WO2021167413A1 - 핵산의 메틸화 차이를 이용한 마커 선별방법, 메틸 또는 탈메틸 마커 및 이 마커를 이용한 진단방법 - Google Patents
핵산의 메틸화 차이를 이용한 마커 선별방법, 메틸 또는 탈메틸 마커 및 이 마커를 이용한 진단방법 Download PDFInfo
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- C12Q2600/158—Expression markers
Definitions
- the present invention relates to a marker selection method using a methylation difference of a nucleic acid, a demethylation marker, and a diagnostic method using the marker, and more particularly, a new method for selecting a disease-specific demethylation marker using a methylation difference in a free nucleic acid,
- the present invention relates to a new cancer diagnosis method by methylation detection for determining cancer by calculating the demethylation marker and the frequency of the marker selected by this method, and a cancer-specific demethylation marker in the selected cfDNA.
- Cancer refers to a group of abnormal cells generated by continuous division and proliferation by disrupting the balance between cell division and death due to various causes, and is also referred to as a tumor or neoplasia. In general, it develops in more than 100 different parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops into serious symptoms through infiltration into surrounding tissues and metastasis to other organs.
- cancer diagnosis is conducted through history taking, physical examination, and clinical pathology, and once suspected, radiological examination and endoscopy are performed, and finally, a biopsy is confirmed.
- the number of cancer cells must be 1 billion cells and the diameter of the cancer must be 1 cm or more to be diagnosed.
- cancer cells already have the ability to metastasize, and in fact, more than half of the cancer has already metastasized.
- tumor markers which find substances directly or indirectly produced by cancer in the blood, are used in cancer screening. Even in the absence of this, it often appears as benign, causing confusion.
- an anticancer agent mainly used for the treatment of cancer there is a problem in that the effect is shown only when the volume of the cancer is small.
- Cancer cells are living organisms with high viability that are caused by mutations in a number of genes. In order for a single cell to turn into a cancer cell and develop into a malignant cancer mass seen in clinical practice, mutations in multiple genes must occur. Therefore, it is necessary to approach at the genetic level in order to fundamentally diagnose and treat cancer.
- DNA methylation mainly occurs in cytosine of CpG island of the promoter region of a specific gene. It is the main mechanism by which the function of the gene is lost without mutation in the protein-specific coding sequence of the gene in vivo, and the cause of the loss of the function of a number of tumor suppressor genes in human cancer is interpreted as Although there is controversy over whether methylation of promoter CpG islands directly induces carcinogenesis or is a secondary change in carcinogenesis, such aberrant methylation/demethylation on CpG islands in various cancer cells such as prostate cancer, colon cancer, uterine cancer, and breast cancer has been reported Therefore, it can be used in various fields, such as early diagnosis of cancer, prediction of cancer risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to chemotherapy.
- MSP methylation-specific PCR
- automatic sequencing or bisulfite pyrosequencing
- the present inventors treated cfDNA with a methylation-sensitive restriction enzyme to cut and decode unmethylated sequences among restriction enzyme target sequences while researching to develop a novel method for accurate diagnosis of cancer in a non-invasive manner. ), if each is classified using sequence information of a certain length from the decoded sequence, the type of cfDNA in the blood can be classified for diseases, particularly diseases such as cancer, and through this, it can act as a cfDNA marker for diseases.
- the present invention was completed by developing a method for screening cancer-specific markers related to methylation in cfDNA, particularly cancer-specific demethylation markers.
- Another object of the present invention is to provide a novel cancer diagnosis method by demethylation detection for determining cancer by calculating the frequency of cancer-specific demethylation markers in selected cfDNA.
- Another object of the present invention is to provide a method for decoding and analyzing sequence information of a predetermined length at the N-terminus of a methylation-sensitive restriction enzyme fragment of cfDNA isolated from blood of an individual in order to provide information necessary for cancer diagnosis. will be.
- N-terminus is a sequence of the cohesive end of the recognition site of a methylation-sensitive restriction enzyme, and consists of a sequence of 25 to 150 bases, and cancer in cfDNA selected by the method of the present invention. To provide a specific demethylation marker.
- the present invention comprises the steps of treating a methylation sensitive restriction enzyme to cfDNA (cell free DNA) isolated from blood; sequencing the sequence of each fragment; obtaining sequence information of a predetermined length from the N-terminus of the fragment; counting the frequency of each sequence information;
- a method for selecting a cancer-specific demethylation marker in cfDNA comprising the step of selecting cancer-specific sequence information as a cancer-specific demethylation marker in cfDNA.
- the present invention provides a new cancer diagnosis method by methylation detection for determining cancer by calculating the frequency of cancer-specific demethylation markers in selected cfDNA.
- the present invention analyzes sequence information of a predetermined length at the N-terminus of a methylation-sensitive restriction enzyme fragment of cfDNA isolated from blood of an individual in order to provide information necessary for cancer diagnosis. provides a way to
- the present invention provides that the N-terminus is a sequence of the cohesive end of the recognition site of a methylation-sensitive restriction enzyme, and consists of a sequence of 25 to 150 bases, and in the method of the present invention cancer-specific demethylation markers in cfDNA selected by
- the present invention comprises the steps of (a) treating a methylation sensitive restriction enzyme in cfDNA (cell free DNA) isolated from blood; (b) sequencing each fragment; (c) obtaining sequence information of a predetermined length from the N-terminus of the fragment; (d) counting the frequency of each sequence information; and (e) selecting the cancer-specific sequence information as a cancer-specific demethylation marker in cfDNA.
- nucleic acid in purified or unpurified form may be used in the present invention, and any nucleic acid containing or suspected of containing a nucleic acid sequence containing a target site (eg, a CpG-containing nucleic acid) may be used.
- a nucleic acid site that can be differentially methylated is the C position of the CpG sequence, and methylation is particularly high in CpG islands where GpG is dense. At certain sites, the density of CpG islands is 10-fold higher compared to other regions of the genome.
- CpG islands have an average G*C ratio of about 60%, whereas normal DNA exhibits an average G*C ratio of 40%.
- CpG islands are typically about 1-2 kb in length, and there are about 45,000 CpG islands in the human genome.
- the sample nucleic acid is DNA.
- nucleic acid mixtures may also be used.
- the specific nucleic acid sequence to be detected may be a fraction of a large molecule, or may exist in the form of isolated molecules in which the specific sequence initially constitutes the entire nucleic acid sequence.
- the nucleic acid sequence need not be a nucleic acid present in pure form, and the nucleic acid may be a small fraction in a complex mixture, such as comprising whole human DNA.
- the nucleic acid contained in the sample used for measuring the degree of methylation of the nucleic acid contained in the sample or used for detecting the methylated CpG island may be extracted by a conventional method known in the art.
- Sequencing methods include, for example, Sanger sequencing, high throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, RNA- Seq (Illumina), digital gene expression [Helicos], next-generation sequencing (NGS), single molecule sequencing by synthesis (SMSS) (Helicos), massively parallel sequencing, clonal single molecule arrays [Solexa )], shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking; sequencing using PacBio, SOLiD, ion torrent, or nanopore platforms.
- the sequencing reaction may be performed in various sample processing units, which may be multiple lanes, multiple channels, multiple wells, or other means of processing multiple sets of samples substantially simultaneously.
- the sample processing unit may also include multiple sample chambers that allow simultaneous processing of multiple executions.
- the sequencing reaction may be performed on one or more types of nucleic acid, at least one of which is known to contain a marker of a disease.
- the sequencing reaction may also be performed on any nucleic acid fragment present in the sample.
- Simultaneous sequencing reactions can be performed using multiplex sequencing.
- cell-free nucleic acids can be sequenced in at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.
- cell-free nucleic acids can be sequenced in less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. Sequencing reactions may be performed sequentially or simultaneously. Subsequent data analysis may be performed on all or part of the sequencing reaction.
- data analysis may be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases, data analysis may be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.
- An exemplary read depth is 1000-50000 reads per locus (base).
- the sample may be any biological sample isolated from a subject.
- the sample may be a body sample.
- the sample may be of body tissue, such as known or suspected solid tumor, whole blood, serum, plasma, feces, leukocytes or lymphocytes, endothelial cells, tissue biopsy, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial fluid or extracellular fluid, intercellular space fluids within the gums (including those of the gums), bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine.
- the sample may be in the form originally isolated from the subject or may be further processed to remove or add components such as cells or to enrich one component compared to another component.
- a sample may be isolated or obtained from the subject and transported to a sample analysis site. Samples can be stored and shipped under a desired temperature, eg, room temperature, 4°C, -20°C, and/or -80°C. A sample may be isolated or obtained from a subject at a sample analysis site.
- the subject may be a human, mammal, animal, pet animal, service animal, or pet.
- the subject may have a disease.
- the subject cannot be free of the disease or detectable disease symptoms.
- the subject may have been treated with one or more therapies, eg, any one or more of surgery, treatment, dosing, chemotherapy, antibody, vaccine, or biologic.
- the subject may or may not be in remission.
- a blood sample may contain varying amounts of nucleic acid containing genomic equivalents.
- a sample of about 33 ng DNA may contain a haploid human genome equivalents of about 10,000 (10 4), in the case of cfDNA has, approximately 200 billion (2x10 11) may contain a separate polynucleotide molecules have.
- a sample of about 100 ng of DNA may contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.
- Exemplary amounts of cell-free nucleic acid in a sample prior to amplification range from about 1 fg to about 1 ⁇ g, eg, from 1 pg to 200 ng, from 1 ng to 100 ng, from 10 ng to 1000 ng.
- the amount may be about 600 ng or less, about 500 ng or less, about 400 ng or less, about 300 ng or less, about 200 ng or less, about 100 ng or less, about 50 ng or less, or about 20 ng or less, or about 10 ng or less. or less, or about 5 ng or less, or about 1 ng or less of a cell-free nucleic acid molecule.
- the amount is at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid. It may be a molecule.
- the amount is 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng or less of a cell-free nucleic acid molecule can The method may comprise obtaining from 1 femtogram (fg) to 200 ng.
- a cell-free nucleic acid is a nucleic acid that is not contained within or otherwise associated with a cell, or that is, a nucleic acid that remains in a sample after removal of an intact cell.
- Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these.
- Cell-free nucleic acids can be double-stranded, single-stranded, or hybrids thereof.
- Cell-free nucleic acids can be released into body fluids through secretion or cell death processes such as cellular necrosis and apoptosis. Some cell-free nucleic acids are released into body fluids from cancer cells, such as circulating tumor DNA (ctDNA). Others are released from healthy cells.
- the cell-free nucleic acid is produced by a tumor cell. In some embodiments, the cell-free nucleic acid is produced by a mixture of tumor cells and non-tumor cells.
- Cell-free nucleic acids exhibit, for example, a length distribution of about 100 to 500 nucleotides, with molecules of 110 to about 230 nucleotides accounting for about 90% of such molecules, with a second minor peak ranging from 240 to 440 nucleotides. do.
- Cell-free nucleic acids can be isolated from bodily fluids through fractionation or cleavage steps, wherein the cell-free nucleic acids as found in solution are separated from intact cells and other non-soluble components of the body fluids. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in a body fluid can be lysed, and cell-free and cellular nucleic acids can be processed together. In general, after addition of buffer and washing steps, the nucleic acid can be precipitated with alcohol. Additional purification steps may remove contaminants or salts, such as using silica-based columns. Non-specific bulk carrier nucleic acids such as Cot-1 DNA, DNA or protein for bisulfite sequencing, hybridization, and/or ligation may be added throughout the reaction to optimize certain aspects of this procedure, such as yield.
- Partitioning may include techniques such as centrifugation or filtration.
- cells in a body fluid can be lysed, and cell-free and cellular nucleic acids can be processed together.
- the sample may contain nucleic acids in various forms, including double-stranded DNA, single-stranded DNA and single-stranded RNA.
- single-stranded DNA and RNA can be converted to a double-stranded form, so that they are included in subsequent processing and analysis steps.
- cfDNA may be derived from human genomic DNA, or it may be derived from DNA of cells, bacteria, fungi or viruses other than humans that coexist with humans or are infected with humans.
- a method for selecting a cancer-specific demethylation marker in cfDNA may include the following steps:
- Step (a) is a step of treating methylation sensitive restriction enzyme in cfDNA (cell free DNA) isolated from blood.
- cfDNA is isolated from the organism.
- cfDNA can be isolated from plasma.
- the isolation method may be performed by a conventional DNA isolation method known in the art that can obtain a purity suitable for treatment with restriction enzymes and sequencing.
- the methylation sensitive restriction enzyme is AatII, AclI, AgeI, Aor13HI, AscI, AsiSI, AvaI, BsaHI, BsiEI, BsiWI, BspDI, BsrFI, BssHII, BstBI, ClaI, CpoI, EagI, FseI, HaeII, HhaI, HinP1I, HpaII (or HapII), HpyCH4IV, Hpy99I, KasI, MluI, NarI, NgoMIV, NotI, PaeR7I, PluTI, PvuI, RsrII, SacII, SalI, SgrAI or TspMI.
- the methylation-sensitive restriction enzyme of the present invention i) selectively cuts the unmethylated target region, ii) the cleaved end creates a cohesive end (not a blunt end), so that the ligation efficiency of the adapter having a complementary cohesive end It is possible to increase the .
- the methylation-sensitive restriction enzyme is preferably an enzyme capable of selectively cleaving CpG methylation, that is, an enzyme capable of specifically cleaving a restriction enzyme recognition site including demethylated CpG.
- an appropriate restriction enzyme can be selected according to the purpose.
- Step (b) is a step of sequencing each fragment.
- sequence translation is performed by a sequence translation method known in the art.
- Sequence translation translates the sequence of each fragment cleaved or uncleaved by a methylation-sensitive restriction enzyme.
- Sequence translation is suitable for translating a large number of fragments, preferably at least 10000 or more, at least 20000 or more, at least 30000 or more, at least 40000 or more, at least 50000 or more, at least 100000 or more, at least 1000000 or more fragments. Detoxification methods are preferred.
- sequence decoding For the sequence decoding, a sequence decoding method known in the art may be used, but any method capable of decoding a large amount of sequence in order to decode the sequence of each fragment in a sufficient quantity may be used without limitation. For example, if the next generation sequencing method (NGS) is used, it has the advantage that a large amount of sequences can be decoded in 18 hours at a low cost. Qualitative and quantitative analysis is possible.
- NGS next generation sequencing method
- an appropriate adapter may be attached so that only the DNA fragment cleaved by a methylation-sensitive restriction enzyme can be translated.
- DNA in the sample may or may not be cleaved by a methylation-sensitive restriction enzyme depending on the methylation state. For example, in the case of detecting cancer DNA that is methylated in normal human cfDNA, but cancer DNA that is darkened and demethylated, it is easy to detect cfDNA mixed in a very low ratio if only the demethylated and cleaved fragment can be decoded.
- Step (c) is a step of obtaining sequence information of a predetermined length from the 5' end of the fragment.
- the term 'predetermined length' indicates the length of a base or base pair from the 5' end in each sequence-translated fragment, preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
- the predetermined length may be one of a natural number less than 25 and a natural number greater than 150 depending on a cancer to be screened or analyzed, a type of a sample, and the like. More preferably the predetermined length may be 30, 60, 80 or 90 bases. Also, in one aspect of the present invention, it may be any natural number from 'predetermined length' to 1000.
- Step (d) is a step of counting the frequency of each sequence information.
- the frequency of each sequence information starting at the 5' end with the cohesive end sequence (CGG in the case of HpaII) generated by restriction enzyme digestion is counted. That is, from all sequences obtained by decoding one sample, the types of sequences of one length (for example, 30) are counted (in the case of 30 nt, theoretically 4 30 types of sequences are possible), and the number of sequences of each type is counted. count the number of times it appears.
- the value of each sequence counted is normalized for comparison with the values of other samples. This normalization is to divide each aggregated value by a value proportional to the decoded amount for a direct quantitative comparison between samples if the amount of readout for each sample is different. In this case, various values are possible, such as the total number of sequences decoded in each sample and the number of sequences mapped to the house keeping gene region, as the value proportional to the amount of translation.
- Step (e) is a step of selecting cancer-specific sequence information as a cancer-specific demethylation marker in cfDNA.
- a sequence of a predetermined length that is significantly higher in the cancer sample group is selected as a marker.
- the difference between the average value of the normal sample group and the cancer sample group is used, or various statistical techniques such as T-test, Mann-Whitney test, Wilcoxon test, or Cohen's D test are used. to select sequences that are significantly different from each other in the two sample groups. In this example, the difference between the average values for breast cancer and lung cancer was analyzed.
- the selected cancer-specific demethylation marker may be a marker customized to the individual providing the sample, and may be a marker commonly applied to cancer type, stage, race, or family.
- the cancer-specific demethylation marker in cfDNA is a sequence of a portion remaining after the N-terminal sequence is cleaved among the recognition sites of the restriction enzyme, and consists of a nucleotide sequence having the same length as the predetermined length.
- the HpaII restriction enzyme recognizes the CCGG base and cuts between C and C.
- the N-terminus of the truncated fragment begins with CGG.
- the cancer-specific demethylation marker has a nucleotide sequence of a predetermined length
- the present invention comprises the steps of isolating cfDNA from blood isolated from an individual; treating the isolated cfDNA (cell free DNA) with a methylation-sensitive restriction enzyme; decoding the sequence of each fragment; obtaining sequence information of a predetermined length from the N-terminus of the fragment; counting the frequency of each sequence information; It relates to a cancer diagnosis method comprising the step of calculating the frequency of cancer-specific demethylation markers in cfDNA and determining cancer.
- the subject is a patient in need of diagnosis of cancer.
- the predetermined length is the same length as the cancer-specific demethylation marker in cfDNA.
- the cancer-specific demethylation marker in cfDNA is a marker set consisting of 1 to 50, preferably 3 to 40, more preferably 5 to 30 markers.
- the present invention relates to a method for deciphering and analyzing sequence information of a predetermined length at the N-terminus of a methylation-sensitive restriction enzyme fragment of cfDNA isolated from blood of an individual to provide information necessary for cancer diagnosis.
- the N-terminus is the sequence of the sticky end of the recognition site of the methylation-sensitive restriction enzyme (eg, the sequence of CGG), and consists of a sequence of 25 to 150 bases consecutively (preferably 30 bases, It consists of a nucleotide sequence of 35 bases, 40 bases, 45 bases or 50 bases, and provides a cancer-specific demethylation marker in the cfDNA selected by the method of the present invention.
- the sequence of the sticky end of the recognition site of the methylation-sensitive restriction enzyme eg, the sequence of CGG
- the indication of the base follows the standard notation, for example, A is adenine, C is cytosine, T is thymine, G is guanine, Y is C or T, W is A or T, R is A or represents G.
- cancer is, but is not limited to, cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, Rectal cancer, gastric cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, It may be prostate cancer, bladder cancer, kidney cancer or ureter cancer.
- the diagnostic method is used to diagnose the presence of a condition, particularly a disease, characterize the condition (eg, stage the cancer or determine the heterogeneity of the cancer) in a subject, or determine the response to treatment of the condition. It can be used to monitor or prognosticate the risk of developing a condition or subsequent course of a condition.
- the present disclosure may also be useful in determining the efficacy of a particular treatment regimen.
- a particular treatment regimen may correlate with the genetic profile of the cancer over time. Such correlations may be useful in choosing a therapy.
- the diagnostic method can be used to monitor residual disease or recurrence of the disease.
- Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage. Genetic profile data may allow for the characterization of specific subtypes of cancer, which may be important in diagnosing or treating cancers of that specific subtype. Such information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer, and may allow the subject or practitioner to adopt treatment options as the disease progresses. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The systems and methods of the present disclosure may be useful in determining disease progression.
- the present invention can use each marker individually as diagnostic or predictive markers, or combine several markers to form a panel display, and some markers improve reliability and efficiency through an overall pattern or list of methylated sites. that can be checked
- the markers identified in the present invention may be used individually or as a combined marker set. Markers can be ranked, weighted, and level of likelihood of developing disease according to the number and importance of markers methylated together. Such an algorithm belongs to the present invention.
- the target nucleic acid site can be hybridized with a known probe immobilized on a solid support (substrate).
- substrate is a substance, structure, surface or material, non-biological, synthetic, inanimate, planar, spherical, or a mixture comprising a specific binding, flat surface material, a hybridization or enzyme recognition site. or numerous other recognition sites beyond the majority of other recognition sites or numerous other molecular species composed of surfaces, structures or materials.
- the substrates may be, for example, semiconductors, (organic) synthetic metals, synthetic semiconductors, insulators and dopants; metals, alloys, elements, compounds and minerals; synthesized, disassembled, etched, lithographed, printed and microfabricated slides, devices, structures and surfaces; industrial, polymers, plastics, membranes, silicones, silicates, glass, metals and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and non-woven fibers, materials and fabrics.
- semiconductors organic synthetic metals, synthetic semiconductors, insulators and dopants
- metals, alloys, elements, compounds and minerals synthesized, disassembled, etched, lithographed, printed and microfabricated slides, devices, structures and surfaces
- industrial, polymers, plastics, membranes, silicones, silicates, glass, metals and ceramics wood, paper, cardboard, cotton, wool, cloth, woven and non-woven fibers, materials and fabrics.
- membranes are known in the art to have adhesion to nucleic acid sequences.
- specific, non-limiting examples of such membranes include nitrocellulose or polyvinyl chloride, diazotized paper and membranes for gene expression detection, such as commercially used membranes under the trade names GENESCREEN, ZETAPROBE, and NYTRAN. have.
- beads, glass, wafers and metal substrates are also included. Methods for attaching nucleic acids to such objects are well known in the art. Alternatively, screening can also be carried out in the liquid phase.
- the method of the present invention can select cancer-specific demethylation markers in cfDNA, and the selected markers can provide information necessary for cancer diagnosis, monitoring treatment regimens, and prognosis of cancer patients, so that it can be used for anticancer treatment. It can be usefully used.
- FIG. 1A is an example of the results of analysis by treatment with HpaII in the lung cancer patient sample group and the normal sample group
- FIG. 1B is a schematic view thereof.
- FIG. 2a shows regions where there is a statistically significant difference between the two sample groups by standardizing (z-score) the number of reads mapped to the enzyme cleavage site treated with SacII in the breast cancer patient sample group and the normal sample group.
- FIG. 2B is a diagram illustrating a difference in probability values between a normal group and a breast cancer group by creating a machine learning model using the breast cancer-specific markers extracted in the process of FIG. 2A to calculate breast cancer prediction probability values (0.0 to 1.0).
- 2c shows the ROC (Receiver Operator Characteristic) curve through the average probability value of each test sample by repeating the model learning 20 times, and AUC (Area Under Curve: 0.0 to 1.0) values are expressed.
- ROC Receiveiver Operator Characteristic
- FIG. 3a is an example of the results of analysis by treatment with HpaII in the breast cancer patient sample group and the normal sample group
- FIG. 3b is a schematic view thereof.
- FIG. 4a shows regions with statistically significant differences between the two sample groups by standardizing (z-score) the number of reads mapped to the enzyme cleavage site treated with SacII in the lung cancer patient sample group and the normal sample group.
- FIG. 4B is a diagram illustrating the difference in probability values between the normal group and the lung cancer group by creating a machine learning model using the lung cancer-specific markers extracted in the process of FIG. 4A to calculate the lung cancer prediction probability values (0.0 to 1.0).
- 4c shows the ROC (Receiver Operator Characteristic) curve through the average probability value of each test sample by repeating the model learning 20 times, and AUC (Area Under Curve: 0.0 to 1.0) values are expressed.
- Figure 4d is a diagram of the ROC curve for each stage (stage) of lung cancer.
- the analysis library for each sample was used as NGS to obtain sequence information for each sequence included in the library. From the decoded sequence of each sample, a sequence starting with a restriction enzyme recognition sequence (CGG for HpaII, GC for SacII) was selected, and the selected sequences from 5' to a certain length (ex. 30, 60, 80, etc.) Sequence information was obtained and sequences were classified according to a predetermined length. The frequencies of sorted sequences in each sample were counted and normalized for comparison between samples.
- CGG for HpaII, GC for SacII restriction enzyme recognition sequence
- a demethylation marker (corresponding to the case of HpaII; Restriction enzymes that cut methylation sites could be selected as methylation markers).
- An average value (DHM score) is obtained for each sample for a given marker, and a reference value of the DHM score that can distinguish a cancer sample from a normal sample is determined. This DHM score was used for the judgment of the sample.
- a DHM score which is the average of values corresponding to the selected markers in the analysis of the sequence information (item 3), was obtained, and if it was higher than the predetermined DHM reference value, it was determined that cancer was present .
- Example 1 Selection of cancer-specific demethylation markers for breast cancer cfDNA and determination accuracy test using the same
- cfDNA isolated from 34 breast cancer sample groups and 53 normal samples was digested with HpaII, one of methylation-sensitive restriction enzymes, and read and analyzed according to the above-described experimental method.
- the sequence of the first 80 nt of the sequence starting with CGG was counted and compared as marker candidates.
- 173 markers with an average of 5 or more and 10 times or more than the average value of the normal group were selected as markers, and the average DHM score of these 173 values was obtained from each sample.
- FIG. 1 a table was made in which normalized scores for each marker of breast cancer and normal samples were recorded, and high numbers were expressed in red and low numbers in blue for easy viewing.
- 1A shows the upper part of the 173 markers.
- 31 samples except for three samples had a value above a certain value in the breast cancer sample, whereas all samples had a value close to 0 in the normal sample. could see
- FIG. 1B is a bar graph showing the DHM score, which is the average of 173 marker values of each sample shown in FIG. 1A . Based on the DHM score of 1, it can be seen that breast cancer and normal samples are clearly distinguished.
- Example 2 Selection of cancer-specific demethylation markers for breast cancer cfDNA treated with SacII and determination accuracy test using the same
- cfDNA isolated from 102 breast cancer sample groups and 139 normal samples was digested with SacII, a methylation-sensitive restriction enzyme, and read and analyzed according to the above-described experimental method.
- SacII SacII
- the sequence of the first 80 nt of the sequence starting with GC was counted and compared as marker candidates.
- Each marker is normalized through the IQR (InterQuartile Range) mean value and normalized through the Z-score to reduce the difference that may appear between sequencing.
- IQR InterQuartile Range
- Z-score Z-score
- a t-test is performed between the breast cancer group and the normal group to select a marker whose p-value is below a specific threshold (eg, 10 -5 ), and the final DHM Score is calculated through the selected marker.
- the final score can be calculated by simply adding the corresponding value for each sample to the selected marker, and can be calculated as a predicted probability value by creating a classification model of machine learning such as logistic regression analysis.
- FIG. 2a shows that a table was created in which normalized / normalized values for each marker of breast cancer and normal samples were recorded, and high numbers were expressed in red and low numbers in green for easy viewing.
- FIG. 2b shows that a machine learning prediction model was created using the selected markers, and the results were generated with probability values ranging from 0 to 1, and it was confirmed that the difference in the distribution of probability values between the breast cancer group and the normal group was clearly shown.
- Fig. 2c using K-Fold Cross Validation among the machine learning model testing methods, random extraction of training and test groups is performed at 8:2 for each cycle, and this operation is repeated 20 times so that one sample is different from each other 20 times.
- the result value was calculated through the training data, and the average value was taken and the ROC (Receiver Operating Characteristic) curve was drawn to measure the performance.
- Example 3 Selection of cancer-specific demethylation markers for lung cancer cfDNA and determination accuracy test using the same
- FIG. 3A a table was made in which normalized scores for each marker of lung cancer and normal samples were recorded, and high numbers were expressed in red and low numbers in blue for easy viewing.
- Figure 3a shows the top part of the 198 markers. In the selected marker, 8 samples except for three samples had values above the reference value in the lung cancer sample, while all samples had a value of 3 or less, which was lower than the reference value 4 in the normal sample.
- FIG. 3b is a bar graph showing the DHM score, which is the average of 198 marker values of each sample shown in FIG. 3a . Based on the DHM score of 4, it could be seen that lung cancer and normal samples were clearly distinguished.
- Example 4 Selection of cancer-specific demethylation markers for lung cancer cfDNA treated with SacII and determination accuracy test using the same
- cfDNA isolated from 75 lung cancer samples and 129 normal samples was digested with SacII, one of the methylation-sensitive restriction enzymes, and read and analyzed according to the above-described experimental method.
- SacII one of the methylation-sensitive restriction enzymes
- Each marker is normalized through the IQR (InterQuartile Range) mean value and normalized through the Z-score to reduce the difference that may appear between sequencing.
- IQR InterQuartile Range
- Z-score Z-score
- a t-test is performed between the lung cancer group and the normal group to select a marker whose p-value is below a specific threshold (eg 10 -5 ), and the final DHM Score is calculated through the selected marker.
- the final score can be calculated by simply adding the corresponding value for each sample to the selected marker, and can be calculated as a predicted probability value by creating a classification model of machine learning such as logistic regression analysis.
- FIG. 4a a table was made in which normalized/standardized values for each marker of lung cancer and normal samples were recorded, and high numbers were expressed in red and low numbers in green for easy viewing.
- FIG. 4b shows that a machine learning prediction model was created through the selected markers and the result values were generated with probability values ranging from 0 to 1, and it was confirmed that the difference in the distribution of probability values was clearly shown in the lung cancer group and the normal group.
- the method of the present invention can select cancer-specific demethylation markers in cfDNA, and the selected markers can provide information necessary for cancer diagnosis, treatment therapy monitoring, and prognosis of cancer patients. It can be usefully used for treatment.
Abstract
Description
효소 | 인식부위 | 서열번호 | 효소 | 인식부위 | 서열번호 |
AatII | GACGT↓C | 1 | HhaI | GCG↓C | 20 |
AclI | AA↓CGTT | 2 | HinP1I | G↓CGC | 21 |
AgeI | A↓CCGGT | 3 | HpaII | C↓CGG | 22 |
Aor13H I | T↓CCGGA | 4 | HpyCH4IV | A↓CGT | 23 |
AscI | GG↓CGCGCC | 5 | Hpy99I | CGWCG↓ | 24 |
AsiSI | GCGAT↓CGC | 6 | KasI | G↓GCGCC | 25 |
AvaI | C↓YCGRG | 7 | MluI | A↓CGCGT | 26 |
BsaHI | GR↓CGYC | 8 | NarI | GG↓CGCC | 27 |
BsiEI | CGRY↓CG | 9 | NgoMIV | G↓CCGGC | 28 |
BsiWI | C↓GTACG | 10 | NotI | GC↓GGCCGC | 29 |
BspDI | AT↓CGAT | 11 | PaeR7I | C↓TCGAG | 30 |
BsrFI | R↓CCGGY | 12 | PluTI | GGCGC↓C | 31 |
BssHII | G↓CGCGC | 13 | PvuI | CGAT↓CG | 32 |
BstBI | TT↓CGAA | 14 | RsrII | CG↓GWCCG | 33 |
ClaI | AT↓CGAT | 15 | SacII | CCGC↓GG | 34 |
Cpo I | CG↓GWCCG | 16 | SalI | G↓TCGAC | 35 |
EagI | C↓GGCCG | 17 | SgrAI | CR↓CCGGYG | 36 |
FseI | GGCCGG↓CC | 18 | TspMI | C↓CCGGG | 37 |
HaeII | RGCGC↓Y | 19 |
Claims (13)
- (a) 개체에서 분리한 cfDNA (cell free DNA)에 메틸화 민감성 제한효소 (methylation sensitive restriction enzyme)를 처리하는 단계;(b) 각 단편의 서열을 분석(sequencing)하는 단계;(c) 단편의 N-말단으로부터 미리 정해진 길이의 서열 정보를 수득하는 단계;(d) 각 서열 정보의 빈도를 계수하는 단계;(e) 암 특이 서열 정보를 cfDNA에서의 암 특이 탈메틸화 마커로 선별하는 단계를 포함하는 cfDNA에서의 암 특이 탈메틸화 마커를 선별하는 방법.
- 제1항에 있어서, 상기 메틸화 민감성 제한효소는 AatII, AclI, AgeI, Aor13H I, AscI, AsiSI, AvaI, BsaHI, BsiEI, BsiWI, BspDI, BsrFI, BssHII, BstBI, ClaI, Cpo I, EagI, FseI, HaeII, HhaI, HinP1I, HpaII, HpyCH4IV, Hpy99I, KasI, MluI, NarI, NgoMIV, NotI, PaeR7I, PluTI, PvuI, RsrII, SacII, SalI, SgrAI 및 TspMI로 이루어진 군에서 선택된 것을 특징으로 하는 방법.
- 제1항에 있어서, 서열을 분석하는 것은 차세대 시퀀싱 (NGS)에 의해서 수행되는 것을 특징으로 하는 방법.
- 제1항에 있어서, 상기 미리 정해진 길이는 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 및 150로 이루어진 군에서 선택된 어느 하나의 길이의 염기인 것을 특징으로 하는 방법.
- 제1항에 있어서, 상기 cfDNA에서의 암 특이 탈메틸화 마커는 N말단 서열이 상기 제한효소의 인식부위의 점착성 말단 (cohesive end)의 서열이며, 상기 미리 정해진 길이와 동일한 길이의 염기서열로 이루어진 것을 특징으로 하는 방법.
- 제1항에 있어서, 상기 암은 자궁경부암, 폐암, 췌장암, 간암, 결장암, 골암, 피부암, 두부 또는 경부암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 질암종, 음문암종, 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 방광암, 신장암 및 수뇨관암으로 이루어진 군에서 선택된 것임을 특징으로 하는 방법.
- (a) 개체에서 분리한 cfDNA (cell free DNA)에 메틸화 민감성 제한효소를 처리하는 단계;(b) 각 단편의 서열을 분석하는 단계;(c) 단편의 N-말단으로부터 미리 정해진 길이의 서열 정보를 수득하는 단계;(d) 각 서열 정보의 빈도를 계수하는 단계;(e) cfDNA에서의 암 특이 탈메틸화 마커의 빈도를 산출하여 암으로 판정하는 단계를 포함하는 암 진단 방법.
- 제7항에 있어서, 상기 개체는 암 진단이 필요한 환자인 것을 특징으로 하는 방법.
- 제7항에 있어서, 상기 미리 정해진 길이는 상기 cfDNA에서의 암특이 탈메틸화 마커와 동일한 길이인 것을 특징으로 하는 방법.
- 제7항에 있어서, 상기 cfDNA에서의 암특이 탈메틸화 마커는 5 내지 50개로 이루어진 마커 세트인 것을 특징으로 하는 방법.
- 암 진단에 필요한 정보를 제공하기 위하여, 개체에서 분리한 cfDNA의 메틸화 민감성 제한효소 단편의 N-말단의 미리 정해진 길이의 서열 정보를 분석하는 방법.
- N말단이 메틸화 민감성 제한효소의 인식부위의 점착성 말단 (cohesive end)의 서열이며, 25염기 내지 150염기의 서열로 이루어지며, 제1항의 방법에 의해서 선별된 cfDNA에서의 암 특이 탈메틸화 마커.
- 제12항에 있어서, 상기 점착성 말단의 서열은 ACGTC(서열번호 39), ATCG(서열번호 40), ATCGC(서열번호 41), CCGGA(서열번호 42), CCGGC(서열번호 43), CCGGCC(서열번호 44), CCGGG(서열번호 45), CCGGT(서열번호 46), CCGGY(서열번호 47), CCGGYG(서열번호 48), CG(서열번호 49), CGAA(서열번호 50), CGAT(서열번호 51), CGC(서열번호 52), CGCC(서열번호 53), CGCGC(서열번호 54), CGCGCC(서열번호 55), CGCGT(서열번호 56), CGG(서열번호 57), CGT(서열번호 58), CGTT(서열번호 59), CGWCG(서열번호 60), CGYC(서열번호 61), GCGCC(서열번호 62), GCGCY(서열번호 63), GCGG(서열번호 64), GGCCG(서열번호 65), GGCCGC(서열번호 66), GTACG(서열번호 67), GWCCG(서열번호 68), RYCG(서열번호 69), TCGAC(서열번호 70), TCGAG(서열번호 71) 및 YCGRG(서열번호 72)로 이루어진 군에서 선택되는 것임을 특징으로 하는 암 특이 탈메틸화 마커.
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