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KR-20260066094-A - Characterizing methylated DNA, RNA, and proteins in the detection of lung neoplasia

KR20260066094AKR 20260066094 AKR20260066094 AKR 20260066094AKR-20260066094-A

Abstract

An invention relating to tumor detection, particularly, non-exclusively, to a method, composition, and related use for detecting neoplasms such as lung cancer is provided.

Inventors

  • 알라위, 하팀
  • 리드가드, 그레이엄 피.
  • 지아코우모파울로스, 마리아
  • 알퀴스트, 데이비드 에이.
  • 테일러, 윌리엄 알.
  • 마호니, 더글라스

Assignees

  • 이그젝트 싸이언스 디블롭먼트 컴패니, 엘엘씨
  • 메이오 파운데이션 포 메디칼 에쥬케이션 앤드 리써치

Dates

Publication Date
20260512
Application Date
20191126
Priority Date
20181127

Claims (20)

  1. A sample characterization method including the following steps: a) a step of measuring the amount of at least one methylation marker gene in the DNA of a sample, wherein the at least one methylation marker gene includes at least one of IFFO1 and HOPX ; b) a step of measuring the amount of at least one reference marker in DNA; and c) a step of calculating the value of the amount of at least one methylation marker measured in DNA as a percentage of the amount of a reference marker measured in DNA, wherein the value represents the amount of at least one methylation marker gene measured in the sample.
  2. A method according to claim 1, wherein the at least one methylation marker gene comprises 1 to 15 methylation marker genes.
  3. In claim 1, at least one methylation marker gene is BARX1, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, ZNF671, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, ZMIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, ANKRD13B, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, HOXA9, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, A method comprising one or more marker genes selected from the group consisting of ZNF781, PTGDR, GRIN2D, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, EMX1, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, B3GALT6, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, ZDHHC1 , and ZNF32 .
  4. The method of claim 1, wherein at least one methylation marker gene is composed of at least one of IFFO1 and HOPX , and further comprises one or more of BARX1, FLJ45983, HOXA9, ZNF781, HOXB2, SOBP, TRH, and FAM59B .
  5. The method of claim 4, wherein at least one methylation marker gene comprises: At least one of IFFO1 and HOPX ; and A group consisting of BARX1, FLJ45983, HOXA9, ZNF781, HOXB2, SOBP, TRH, and FAM59B.
  6. A method according to any one of claims 1 to 5, wherein at least one reference marker comprises one or more reference markers selected from B3GALT6 DNA and β-actin DNA.
  7. A method according to any one of claims 1 to 6, wherein the DNA is treated with a reagent that selectively modifies the DNA in a manner specific to the methylation state of the DNA.
  8. The method of claim 7, wherein the reagent comprises a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme.
  9. A method according to any one of claims 1 to 8, wherein the sample comprises one or more of tissue, blood, serum, plasma, and sputum.
  10. A method according to any one of claims 1 to 9, wherein DNA is extracted from a sample.
  11. A method according to any one of claims 1 to 10, wherein DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA.
  12. A method according to any one of claims 1 to 11, wherein the step of measuring the amount of a methylation marker gene comprises using one or more of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture.
  13. The method of claim 12, wherein the measurement step comprises multiplex amplification.
  14. A method according to any one of claims 1 to 13, wherein the step of measuring the amount of at least one methylation marker gene comprises using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme assay, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genome sequencing PCR.
  15. As a method for characterizing at least one sample of an object, a) A step of measuring the amount of at least one methylation marker gene in the DNA of a sample obtained from a subject, comprising: i) a step of measuring the amount of at least one reference marker in DNA; and iii) a step of calculating the value of the amount of at least one methylation marker gene measured in DNA as a percentage of the amount of a reference marker measured in DNA, wherein the value represents the amount of at least one methylation marker gene measured in the sample; And a method including one or more of the following steps: b) a step of measuring the amount of at least one RNA marker in a sample obtained from a subject; and c) A step of analyzing the presence or absence of at least one protein marker in a sample obtained from a subject.
  16. The method of claim 15, wherein the step of measuring the amount of at least one RNA marker in a sample comprises: i) a step of measuring the amount of reference RNA in the sample; and ii) a step of calculating the value of the amount of at least one RNA marker measured in the sample as a percentage of the reference RNA amount measured in the sample, wherein the value represents the amount of at least one RNA marker measured in the sample, and the amount of at least one RNA marker in the sample represents the expression level of the gene for said at least one RNA marker.
  17. The method of claim 15 or claim 16, wherein at least one RNA marker comprises mRNA.
  18. The method of claim 17, wherein at least one RNA marker comprises an mRNA selected from the group consisting of GAGE12D, FAM83A, LRG1, XAGE-1 d, MAGEA4, SFTPB, AKAP4, and CYP24A1 .
  19. A method according to any one of claims 15 to 18, wherein the reference RNA is selected from the group consisting of CASC3 mRNA, β-actin mRNA, U1 snRNA and U6 snRNA.
  20. In any one of claims 15 to 19, at least one methylation marker gene is BARX1, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, ZNF671, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, ZMIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, ANKRD13B, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, HOXA9, TRH, SP9, DMRTA2, ARHGEF4, A method comprising one or more marker genes selected from the group consisting of CYP26C1, ZNF781, PTGDR, GRIN2D, MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, EMX1, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, B3GALT6, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, ZDHHC1, ZNF32, IFFO1 , and HOPX .

Description

Characterizing methylated DNA, RNA, and proteins in the detection of lung neoplasia This application claims priority to U.S. Provisional Application No. 62/771,965 filed on November 27, 2018, which is incorporated herein by reference. Field of invention An invention relating to tumor detection, particularly, non-exclusively, to a method, composition, and related use for detecting neoplasms such as lung cancer is provided. Background of the Invention Lung cancer remains the number one cancer in the United States, and effective screening methods are urgently needed. Lung cancer alone claims 221,000 lives annually. DNA methylation profiling has revealed unique patterns in DNA promoter regions associated with cancer and holds significant applicability for the detection of lung malignancies. However, optimal discriminative markers and marker panels are required. Summary of the Invention The present invention provides a collection of methylation markers analyzed in tissue or plasma that distinguish all types of lung cancers very well while remaining negative in normal lung tissue and benign nodules. Markers selected from the collection may be used alone or in a panel, for example, to characterize blood or body fluids, and have the use of screening for lung cancer and identifying malignant tumors in benign nodules. In some embodiments, markers in this panel are used to distinguish one type of lung cancer from another, for example, to distinguish the presence of lung adenocarcinoma or large cell carcinoma from the presence of lung small cell carcinoma, or to detect mixed pathological carcinomas. The present invention provides a technique for screening markers that provide a high signal-to-noise ratio and low background levels when detected from samples taken from subjects. BARX1, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, ZNF671, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, ZMIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, ANKRD13B, DOCK2, MAX_chr19.163, ZNF132, MAX chr19.372, HOXA9, TRH, SP9, DMRTA2, ARHGEF4, CYP26C1, ZNF781, PTGDR, GRIN2D, Methylation markers and/or panels of markers (e.g., chromosomal region(s)) having annotations selected from MATK, BCAT1, PRKCB_28, ST8SIA_22, FLJ45983, DLX4, SHOX2, EMX1, HOXB2, MAX.chr12.526, BCL2L11, OPLAH, PARP15, KLHDC7B, SLC12A8, BHLHE23, CAPN2, FGF14, FLJ34208, B3GALT6, BIN2_Z, DNMT3A, FERMT3, NFIX, S1PR4, SKI, SUCLG2, TBX15, ZDHHC1, ZNF329, IFFO1, and HOPX were identified in this study by comparing the methylation status of methylation markers obtained from lung cancer samples with corresponding markers in normal (non-cancerous) samples. As described herein, the present invention provides a number of methylation markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more markers) for high discrimination against lung cancer and, in some embodiments, for discrimination against lung cancer types. Experiments applied selection filters to candidate markers to identify markers that provide a high signal-to-noise ratio and a low background level to provide high specificity and selectivity for the purpose of characterizing biological samples, for example, for cancer screening or diagnosis. For example, as described below, a methylation analysis of a combination of eight markers, SLC12A8, KLHDC7B, PARP15, OPLAH, BCL2L11, MAX.chr12.526, HOXB2, and EMX1, showed 98.5% sensitivity (134/136 cancers) and 100% specificity for all cancer tissues tested. In another specific example, the panel of six markers (SHOX2, SOBP, ZNF781, CYP26C1, SUCLG2, and SKI) showed 92.2% sensitivity with 93% specificity, and the panel of four markers (ZNF781, BARX1, EMX1, and HOXA9) showed 96% overall sensitivity and 94% specificity. Accordingly, the present application provides a method for processing a sample obtained from a subject, the method comprising the step of analyzing the methylation status of one or more marker genes in the sample. In a preferred embodiment, the methylation status of the methylation marker is determined by measuring the amounts of the methylation marker and the reference marker in the sample, and determining the methylation status for the methylation marker in the sample by comparing the amount of the methylation marker in the sample with the amount of the reference marker. Without limiting the invention to specific applications or applications, the method finds a use, for example, to characterize a sample obtained from a subject with or suspected of having lung cancer when the methylation status of the methylation marker differs from the methylation status of the corresponding marker analyzed in a subject without a neoplasm. In a preferred embodiment, the methylation marker is BARX1, LOC100129726, SPOCK2, TSC22D4, MAX.chr8.124, RASSF1, ZNF671, ST8SIA1, NKX6_2, FAM59B, DIDO1, MAX_Chr1.110, AGRN, SOBP, MAX_chr10.226, ZMIZ1, MAX_chr8.145, MAX_chr10.225, PRDM14, ANGPT1, MAX.chr16.50, PTGDR_9, ANK