing DFFA like effector B (CIDEB) inhibitors
CN113215264A (zh) * 2021-07-07 2021-08-06 上海伯豪生物技术有限公司 用于子宫内膜癌早期筛查人外周血循环肿瘤dna中tmem101基因甲基化检测试剂盒
CN113215264B (zh) * 2021-07-07 2021-10-01 上海伯豪生物技术有限公司 用于子宫内膜癌早期筛查人外周血循环肿瘤dna中tmem101基因甲基化检测试剂盒
CN114990213A (zh) * 2022-07-11 2022-09-02 重庆大学附属肿瘤医院 一种基于znf334基因的检测试剂盒及应用

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