KR101990954B1 - Method for Screening Cancer By Analyzing Index of Tumor-Derived DNA - Google Patents

Abstract

The present invention relates to a method of providing information, a composition and a kit for analyzing a tumor or a lesion thereof. Specifically, the present invention relates to a method of providing information, a composition and a kit for analyzing a tumor or a lesion thereof by quantitatively analyzing the concentrations of tumor or lesion-derived DNA fragments and normal cell-derived DNA fragments, and then determining the reference value using the concentration ratio as an index, through oligonucleotides capable of amplifying normal cell-derived DNA fragments and oligonucleotides capable of amplifying DNA fragments derived from a tumor or lesion thereof in DNA isolated from biological samples.

Description

[0001] The present invention relates to a cancer screening method,

The present invention relates to a method for providing information for analyzing a tumor or a lesion thereof, a composition and a kit for analyzing a tumor or a lesion thereof, and more particularly to a composition and a kit for analyzing an oligosaccharide or oligosaccharide capable of amplifying a tumor or a DNA fragment derived from the lesion from DNA isolated from a biological sample Nucleotide and a normal cell-derived DNA fragment are quantitatively analyzed through an oligonucleotide capable of amplifying the DNA fragment and the DNA fragments derived from the lesion and the normal cell, and then the reference value is determined by setting the concentration ratio as an index , A method for providing information for analyzing a tumor or a lesion thereof, and a kit and a composition for analyzing a tumor or lesion thereof.

The World Cancer Research Institute (WHO) International Cancer Research Institute estimates that 14 million new cancer patients will develop annually worldwide by 2012, with a steep increase to 20 million annually in 2030 (World Cancer Report 2014). In Korea, 210,000 new cancer patients develop annually and 70,000 people die (National Cancer Registry Statistics in 2015). The incidence of cancer is increasing, but the 5 year survival rate is stagnating or increasing slowly. This is because cancer diagnosis is the most important reason for effective diagnosis, and after the cancer mass has grown to a size that can be confirmed, there is a risk of micro metastasis and gene expression and mutation among cancer cells in the same tumor tissue (Intra-tumor heterogeneity) due to the difference in the analysis results may be different depending on which part of the biopsy is accompanied by the disadvantage.

The development of cancer biopsy (or biopsy) technology for the commercialization of early diagnosis of cancer has led to the development of cancer-related biomarkers, including cancer development, therapeutic effect monitoring, prognostic evaluation, The development of cancer diagnosis and treatment, including selection of therapeutic drugs, is also expected.

However, there are technical issues related to the sensitivity and reproducibility of analytical indicators for liquid biopsies. Circulating tumor cells are present in the blood in trace amounts as they assess the survival prognosis based on an average of 5 to 7.5 mL of blood in cancer patients (Molecular oncology 10 (2016) 395-407). In addition, non-cell DNA is DNA that is liberated in the primary lesion or actively released from the tumor by necrosis and cell death of the tumor cell. The DNA and tumor DNA derived from normal cell death should be discriminated with high sensitivity and the half-life 10 hours on average at refrigeration temperature) (Cell 164 (2016) 57-68).

In order to overcome these technical limitations, digital PCR (Proc. Natl. Acad. Sci. 96 (1999) 9236-9241), BEAMing analysis (Nat.Method 3 (2006) 95-97), deep sequencing PLoS ONE 11 (2016) e0146638) have been developed to improve the analytical sensitivity and reproducibility of liquid biopsies. However, it has been shown that a DNA fragment containing trace amounts (<1%) of plasma or its lesion And a technique capable of rapidly and highly-sensitively analyzing the presence or absence of cancer or the cancer risk is required.

Under these technical backgrounds, the inventors of the present application conducted quantitative analysis of the concentration of DNA fragments derived from tumors or their lesions and DNA fragments derived from normal cells through oligonucleotides capable of amplifying DNA fragments derived from tumors or lesions thereof and DNA fragments derived from normal cells And then determining the concentration ratio as an index, it is possible to diagnose whether or not the tumor or cancer is present or not, and the present invention has been completed.

It is an object of the present invention to provide a method for providing information for analyzing a tumor or a lesion thereof.

It is an object of the present invention to provide a method for providing information for cancer diagnosis.

It is an object of the present invention to provide compositions and kits for the analysis of tumors or lesions thereof.

It is an object of the present invention to provide a cancer diagnostic composition and a kit.

In order to achieve the above object, the present invention provides a method for producing a DNA fragment comprising: (a) i) DNA isolated from a biological sample; ii) an oligonucleotide capable of amplifying a tumor or a lesion-derived DNA fragment thereof; And iii) oligonucleotides capable of amplifying a normal cell-derived DNA fragment are mixed and subjected to real-time quantitative amplification; (b) measuring the Ct value of the DNA fragment derived from the tumor or its lesion and the DNA fragments derived from the normal cell in the step (a) and determining the concentration; And a step of determining an index which is a ratio of the concentration of the DNA fragments derived from the tumor or the lesion to the amplified normal cell-derived DNA fragment, and judging the tumor as a tumor when the determined index is not less than a cut-off value And a method for providing information for analyzing a tumor or lesion thereof.

The present invention also relates to a method of amplifying a tumor or a lesion thereof, comprising: i) an oligonucleotide capable of amplifying a DNA fragment derived from a tumor or a lesion thereof, and ii) an oligonucleotide capable of amplifying a normal cell- Thereby providing a composition for lesion analysis.

The present invention also relates to a method of amplifying a tumor or a lesion thereof, comprising: i) an oligonucleotide capable of amplifying a DNA fragment derived from a tumor or a lesion thereof, and ii) an oligonucleotide capable of amplifying a normal cell- A kit for analyzing a lesion is provided.

According to the present invention, quantitatively analyzed concentrations and indexes of tumor or lesion-derived DNA fragments and normal cell-derived DNA fragments contained in the circulating non-cell DNA and exosome DNA extracted from a biological sample are compared with the control group The presence or absence of the tumor and the risk thereof can be easily discriminated from the statistical value.

If the DNA concentration and / or the index of the analytical sample exceeds the reference value when the tumor or lesion-derived DNA, the range of the DNA concentration from the normal cell, and the index range thereof are different and is used as a parameter, And the risk of developing solid tumors.

In addition, after therapeutic treatment has been performed on cancer patients, quantitative analysis and / or indexes of the DNA fragments or lesion-derived DNA fragments and DNA fragments derived from normal cells in the blood are analyzed to determine whether or not cancer has passed and whether residual cancer is present Can be predicted.

Furthermore, the quantitative analysis and / or indexes of the tumor or lesion-derived DNA fragments and the normal cell-derived DNA fragments contained in the circulating non-cell DNA and exosome DNA extracted from the biological sample were analyzed to select solid tumors Second, the cancer mutation and methylation abnormality can be secondary analyzed from these circulating non-cell DNAs and used as diagnosis and treatment prognostic indicators of solid tumors.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram illustrating a method for collecting and purifying septate DNA and circulating tumor cells and / or lesion-derived non-cell DNA in blood according to an embodiment of the present invention; Real-time quantitative amplification of tumor and / or lesion-derived DNA fragments and normal cell-derived DNA fragments; And analyzing the tumor / normal and lesion / normal DNA indices from the analysis data.
FIG. 2 is a graph showing the distribution of tumor and / or lesion-derived DNA and normal cell-derived DNA fragments in a real-time PCR using circulating tumor cells and / or lesion-derived non-cell DNAs isolated from the blood of colon, colon, Time quantitative amplification. 2A shows a quantitative amplification curve of the DNA fragments according to SYBR Green fluorescence dye binding, 2B shows a result of standard curve generation of DNA fragments derived from normal cells in combination with SEQ ID NOS: 1 and 3 using human control genomic DNA, and 2C Shows the results of standard curve setting of the tumor and / or lesion-derived DNA fragments of the combination of SEQ ID NOS: 4 and 5 using human control genomic DNA, and 2D shows the statistical significance (Mann-Whitney test = 0.000) is confirmed.
FIG. 3 is a graph showing the results of real-time quantitative amplification of tumor and / or lesion-derived DNA and normal cell-derived DNA fragments using circulating tumor cells and / or lesion-derived exosomal DNA purely isolated from the blood of cancer patients and non- The results are shown. 3A shows a quantitative amplification curve of the DNA fragments according to SYBR Green fluorescence dye binding, 3B shows a result of setting a standard curve of DNA fragments derived from normal cells in combination with SEQ ID NOS: 2 and 3 using human control genomic DNA, and 3C Shows the results of standard curve setting of the tumor and / or lesion-derived DNA fragments of the combination of SEQ ID NOS: 4 and 5 using human control genomic DNA, and 3D shows statistical significance (Mann-Whitney test = 0.004).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a method for detecting a protein comprising: (a) i) DNA isolated from a biological sample; ii) an oligonucleotide capable of amplifying a tumor or a lesion-derived DNA fragment thereof; And iii) oligonucleotides capable of amplifying a normal cell-derived DNA fragment are mixed and subjected to real-time quantitative amplification; (b) measuring the Ct value of the DNA fragment derived from the tumor or its lesion and the DNA fragments derived from the normal cell in the step (a) and determining the concentration; And a step of determining an index which is a ratio of the concentration of the DNA fragments derived from the tumor or the lesion to the amplified normal cell-derived DNA fragment, and judging the tumor as a tumor when the determined index is not less than a cut-off value And a method for providing information for analyzing a tumor or lesion thereof.

The biological sample may be, for example, a liquid biopsy obtained from a tumor-bearing patient, a cancer patient, or a suspected cancer patient. The DNA isolated from the biological sample may be a blood or plasma circulating tumor cell or lesion-derived non-cellular DNA and / or exosome DNA, and may be a liquid biopsy for detection and isolation of non-cellular DNA and / For example, blood, serum or plasma may be obtained in a tumor-bearing patient, a cancer patient, or a suspected cancer-susceptible patient.

On the other hand, exosomes are micro vesicles released from various various cells including cancer cells, and can be obtained in biological samples such as blood, serum or plasma. Exosomes can be separated from biological samples by size exclusion chromatography, density gradient centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradient, cell organotactic electrophoresis, magnetic activated cell sorting (MACS) Can be concentrated and separated through membrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, and the like.

In addition, when the exosome is separated from a specific cell type, it can be separated by using an antibody against an exosomal surface antigen, an extramammer, a polymer, or the like. In the case of cancer cells, cancer cell-specific surface antigens can be selected and separated using an antibody, aptamer, or a polymer specifically binding thereto.

DNA can be extracted from exosomes and isolated. In order to isolate the DNA from the exosome, the DNAse may be pretreated in order to remove the DNA present on the surface or outside of the exosome, as the case may be. This enzyme treatment can enrich internal exosome DNA.

There are analytical limitations because the tumor and / or lesion-derived marker DNA to be amplified contained in non-cellular DNA of plasma is often present in less than 1% of the normal-derived DNA. Thus, in addition to non-cellular DNA, it may also include the case of secretion of tumor and / or lesion-derived DNA in a cell organelle.

DNA fragments that have been transferred to the bloodstream by abnormal apoptosis and / or necrosis process from abnormal cells in the cancer tissues of cancer patients are used to remove the tumor cell free- It is known that the non - cellular DNA concentration existing in the cellular DNA state and the non - cellular DNA concentration in the blood flow state is increased compared with the normal one.

The separation of the non-cell DNA can be performed according to the manufacturer's instructions using a commercially available kit through a known method, for example, a magnetic bead-based separation method or a column-based separation method, an isopropanol precipitation- Phenol: chloroform-based separation method or the like. In one embodiment, the non-cellular DNA can be isolated using a MagMax kit that separates from the biological sample using magnetic beads.

DNA fragments detected in plasma in the blood may include non-cell DNA derived from tumor and / or lesion and DNA fragments originating from apoptosis of normal cells. Of these, the length distribution of noncircular DNA derived from circulating tumor cells or lesions in blood varies. The nucleosome, the basic unit of the chromatin structure, is composed of histone octamers, with about 147 bases wrapped around a histone octamer, with a linker DNA length of 162 to 167 base pairs. When a tumor is present, the length of the tumor is often less than 100 basepairs.

In particular, the tumor or a lesion-derived DNA fragment thereof may comprise a short interspersed nuclear element (SINE), for example, an ALU. Specifically, the tumor or a lesion-derived DNA fragment thereof may comprise a consensus sequence comprising the Alu Ya5 and / or Yb8 subfamily. In this case, the consensus sequence including the Alu Ya5 and / or Yb8 subfamily may include the sequence of SEQ ID NO: 6, and the sequence of SEQ ID NO: 6 may include any nucleotide.

According to the present invention, the consensus sequence containing the Ya5 and Yb8 sub-family of ALU repeat sequences based on the human standard genome (GRCh37 / hg19) base sequence can be analyzed.

The ALU repeating sequence units (about 280 bases), which account for about 13% of the human genome sequence and act on the molecular mechanism of disease development including solid tumors, are truncated in the normal killing process, 167 base pairs or less. Taking this into consideration, oligonucleotides capable of amplifying non-cell DNA with high sensitivity and reproducibility were prepared. An oligonucleotide capable of amplifying the tumor or a lesion-derived DNA fragment thereof and an oligonucleotide capable of amplifying the normal-cell-derived DNA fragment can be selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, for example.

Specifically, the oligonucleotide capable of amplifying the normal cell-derived DNA fragment can be selected from the group consisting of SEQ ID NOS: 1 to 3, and the oligonucleotide capable of amplifying the tumor or DNA fragment derived from the lesion thereof is represented by SEQ ID NO: 4, or 5.

The oligonucleotides may comprise, for example, the sequences set forth in Table 1.

[Table 1]

^* Y = C / T, ^† W = A / T, ^‡ K = G / T, ^** R = G / A

At this time, the sequence of the amplicon amplified by the oligonucleotide is shown in Table 2 below.

[Table 2]

^{* Y = C / T, †} W = A / T, ‡ K = G / T, ** R = G / A, N is any nucleotide

1) Among the 1X 10 ^ 6 copy number distributed on the human chromosome, only the specific position of chromosome 1 is indicated by reference.

The real-time quantitative amplification can be performed, for example, by real-time PCR, and the amount of PCR amplification product in real-time PCR can be detected by fluorescence signal. An intercalating method using an intercalator that binds to double helix DNA and detects fluorescence using an oligonucleotide labeled with a fluorophore at the 5 'end and a quencher at the 3' end. And the like.

As the real-time PCR proceeds, the intensity of the fluorescence signal increases according to the amount of the polynucleotide that is increased, and an amplification profile curve showing the intensity of the fluorescence signal according to the number of amplification cycles is obtained.

The amplification profile curve generally includes a baseline region in which a background fluorescence signal is not reflected, an exponential region in which a fluorescence signal increases with an increase in the amount of polynucleotide generation, and a PCR The reaction is divided into a plateau region where the intensity of the fluorescence signal is not increased by saturation. The baseline region appears early in the PCR reaction because the amount of PCR amplification has not yet reached a detectable amount.

A fluorescence signal intensity at a point where the amount of PCR amplification reaches a detectable amount by fluorescence is generally referred to as a threshold and a value corresponding to a threshold value in the amplification profile curve The number of amplification cycles is called the threshold cycle (Ct) value.

When the initial polynucleotide concentration is different and the initial polynucleotide concentration is different, the number of amplification cycles to reach the detectable amount of the amplification product becomes smaller and the critical cycle value becomes smaller as the initial amount of the polynucleotide increases. Thus, the logarithmic value of the initial polynucleotide amount and the critical cycle value are in strong inversely proportional relationship, and the desired polynucleotide quantification is usually performed using the critical cycle value. The quantification of polynucleotides using real - time polymerase chain reaction is largely an absolute quantitative and a relative quantitative. Absolute quantitation is a method in which real-time PCR is performed on a sample of which the amount of the polynucleotide is known to generate a standard curve of the critical cycle value with respect to the amount of the polynucleotide to perform quantification, Even if you do not know the amount of nucleotides, it is a way to compare the relative amounts with other samples. Even if any method is used, the threshold value should be determined first to calculate the critical cycle value in order to perform the quantification.

According to the present invention, the step (a) comprises measuring the Ct value of the amplified tumor or its lesion-derived DNA fragment and the normal cell-derived DNA fragment and determining the concentration.

The concentration of the DNA fragment may be determined by analyzing a standard curve in which the concentration is determined based on a threshold cycle (Ct) value for a DNA standard material.

Thereafter, an index, which is a ratio of the concentration of the DNA fragments derived from the tumor or the lesion thereof to the concentration of the amplified normal cell-derived DNA fragment, is determined, and when the determined index is not less than a cut-off value, .

In order to derive the reference value using the concentration ratio as an index, the Ct value of the DNA fragments derived from the real-time quantitative amplified tumor or its lesions and the DNA fragments derived from the normal cells are measured, Or the lesion-derived DNA fragment is calculated and then compared with the concentration ratio of the control DNA fragment to determine the presence or absence of the tumor and the risk thereof.

The "index" is calculated based on the concentration of the DNA fragments derived from the tumor or its lesions / the concentration of DNA fragments derived from normal cells based on the concentration of the DNA fragments derived from the normal cells and the DNA fragments derived from the lesions .

A "reference value" is a numerical value used to distinguish between a tumor or its lesion state and a normal state. If the index is larger than the reference value, it is classified as a tumor or its lesion state. If the index is smaller than the reference value, the reference value can be classified as a normal state. Statistical analysis methods such as average value calculation or ROC curve analysis can be used to calculate the reference value.

Specifically, the index based on the concentration ratio of the tumor-derived DNA fragment or the lesion-derived DNA fragment to the normal cell-derived DNA fragment is calculated based on the data on the distribution range of the tumor-type and / or lesion- . The reference value is expressed and evaluated by the curve of the Receiver Operation Characteristic (ROC) curve, in which the index distinguishes between the tumor group and the normal group, that is, the accuracy is expressed by a curve of sensitivity and 1-specificity. ROC curve analysis is a curve representing the performance of the diagnosis. It consists of sensitivity, specificity, and accuracy. Sensitivity and specificity are trade-off between each other, so the sum of the two values is always 1, so the specificity decreases when the sensitivity increases. In the ROC curve, the area under curve (AUC) represents the area under the curve where the x-axis is 1-specific and the y-axis is sensitive and accucracy is indicated.

ROC curve analysis showed that the sensitivity and specificity change with changes in the index cut-off, and thus the reference value with optimal sensitivity, specificity, positive predictive value, negative predictive value, positive bias ratio, negative likelihood ratio (cut -off), and the accuracy is evaluated by Area Under the ROC Curve (AUC).

The reference value is a threshold value showing a high effect in predicting the tumor or lesion thereof. According to an embodiment of the present invention, when the reference value is 0.284 - 0.688, for example, 0.25 in the 95% confidence interval range for tumor or cancer And it can be distinguished from normal group.

Specifically, in Example 5, each DNA concentration was quantified by substituting tumor grade and / or lesion-derived DNA Ct value and normal dead DNA Ct value into a concentration gradient standard curve of a DNA standard material, and the index was calculated therefrom ROC curve analysis was performed using SPSS Statistics 22 (IBM, USA) and MedCalc 14.8.1 (MedCalc Software, Belgium) statistical program. AUC was 0.82, sensitivity was 80%, specificity was 84.62%, positive predictive value was 66.67%, negative predictive value was 91.67%, positive biometric ratio was 5.20, negative likelihood ratio was 0.24, statistical significance level was P = 0.0019 when the cut-off was 0.250 . Increasing the cut-off to more than 0.250 increased specificity but decreased sensitivity and accuracy. Sensitivity increased with less than 0.250, but specificity and accuracy decreased. The 95% confidence interval range for the normal group according to Example 5 was 0.205 - 0.299 and 0.284 - 0.688 for tumor or cancer.

Specifically, the method according to the present invention is shown in Fig. According to Fig. 1, CTCs and / or lesion-derived non-cell DNA and exosome DNA are captured and separated, and the tumor and / or lesion-derived DNA fragment and the normal dead DNA fragment are qPCR , Tumor / normal and lesion / normal DNA indices can be analyzed from the analysis data to determine tumor presence and risk.

In the present invention, the tumor may comprise cancer, which may be, for example, solid cancer or sporadic solid cancer. The solid cancer or sporadic solid cancer may be selected from the group consisting of colon cancer, stomach cancer, pancreatic cancer, lung cancer, gall cancer, breast cancer, liver cancer, blood cancer, thyroid cancer, bladder cancer, lymphoma, ovarian cancer, endometrial cancer, brain tumor, have. Specifically, according to one embodiment of the present invention, the cancer may be, for example, colorectal cancer, breast cancer, ovarian cancer, lung cancer or lymphoma.

A lesion refers to a tissue that has been altered by the pre-onset state of a tumor or cancer or by the onset of a tumor or cancer. But may be, but not limited to, precancerous lesions, benign lesions or malignant lesions, depending on the severity of the tumor or cancer.

The present invention also relates to a method of amplifying a tumor or a lesion thereof, comprising: i) an oligonucleotide capable of amplifying a DNA fragment derived from a tumor or a lesion thereof, and ii) an oligonucleotide capable of amplifying a normal cell- To a composition for analyzing a lesion.

The present invention also relates to a cancer diagnostic composition comprising DNA isolated from a biological sample, the oligonucleotide capable of amplifying a DNA fragment derived from the tumor or a lesion thereof, and ii) an oligonucleotide capable of amplifying a DNA fragment derived from a normal cell .

The present invention also relates to a method of amplifying a tumor or a lesion thereof, comprising: i) an oligonucleotide capable of amplifying a DNA fragment derived from a tumor or a lesion thereof, and ii) an oligonucleotide capable of amplifying a normal cell- And a kit for analyzing a lesion.

The present invention further provides a cancer diagnostic kit comprising an oligonucleotide capable of amplifying a DNA fragment derived from a tumor or a lesion thereof from DNA isolated from a biological sample, and ii) an oligonucleotide capable of amplifying a DNA fragment derived from a normal cell, .

The description of the composition according to the present invention and the constitution related to each of the kit can be applied in the same manner as the constitution described in carrying out the method.

The kits may contain various buffers and reagents, and the optimal amounts of reagents, buffers or reagents used for a particular reaction may be determined by those skilled in the art. Each configuration included in the kit may be made in a separate package or compartment.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Purification of non-cell DNA from plasma of colon cancer, colon polyp, and non-tumor (normal) group

A total of 72 patients with gastrointestinal endoscopy and pathologists were classified into colorectal cancer, colorectal cancer, and non - tumor (normal) group based on colonoscopy and cytology / histopathology. Colon cancer patients were diagnosed as adenomas in colonoscopy and diagnosed as adenomas in histologic examination. Patients diagnosed or treated for cancer in other organs were excluded. The colon adenocarcinoma was diagnosed as having a size of 1 ㎝ or more in colonoscopy and excluded those who were found to have dysplasia or tumor in polypectomy. The non - tumor group was diagnosed with colorectal endoscopy, normal diagnosis or irritable bowel syndrome within 1 year, and was excluded from the history of chemotherapy or treatment. After obtaining the approval of MD17051, 2017.10.25.), The study consent was obtained and the clinical tissue sample and the blood sample were collected, and the clinical sample was taken to the human origin bank of the hospital. Was deposited. Subsequently, samples containing plasma samples (average of 5 cc plasma per 1 study group / total of 351 tubes divided by 1 mL / tube) were distributed according to institutional procedures (approval number KU Guro Gene Bank 2018-005). In order to isolate the tumor-derived DNA from the plasma sample first, the cells were centrifuged at 16,000 xg for 5 minutes for 5 minutes, and then the QIAamp circulating nucleic acid kit (Qiagen, Cat. No 55114) was prepared according to the manufacturer's manual Pure separation. The purely isolated tumor-derived DNA and genomic DNA concentrations were quantified using Quantus fluorometer instrument and QuantiFluor dsDNA system reagent (Promega, Cat. No. E2670).

Example 2. Pure isolation of exosome DNA from plasma of colon cancer, colon polyp, and non-tumor (normal) group

100 [mu] L of 1X PBS buffer was added to 200 [mu] l of the plasma sample prepared in the same manner as in Example 1 and stirred. After addition of 10 μL of protease K (20 mg / mL), the plasma protein was degraded at 37 ° C for 10 minutes. Thereafter, 300 μL of exosomal precipitation solution (final 16% of polyethylene glycol (molecular weight 6,000), 1M sodium chloride) was added to the reaction solution, and homogeneously mixed with stirring, followed by precipitation reaction at 4 ° C. for 15 hours. The supernatant was discarded by centrifugation at 12,000 x g for 10 minutes, and the exosome precipitate was recovered. Exosome DNA from each precipitate was purified by MagmaX cell-free DNA isolation kit (Thermofisher, Cat. No A29319) according to the manufacturer's manual.

Example 3. Tumor and / or lesion-derived DNA and normal killing DNA analysis Oligonucleotide design

DNA fragments detected in blood plasma include non-cell DNA derived from tumor and / or lesion and DNA fragments originating from apoptosis of normal cells. The ALU repeating sequence units (about 280 bases), which account for about 13% of the human genome sequence and act on the molecular mechanism of disease development including solid tumors, are truncated in the normal killing process, Oligonucleotides were designed to amplify them with high reproducibility in the presence of less than 167 basepairs. In addition, the tumor and / or lesion-derived DNA is introduced into the bloodstream through cell necrosis rather than the normal killing process, which is longer than the normal killing DNA fragment, so that a plurality of oligonucleotides having a length corresponding to the ALU repeat sequence unit are designed Respectively. The human standard genomic sequence was based on GRCh37 / hg19, the consensus sequence containing the Ya5 and Yb8 sub-family of the ALU repeat sequence as a template, the oligonucleotide for analysis as primer primer 6.0 (Premier BioSoft, USA) Designed using software Primer 3, Table 3 shows.

[Table 3]

^* Y = C / T, ^† W = A / T, ^‡ K = G / T, ^** R = G / A

Example 4. Quantitative PCR analysis of tumor and / or lesion-derived DNA and normal-dead DNA fragments from plasma of colorectal, colon, non-tumor (normal)

Using the circular non-cell DNA or exosome DNA of colon cancer, polyp, and non-tumor group purely isolated through Examples 1 and 2 as amplification templates and using the oligonucleotides of Table 1, the DNA of tumor and / or lesion- Normal dead DNA fragments were analyzed by real-time quantitative PCR (polymerase chain reaction). Normal dead DNA fragments were amplified with oligonucleotide 1 and 3 (118 base pair) combinations and tumor and / or lesion-derived DNA fragments were amplified with oligonucleotide 4 and 5 (270 base pair) combinations. The DNA standard material established a standard curve using human control genomic DNA (Thermofisher, Cat. No. 4312660) in the 1.0, 0.1, 0.01, 0.001, 0.0001, 0.00001 pg / μL concentration range. The detailed procedure of quantitative PCR is as follows. 3.0 μL of circulating non-cellular DNA (at least 5 pg), 10.0 μL of PowerSYBR Green PCR Master Mix (Thermo Fisher, Cat. No. 4367659), 0.48 μL of oligonucleotide combination (final 10 μM) for each DNA fragment amplification, The reaction mixture was mixed with sterilized water (6.52 μL) to prepare a reaction solution. The reaction mixture was placed in a real-time quantitative PCR instrument (Thermo Fisher, AB 7500 FAST model) and incubated at 95 ° C for 10 minutes, once at 95 ° C for 15 seconds, Amplification reaction was performed. The presence or absence of nonspecific reaction products was confirmed by the melting curve analysis after the amplification reaction. Representative examples of quantitative analysis results of tumor and / or lesion-derived DNA and normal-dead DNA fragments from circulating non-cellular DNA of colon cancer and non-tumor normal group are shown in FIG. 2, Table 4 and Table 5. Based on the descriptive statistics of Mann-Whitney test, we have tried to find the reference value for discriminating colorectal cancer and non-tumor normal group and obtained the accuracy results as shown in Table 5 at cut-off 0.330.

[Table 4]

* Sample: Plasma sample of cancer patient

[Table 5]

Example 5. Quantitative PCR analysis of tumor and / or lesion-derived DNA and normal-dead DNA fragments from plasma of cancer patient and non-tumor control group

Using the exosome DNA of cancer patients (colon cancer, breast cancer, ovarian cancer, lung cancer, lymphoma) and non-tumor control which were purely isolated in the same manner as in Examples 1 and 2 as amplification templates and using the oligonucleotides of Table 1, / Or lesion-derived DNA and normal-killing DNA fragments were quantitatively analyzed by PCR. Genomic DNA fragments were amplified in combination with oligonucleotides 2 and 3 (107 base pairs), and tumor and / or lesion-derived DNA fragments were amplified in combination with oligonucleotides 4 and 5 (270 base pairs). The DNA standard material established a standard curve using human control genomic DNA (Thermofisher, Cat. No. 4312660) in the 1.0, 0.1, 0.01, 0.001, 0.0001, 0.00001 pg / μL concentration range. The detailed procedure of quantitative PCR is as follows. 3.0 μL of exosome DNA (minimum 5 pg or more), 10.0 μL of PowerSYBR Green PCR Master Mix (Thermo Fisher, Cat. No. 4367659), 0.48 μL of oligonucleotide combination (final 10 μM) for amplification of each DNA fragment, deionized sterilized water The reaction mixture was placed in a real-time quantitative PCR instrument (Thermo Fisher, AB 7500 FAST model) by mixing 6.52 μL of the reaction mixture, followed by 45 cycles of 95 ° C. for 10 minutes, 95 ° C. for 15 seconds, Respectively. The presence or absence of nonspecific reaction products was confirmed by the melting curve analysis after the amplification reaction. Representative examples of quantitative analysis of tumor and / or lesion-derived DNA and normal-dead DNA fragments from exosome DNA isolated from peripheral blood plasma of cancer patient and non-tumor control group are shown in FIG. 3, Table 6 and Table 7. Based on the descriptive statistics of the Mann-Whitney test, the reference values for discriminating between cancer patients and non-tumor controls were searched and the accuracy results as shown in Table 7 were obtained at a cut-off of 0.250.

[Table 6]

* Sample: Plasma sample of cancer patient

[Table 7]

Example 6. Quantitative concentration and index analysis of tumor and / or lesion-derived DNA and normal-dead DNA fragments from quantitative PCR data

The quantitative analysis of the quantities of the tumor or its lesion-derived DNA and the normal cell-derived DNA fragment from the PCR data of the tumor according to an embodiment of the present invention and the analysis of the index thereof are as follows. Once the real-time quantitative PCR analysis is complete, amplification data containing the Ct value is extracted from the SDS analysis program of the PCR machine. The concentration standard curve of the DNA derived from the tumor or its lesion and the DNA derived from normal cells was set using the Ct value of the 6 dilution concentration sections (3-repeat) of the DNA standard material, and the ratio - Converting cell DNA or exosome DNA concentration to a quantitative concentration (pg / uL). The index based on the concentration ratio of the DNA fragments derived from the tumor or the lesion thereof to the DNA fragments derived from normal cells is calculated on the basis of the data on the distribution range of the tumor type and / or the lesion-derived DNA distribution range and the normal killing DNA concentration. (Receiver Operation Characteristic) curve analysis is used to express and evaluate the characteristics of the index to discriminate between the tumor group and the normal group, ie, the sensitivity and the 1-specificity curve. Sensitivity and specificity are trade-off between each other, so the sum of the two values is always 1, so the specificity decreases when the sensitivity increases. ROC curve analysis showed that the sensitivity and specificity change with changes in the index cut-off, and thus the reference value with optimal sensitivity, specificity, positive predictive value, negative predictive value, positive bias ratio, negative likelihood ratio (cut -off), and the accuracy is evaluated by Area Under the ROC Curve (AUC).

In the case of Example 5, each DNA concentration was quantified by substituting the tumor grade and / or lesion-derived DNA Ct value and the normal death DNA Ct value into the concentration gradient standard curve of the DNA standard material. After calculating the index, ROC curve analysis was performed using SPSS Statistics 22 (IBM, USA) and MedCalc 14.8.1 (MedCalc Software, Belgium) statistical program. AUC was 0.82, sensitivity was 80%, specificity was 84.62%, positive predictive value was 66.67%, negative predictive value was 91.67%, positive evil ratio was 5.20, and negative likelihood ratio was 0.24 when the cut-off was 0.250. The 95% confidence interval range for the normal group according to Example 5 was 0.205 - 0.299 and 0.284 - 0.688 for tumor or cancer. There may be differences in the DNA concentration interval and index cut-off (threshold) values between tumor and malignant tumor types, tumor-derived or lesion-derived DNA, normal dead DNA concentration range and index range.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents

<110> EROM co., Ltd <120> Method for Screening Cancer By Analyzing Index of Tumor-Derived          DNA <130> 267 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 ygaggtcagg agwtcgagac ca 22 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gwtcgagacc akccyggc 18 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 agcctcccya gtagctggga r 21 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 cgcrgtggct cacgcc 16 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ggagtctcgc tctgtcgcc 19 <210> 6 <211> 298 <212> DNA <213> Artificial Sequence <220> <223> Alu consensus sequence <400> 6 ggccgggcgc rgtggctcac gcctgtaatc ccagcacttt gggaggccga ggcgggcgga 60 tcannygagg tcaggagwtc gagaccakcc yggctaacan ggtgaaaccc cgtctctact 120 aaaaatacaa aaaattagcc gggcgtggtg gcgggcgcct gtartcccag ctactyggga 180 ggctgaggca ggagaatggc gtgaacccgg gaggcggagc ttgcagtgag ccgagatcgc 240 gccactgcac tcnncannnn ngcctgggcg acagagcgag actccgtctc aaaaaaaa 298 <210> 7 <211> 119 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 7 ygaggtcagg agwtcgagac cakccyggct aacanggtga aaccccgtct ctactaaaaa 60 tacaaaaaat tagccgggcg tggtggcggg cgcctgtart cccagctact ygggaggct 119 <210> 8 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 8 gwtcgagacc akccyggcta acanggtgaa accccgtctc tactaaaaat acaaaaaatt 60 agccgggcgt ggtggcgggc gcctgtartc ccagctacty gggaggct 108 <210> 9 <211> 278 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 9 cgcrgtggct cacgcctgta atcccagcac tttgggaggc cgaggcgggc ggatcannyg 60 aggtcaggag wtcgagacca kccyggctaa canggtgaaa ccccgtctct actaaaaata 120 ggggggggg gcaggagaat ggcgtgaacc cgggaggcgg agcttgcagt gagccgagat cgcgccactg 240 cactcnncan nnnngcctgg gcgacagagc gagactcc 278

Claims (18)

A method of providing information for the analysis of a tumor or lesion thereof comprising the steps of:
(a) i) DNA isolated from a biological sample;
ii) an oligonucleotide capable of amplifying a tumor comprising a consensus sequence comprising the Alu Ya5 or Yb8 subfamily or a DNA fragment derived from the lesion thereof selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and normal cell- Real-time quantitative amplification of oligonucleotides capable of amplifying the fragments;
(b) measuring the Ct (threshold cycle) value of the DNA fragment derived from the tumor or its lesion and the DNA fragments derived from the normal cell in the step (a) and determining the concentration; And
(c) determining an index, which is a ratio of the concentration of the DNA fragments derived from the tumor or a lesion thereof, to the amplified DNA fragments derived from normal cells, and determining the tumor as a tumor with the determined index being equal to or greater than a reference value (cut-off). 2. The method of claim 1, wherein the DNA isolated from the biological sample comprises circulating non-cellular DNA and / or exosome DNA. delete The method according to claim 1, wherein the concentration of the DNA fragment in the step (b) is determined by comparing the concentration of the DNA fragment with a standard curve determined based on the Ct value for the DNA standard material. delete delete The method of claim 1, wherein the reference value is 0.284 - 0.688 obtained through ROC (Receiver Operation Characteristic) curve analysis. 2. The method of claim 1, wherein the tumor comprises solid cancer or sporadic solid cancer. 1) to SEQ ID NO: 5 in a DNA isolated from a biological sample, i) an oligo Ya5 or Yb8 subfamily comprising a consensus sequence or an oligo Nucleotide and ii) an oligonucleotide capable of amplifying a DNA fragment derived from a normal cell. 10. The composition of claim 9, wherein the DNA isolated from the biological sample comprises circulating non-cellular DNA and / or exosome DNA. delete delete delete (I) a consensus sequence comprising the Alu Ya5 or Yb8 subfamily selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5 in DNA isolated from the biological sample, i) amplifying the tumor or DNA fragment derived from the lesion thereof And ii) an oligonucleotide capable of amplifying a normal cell-derived DNA fragment. 15. The kit of claim 14, wherein the DNA isolated from the biological sample comprises circulating non-cellular DNA and / or exosome DNA. delete delete delete

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