KR101751962B1 - Use of Cell-Free DNA for Diagnosing Gastric Cancer - Google Patents
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- KR101751962B1 KR101751962B1 KR1020150061790A KR20150061790A KR101751962B1 KR 101751962 B1 KR101751962 B1 KR 101751962B1 KR 1020150061790 A KR1020150061790 A KR 1020150061790A KR 20150061790 A KR20150061790 A KR 20150061790A KR 101751962 B1 KR101751962 B1 KR 101751962B1
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Abstract
The present invention relates to a new use for the diagnosis of gastric cancer of certain cell-free DNA. The cell-free DNA of the present invention can be easily obtained from body fluids and can be used as a new biomarker for gastric cancer diagnosis which can be easily detected by a gene amplification method. The cell-free DNA of the present invention can accurately diagnose stomach cancer in early stage, especially as a biomarker specific to early-stage gastric cancer. The cell-free DNA of the present invention can be usefully used for early stage gastric cancer diagnosis, evaluation of gastric cancer progression, and evaluation of the performance and suitability of anticancer drugs for gastric cancer treatment.
Description
The present invention relates to the use of cell-free DNA for gastric cancer diagnosis.
Cancer is the number one cause of death in Koreans, despite the rapid development of cancer diagnosis and treatment methods in recent years. Gastric cancer is the most common cancer in Korea, the second most common cancer. According to the cancer death rate surveyed in 2012, 33.1 people died of lung cancer per 100,000 population; 22.5 deaths from liver cancer; 18.6 patients died from gastric cancer; And 16.3 patients died of colon cancer. Among cancer deaths, the number of patients who died of gastric cancer was the second most common. The economic loss due to cancer treatment is known to be 1.1 trillion won, which is 1.5% of gross domestic product. As social costs increase, individuals and societies are in a situation where it is difficult to cover costs. During the past 30 years, research on cancer has been rapidly advancing and many cancer-specific oncogenes or cancer-suppressing genes have been identified and new therapeutic methods have been proposed through research on the function / mechanism of these genes. However, Homework remains. The prevalence of gastric cancer in Korea is increasing every year. Therefore, it is required to develop a diagnostic method for screening the genetic factors of gastric cancer. Gastric cancer is a malignant (cancerous) cell found in the stomach tissue. It is a decline in the western world, but it is the most prevalent malignant tumor in the Asian region, especially in Korea.
Exosomes present in the blood contain small amounts of protein, RNA, and DNA, so that changes in the genome and proteome of the cells can be identified by analyzing the fractions. Recently, peptides, lipids, microRNAs, mRNA and cell-free DNA (cfDNA) have been actively studied in the secretory body, and the onset of cancer, inflammation and heart There are many reports that it can be applied as a biomarker of disease. In particular, cfDNA is more stable at room temperature than RNA or microRNA and is suitable for diagnostic tests. The cfDNA may be used in the treatment of pediatric lymphoma, breast cancer, non-small-cell lung carcinoma, prostate cancer, ovarian cancer, glial tumor, It has been reported that the level of rectal cancer is higher than that of rectal cancer. However, no studies have been reported on the level of cfDNA in gastric cancer.
The patent documents and references cited herein are hereby incorporated by reference to the same extent as if each reference was individually and clearly identified by reference.
The present inventors have sought to find new biomarkers useful for the early diagnosis of gastric cancer from biological substances present in blood exosomes. As a result, it has been experimentally confirmed that a specific cell-free DNA present in the blood circulation appears to be a significantly higher level in gastric cancer patients, and it is proved that the gastric cancer can be used as a biomarker for diagnosis Thereby completing the present invention.
It is an object of the present invention to provide a composition for the diagnosis of gastric cancer, which comprises, as an active ingredient, a preparation capable of detecting a specific cell-free DNA.
Another object of the present invention is to provide a diagnostic kit for gastric cancer comprising the composition as an active ingredient.
It is still another object of the present invention to provide a biomarker for diagnosis of gastric cancer including cell-free DNA.
It is another object of the present invention to provide a method for detecting cell-free DNA to provide information necessary for diagnosis of gastric cancer.
Other objects and technical features of the present invention will be described in more detail with reference to the following detailed description, claims and drawings.
According to one aspect of the present invention, the present invention includes an agent capable of detecting cell-free DNA having the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, or 9 as an active ingredient The composition for gastric cancer diagnosis.
As used herein, the term " cell-free DNA, cfDNA " refers to a fragment DNA that circulates in human body fluids. The cell-free DNA is mostly double-stranded DNA and may exist in the form of a nuclear protein complex. Since the cell-free DNA is observed in various physiological and pathological conditions such as inflammatory disorder, oxidative stress, and malignant tumor, it can be used as a useful biomarker for diagnosing disease and predicting prognosis.
In the present invention, the cell-free DNA having the nucleotide sequence disclosed in SEQ ID NO: 5 is a cell-free DNA derived from the ENTPD6 (ectonucleoside triphosphate diphosphohydrolase 6) gene. As used herein, the term "ENTPD6 cfDNA &Quot;
In the present invention, the cell-free DNA having the nucleotide sequence shown in SEQ ID NO: 6 was detected by the microarray probe A_16_P0178165, and the
In the present invention, the nucleotide sequence shown in SEQ ID NO: 7 is a sequence in which the 14th nucleotide in the nucleotide sequence shown in SEQ ID NO: 5 is deleted.
In the present invention, the nucleotide sequence shown in SEQ ID NO: 8 is a sequence in which the 14th nucleotide and the 24th nucleotide are deleted in the nucleotide sequence shown in SEQ ID NO: 5.
In the present invention, the nucleotide sequence shown in SEQ ID NO: 9 is a sequence in which the 14th nucleotide in the nucleotide sequence shown in SEQ ID NO: 5 is deleted and the 105th nucleotide is [T / A] (W).
According to an embodiment of the present invention, the agent capable of detecting the cell-free DNA is a primer or a probe having a sequence complementary to the nucleotide sequence.
The term " complementary " as used herein means having complementarity enough to selectively hybridize to the nucleotide sequence under any particular hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from perfectly complementary, and the primer or probe of the present invention may optionally hybridize to any one of the nucleotide sequences set forth in SEQ ID NOS: 5 to 0 If possible, one or more mismatch nucleotide sequences may be present.
As used herein, the term " probe " refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, which can specifically hybridize to a target nucleotide sequence, Or artificially synthesized. The probe is preferably single-stranded. Preferably, the probe of the present invention is oligodeoxyribonucleotide. The probes can include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogs or derivatives. In addition, the probe of the present invention may also include a ribonucleotide. For example, the probes of the present invention can be used in a variety of ways, including but not limited to skeletal modified nucleotides such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA 2-O-methyl RNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides with nucleotide modifications, such as C-5 substituted pyrimidines, wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, (Including fluorine, bromine, chlorine, bromine, chlorine, bromine, and iodine) with a C-7 substituent, Ah Goto -, methyl-, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine. The probe may additionally be labeled by a radioactive label, a fluorescent label, an enzyme label, a sequence label, or biotin.
As used herein, the term " primer " refers to a single-stranded DNA strand that can act as a starting point for template-directed DNA synthesis under suitable conditions (i. E., Four other nucleoside triphosphates and polymerase) Means an oligonucleotide. The suitable length of the primer varies depending on various factors such as temperature and use of the primer. The primer sequence need not have a sequence completely complementary to a part of the template, and it is sufficient that the primer sequence has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer of the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence as a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing to the nucleotide sequence and acting as a primer. The primers of the present invention may additionally be labeled with a radiolabel, a fluorescent label, an enzyme label, a sequence label, or biotin.
According to one embodiment of the present invention, the primer is used in an amplification reaction. The amplification reaction refers to a reaction for amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art and include polymerase chain reaction, reverse transcription polymerase chain reaction, real-time polymerase chain reaction, Ligase chain reaction, repair chain reaction, transcription-mediated amplification, self sustained sequence replication, selective amplification of the target polynucleotide sequence, amplification of the target polynucleotide sequence, a selective amplification of target polynucleotide sequences, a consensus sequence primer polymerase chain reaction, an arbitrarily primed polymerase chain reaction, a nucleic acid sequence based amplification ), Strand displacement amplification ), And loop-mediated isothermal amplification.
According to another embodiment of the present invention, the probe of the present invention is used in a microarray chip.
According to another embodiment of the present invention, the gastric cancer to be diagnosed in the present invention is an early stage gastric cancer. The stage (stage) of stomach cancer was determined based on the degree of invasion of the stomach wall, the degree of metastasis to the surrounding lymph nodes, and metastasis to other organs such as the liver, peritoneum or lung, Or four. According to the International Staging of Gastric Cancer (American Joint Committee on Cancer 7th edition, 2010), stomach cancer is classified as T0 if there is no evidence of tumor according to the depth of involvement of primary cancer. When the tumor invades the mucosal layer or submucosal layer of the gastric wall, T1; T2 when the tumor invades the muscle layer or subserosa; T3 when the tumor invaded the serous layer but did not invade the surrounding organs; And T4 if the tumor penetrates the serous layer and invades organs around the spleen, transverse colon, liver, diaphragm, pancreas, abdominal wall, adrenal gland, kidney, small intestine, or retroperitoneum. In the absence of stomach-related lymph node metastasis according to lymph node metastasis, N0; N1 if there is 1 to 2 perigastric lymph node metastases; 3 to 6 N2 if there is gastric lymph node metastasis; If there is more than 7 gastric lymph node metastases, it is classified as N3a or N3b. If there is no distant metastasis, it is classified as M0. If distant metastasis is present, it is classified as M1.
In the present invention, stomach cancer at an early stage refers to stomach cancer corresponding to stage T1.
Gastric cancers classified as T1 are early gastric carcinoma (T1) and advanced gastric carcinoma (T1). The T1 advanced gastric cancer refers to a gastric cancer which only invades the submucosal layer unlike the common advanced gastric cancer of T2, T3 or T4 which infiltrates into the muscle layer.
According to another aspect of the present invention, there is provided a diagnostic kit for gastric cancer comprising the composition described above.
The diagnostic kit of the present invention may be a kit for polymerase chain reaction (PCR) or a microarray chip.
The kit for polymerase chain reaction may contain reagents necessary for PCR amplification, such as a buffer, a DNA polymerase, a DNA polymerase enzyme, and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.
The probe of the present invention can be used in a microarray chip. In the microarray chip, the probe is used as a hybridizable array element and immobilized on a substrate. Such supports include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the support. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element may be coupled to a support via linkers (e.g., ethylene glycol oligomers and diamines).
According to another aspect of the present invention, there is provided a diagnostic biomarker for gastric cancer comprising cell-free DNA having the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, or 9 do.
According to another aspect of the present invention, the present invention provides a method for producing a cell-free DNA having the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, or 9 Is detected.
According to an embodiment of the present invention, the method for detecting cell-free DNA in the present invention comprises the following steps:
(a) separating cell-free DNA from a biological sample; And
(b) a primer set consisting of the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 or a primer set consisting of oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4, using the separated cell- And performing a polymerase chain reaction (PCR).
Hereinafter, the method of the present invention will be described step by step.
Separating the cell-free DNA from the biological sample
First, prepare a biological sample for the isolation of cell-free DNA. The biological sample is a body fluid, and the cell-free DNA may be contained in exosome in the body fluid. The fraction is a 30-100 nm sized vesicle containing DNA, RNA, or protein. The body fluid may be a blood, urine, feces, saliva, lymph fluid, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, amniotic fluid, chorionic villus sample, placenta sample, lavage, ocular fluid). Most preferably, the body fluid is blood, plasma or serum.
The cell-free DNA is isolated from the biological sample. The cell-free DNA can be isolated by separating the exosome from the body fluid and extracting, separating and purifying the nucleic acid from the separated fractions.
Methods for detecting cell-free DNA
After cell-free DNA is isolated and purified, cell-free DNA having the nucleotide sequence disclosed in SEQ ID NOS: 5, 6, 7, 8, or 9 is detected.
In the present invention, the cell-free DNA may be detected by a gene amplification method or a microarray method.
When the cell-free DNA is detected by the gene amplification method, the separated cell-free DNA is used as a template and a primer set consisting of the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, 3 and the oligonucleotide of SEQ ID NO: 4 is used to perform a polymerase chain reaction (PCR).
According to another embodiment of the present invention, additional sequence analysis can be performed on the PCR product after the PCR.
The features and advantages of the present invention are as follows.
(i) The present invention provides a new use for the diagnosis of gastric cancer of certain cell-free DNA.
(Ii) The cell-free DNA of the present invention is a novel biomarker for gastric cancer diagnosis which can be easily obtained from body fluids and can be easily detected by a gene amplification method.
(Iii) The cell-free DNA of the present invention can accurately diagnose stomach cancer in early stage, especially as a biomarker specific to early gastric cancer.
(Iv) The cell-free DNA of the present invention can be usefully used for early stage gastric cancer diagnosis, evaluation of gastric cancer progression, and evaluation of the performance and suitability of anticancer drugs for gastric cancer treatment.
The present invention relates to a new use for the diagnosis of gastric cancer of certain cell-free DNA. The cell-free DNA of the present invention can be easily obtained from body fluids and can be used as a new biomarker for gastric cancer diagnosis which can be easily detected by a gene amplification method. The cell-free DNA of the present invention can accurately diagnose stomach cancer in early stage, especially as a biomarker specific to early-stage gastric cancer. The cell-free DNA of the present invention can be usefully used for early stage gastric cancer diagnosis, evaluation of gastric cancer progression, and evaluation of the performance and suitability of anticancer drugs for gastric cancer treatment.
Figure 1 shows CGH array hybridization and comparison of cfDNA in gastric cancer patients. Panel 1) shows the CGH array hybridization comparative analysis scheme. Panel 2) shows the DNA region classification commonly observed in stages of stomach cancer progression. Panel 3) shows a common DNA region classification in Dye swap analysis. The cutoff of Dye swap analysis is log ratio ≥ 1 or ≤ -1. s is serum; p is plasma; T1_e is T1 early carcinoma; T1_a is T1 advanced carcinoma; T2_a is T2 advanced carcinoma; And T3_a: T3 advanced carcinoma, respectively.
Fig. 2 shows the melting curve analysis result of PCR-amplified ENTPD6 cfDNA. x-axis is the melting temperature; The y-axis is the first derivative of the change in fluorescence (dF / dT) with respect to the change in fluorescence intensity with time; And Ct are cycle thresholds, respectively.
Fig. 3 shows the melting curve analysis result of PCR-amplified A_16_P01781625 cfDNA. x-axis is the melting temperature; The y-axis is the first derivative of the change in fluorescence (dF / dT) with respect to the change in fluorescence intensity with time; And Ct are cycle thresholds, respectively.
Figure 4 shows nucleotide sequence analysis results for the ENTPD6 cfDNA of the present invention.
Figure 5 shows nucleotide sequence analysis results for the A_16_P01781625 cfDNA of the present invention.
Example
Materials and Methods
1. Approval of clinical trial screening committee and test sample distribution
The experiments of the present invention were approved by the Institutional Review Board of Chungbuk National University (CBNU-201412-BMBR-108-01). All experimental samples were given sera or plasma from stage 1 (T1), 2 (T2) and 3 (T3) gastric cancer patients from Severance Hospital Gene Bank.
2. Extraction and quantification of cell-free DNA of blood
1, 2, or 3 stomach cancer patients by precipitation of blood exosomes using miRCURY ™ RNA Isolation Kits - Exosome Isolation (Exiqon, Denmark) from 100 μl of serum or 100 μl of plasma. The obtained blood fraction was suspended in a resuspension buffer, and a DNA extraction solution prepared by mixing phenol, chloroform and isoamyl alcohol in a ratio of 25: 24: 1 was added, Cell-free DNA (cfDNA) was extracted from the body. The extracted cfDNA was subjected to qualitative and quantitative analysis using a NanoDrop spectrometer (ND-1000, Micro-Spectrophotometer).
3. Amplification of cell-free DNA
CfDNA was amplified using a genomePlex whole genome amplification kit (Sigma-Aldrich, USA). 10Ox fragmentation was added to 500 ng of the amplified cfDNA, reacted at 95 DEG C and placed on ice. 1x library preparation buffer, library stabilization solution and library preparation enzyme were added to the reaction solution of the above reaction, and the mixture was sequentially incubated at 16 ° C for 20 minutes; 20 minutes at 24 占 폚; 20 minutes at 37 占 폚; And an isothermal reaction was performed under the condition that the reaction was allowed to proceed at 75 ° C for 5 minutes and then the reaction solution was held at 4 ° C. The PCR reaction was carried out by adding 10x amplification master mix and whole genome amplification DNA polymerase to the reaction solution. The PCR reaction was sequentially performed at 95 캜 for 3 minutes; 15 seconds at 94 占 폚; And 65 ° C for 5 minutes for 1 cycle, and the PCR reaction solution was maintained at 4 ° C. The PCR product was purified using a GenElute PCR Clean-up kit and quantitated using a NanoDrop spectrometer (ND-1000).
4. Comparative Genomic Hybridization (CGH) Microarray
SurePrint G3 human genome comparison A genomic hybridization (CGH) microarray was used to determine from which chromosome the amplified cfDNA was derived. The SurePrint G3 human genome CGH microarray (4 x 180K) contains 170,334 biological features; Replicates (X5) of 1,000 biological probes; And 6,539 internal quality control features and uses the genome source of UCSC hg18 (NCBI Build 36, March 2006) and 13 kb (11 KB in Ref. Seq genes) probe spacing probe spacing. The fluorescent labeling for the cfDNA was performed as follows. A random primer, 5 × reaction buffer, 10 × dNTPs, cyanine 3-dUTP or cyanine 5-dUTP and Exo (-) cleavage enzyme were added to 2 μg of the cf DNA, At 37 [deg.] C for 2 hours; And reacted at 65 ° C for 10 minutes, and then maintained at 4 ° C. The fluorescently labeled cfDNA was purified and quantified using a NanoDrop spectrometer. The SurePrint G3 human genome CGH microarray reaction was performed as follows. Cot-1 DNA (1.0 mg / ml), 10x aCGH blocking reagent, and 2x HI-RPM hybridization buffer were added to the fluorescent-labeled cfDNA, followed by sequencing at 95 ° C for 3 minutes; And allowed to react at 37 ° C for 30 minutes and then maintained at 4 ° C. The reaction solution was injected into an array gasket, and the hybridization reaction was carried out at 65 ° C for 24 hours. The hybridized chip was washed using Agilent Oligo-aCGH / ChIP-on-
5. Design of real-time PCR primer for classification of DNA region and analysis of melting curve according to progression stage of gastric cancer
Classification of DNA regions according to the progression stage of gastric cancer was performed through screening of genes commonly observed from samples of male and female patients of serum and plasma or stage of progression of gastric cancer for each stage of gastric cancer. For example, the CGH microarray results were analyzed by the Dye-swap method, and the genes of the stomach cancer progressing stages were compared with each other using a venn diagram to select commonly observed genes. The selection of genes for T2 and T3 was based only on the genes that were detected in the middle stage of gastric cancer and commonly observed in T2 and T3 stages. Real-time PCR primers for melting curve analysis of the selected genes were designed based on the probe nucleotide sequence (60mer) of the genes identified in the CGH microarray. A gene corresponding to the probe nucleotide sequence was searched using GenBank, and sequence alignment was performed on the nucleotide sequence of the searched gene and the nucleotide sequence of the probe. A primer was designed to have an amplicon size of 113 bp-183 bp centered on the nucleotide sequence of the probe. In addition, M13 forward (5'-GTTTTCCCAGTCACGAC-3 ') was inserted forwardly at the 5' It was designed to enable sequencing of nucleotides of ricons.
6. Real-time PCR and analysis of the melting curve for cfDNA
Sex, serum, plasma, and gastric cancer stage, and performed melting curve analysis. Real-time PCR was performed by mixing 50ng of cfDNA, 2x HRM PCR master mix and 10 mu M primer and sequentially sequencing at 95 ° C for 5 minutes; 95 ° C for 10 seconds; 30 seconds at 55 占 폚; And 72 < [deg.] ≫ C for 30 seconds as one cycle. The high resolution melting (HRM) confirmation step was performed by increasing the temperature from 70 ° C to 85 ° C by 0.1 ° C in a Rotorgene Q instrument. The result is a Ct (threshold cycle) value of 30 or less; A single peak melting curve; And a first derivative of the change in fluorescence F / dT with respect to a change in fluorescence intensity over one or more times was analyzed as normal amplification.
7. Analysis of DNA nucleotide sequences
For melting curve analysis, real - time PCR amplicons were purified by sex or staging stage of gastric cancer. Nucleotide sequencing of the amplicon was performed using M13 forward. The nucleotide sequence analysis may include purification of the amplicon; A cycle sequencing reaction step; Dye terminator Purification of the reaction product; And an ABI3730XL sequencer analysis step. The nucleotide sequence analysis was performed as follows. A
8. Statistical Analysis
The significance of cfDNA extraction by sex, serum, plasma and stomach progression stage was analyzed by Student T test.
Experiment result
1. Blood samples from gastric cancer patients
The patients were divided into two groups according to the age of the patients: T1 early gastric carcinoma (T1_e), T1 advanced gastric carcinoma (T1_a), T2 advanced gastric carcinoma (T2_a) A total of 100 cases of T3 advanced gastric carcinoma (T3_a) patients with serum and plasma were randomly assigned (Table 1). T1_e stage male patients were 54 to 74 years old, T1_e stage female patients were 39 to 50 years old, T1_a stage male patients were 56 to 83 years, T1_a stage female patients were 63 to 76 years, T2_a stage Of male patients are between the ages of 43 and 77 years, those of T2_a stage are between 39 and 81 years, those of T3_a stage are between 40 and 74 years, and those of T3_a stage are between 34 and 72 years old.
2. Quantification of cfDNA
Quantitative analysis of cfDNA was performed. The amount of purified cfDNA from T1_e stage gastric cancer patient is as follows. Serum of male patients: 444.2 ± 264.6ng; Serum of female patients: 3795.1 ± 710.3ng; Plasma of male patients: 486.2 ± 648.4ng; And plasma of female patients: 6131 ± 646.9ng. The amount of purified cfDNA from gastric cancer patient in T1_a stage is as follows. Serum of male patients: 385.0 ± 331.1ng; Serum of female patients: 4048.9 ± 3666.3ng; Plasma of male patients: 30.6 ± 293.8ng; And plasma of the female patient: 1627.5 ± 1583.2ng. The amount of cfDNA purified from gastric cancer patient in T2_a stage is as follows. Serum of male patients: 578.5 ± 282.2ng; Serum of female patients: 387.6 ± 92.2ng; Plasma of male patients: 497.9 ± 306.2ng; And plasma of female patients: 453.7 ± 182.5ng. The amount of cfDNA measured from patients with gastric cancer at stage T3_a is as follows. Serum of male patients: 704.8 ± 243.8ng; Serum of female patients: 789.7 ± 140.4ng; Plasma of male patient: 583.7 ± 341.2ng; And plasma of female patients: 2816.5 ± 1348.2ng. As a result of the above results, there was no statistically significant difference in cfDNA release of blood between male and female, serum and plasma or gastric cancer progression stage, but in case of male patient serum, cfDNA emission was increased with progression of gastric cancer.
3. Analysis of DNA region by CGH microarray analysis
For the CGH microarray analysis, 50 ng of cfDNA isolated from each sample was amplified by applying the whole gene amplification method. For 1 μg of the amplified cfDNA,
DNA regions commonly observed at each step were compared and analyzed in the form of Dye-swap. We classified DNA regions that showed significant differences in the progression stage of gastric cancer, serum and plasma, and patient gender by log ratio ≥ 1 or ≤ -1. Using the Van diagram analysis, the separated DNA regions were divided into DNA regions commonly observed in serum, plasma, gastric cancer stage and patient's sex in each chip (FIG. 1).
From the CGH microarray chip comparing T1_e with T1_a, 166 DNA regions of T1_e stage and 115 DNA regions of T1_a stage were observed. In the CGH microarray chip comparing T1_a and T2_a, DNA region of T1_a stage was 264 And 227 DNA regions in the T2_a stage. From the CGH microarray chip comparing T3_a and T1_e, 673 DNA regions in the T3_a stage and 812 DNA regions in the T1_e stage were observed. Dye-swap comparative analysis was performed on the DNA regions classified on each chip. As a result, four DNA regions were observed in the T1_e phase, 37 (T1_a), 5 (T1_a), and T2_a and T3_a phases (Tables 2, 3 and 4). Table 2 below shows the DNA regions commonly observed at the T1_e stage. Table 3 shows the DNA regions commonly observed in the T1_a step. Table 4 shows the DNA regions commonly observed in the T2_a and T3_a phases. In Table 2, 3 and 4, a region not including a gene symbol, a gene name, and an accession # means an intron region.
4. Real-time PCR analysis of cfDNA melting curve
ENTPD6 cfDNA in the T1_e step of the analyzed DNA region; And A_16_P01781625 cfDNA was selected in step T1_a to perform melting curve real time PCR analysis. Real-time PCR primers for melting curve analysis were designed such that the size of the amplicon was 113 bp to 183 bp centered on the probe nucleotide sequence of each DNA region, and M13 froward was added at the 5 'end to be used for nucleotide sequence analysis (Table 5).
Melting curve Real-time PCR analysis was performed by assuming that the cycle threshold (Ct) value was 30 or less and that a single melting curve was observed as a normal PCR product. Melting curve real-time PCR analysis was performed on the ENTPD6 DNA region selected from the DNA regions observed in step T1_e (FIG. 2).
The melting curve analysis (melting peak) of ENTPD6 is as follows.
For serum of male patients, T1_e = 79.37 ℃; T1_a = 79.4 DEG C; And T2_a = 79.45 ℃, and the T3_a step was not amplified and could not be analyzed. For serum of female patients, T1_e = 79.35 ℃; And T2_a = 79.4 ℃, and T1_a and T3_a phases were not amplified and could not be analyzed. For plasma of the male patient, T1_e = 79.27 ℃; And T3_a = 79.47 ℃, and T1_a and T2_a phases were not amplified and could not be analyzed. Plasma of female patients was analyzed at T1_e = 79.37 ℃ and T1_a, T2_a, and T3_a stages were not amplified because they were not amplified. Among the above results, the analysis of the samples of T1_a, T2_a and T3_a stages satisfied the condition of Ct = 30 or less.
Melting curve real-time PCR analysis was performed on A_16_P01781625 selected from the DNA regions observed in step T1_a (FIG. 3).
The melting curve analysis result (melting peak) of A_16_P01781625 is as follows.
For serum of male patients, T1_e = 78.83 ℃; T1_a = 78.77 DEG C; T2_a = 78.75 DEG C; And T3_a = 78.65 [deg.] C. For serum of female patients, T1_e = 78.8 ℃; T1_a = 78.75 DEG C; T2_a = 78.6 DEG C; And T3_a = 78.5 [deg.] C. For plasma of male patients, T1_e = 78.63 ℃; T1_a = 78.55 DEG C; T2_a = 78.65 DEG C; And T3_a = 78.45 [deg.] C. For plasma of female patients, T1_e = 78.78 ℃; T1_a = 78.75 DEG C; T2_a = 78.78 DEG C; And T3_a = 78.65 [deg.] C. Among the above results, Ct = 30 or less was satisfied in T1_e, T1_a and T2_a stages.
5. cfDNA nucleotide sequence analysis result
Nucleotide sequencing was performed on the melting curve real-time PCR products (FIGS. 4 and 5 and Tables 6 and 7). Table 6 shows the nucleotide sequences of ENTPD6 cfDNA, ENTPD6 cfDNA, and A_16_P01781625 cfDNA confirmed by nucleotide sequence analysis, and Table 8 shows the nucleotide sequence characteristics of the chromosomes and cfDNA corresponding to ENTPD6 and A_16_P01781625. In Table 7, " - " means deletion of A, and " W "means A or T substitution.
Nucleotide sequence analysis for ENTPD6 cfDNA revealed that the ENTPD6 cfDNA nucleotide sequence was identical to the
A_16_P01781625 The nucleotide sequence analysis for cfDNA confirmed that the nucleotide sequence of A_16_P01781625 cfDNA was located in the 5'region 7035bp of
conclusion
In the present invention, the level of cfDNA released into the blood and the dielectric characteristics were analyzed, and it was observed that the level of specific cfDNA increased with progression of the stomach. Melting curve In real-time PCR analysis, ENTPD6 cfDNA and A_16_P01781625 cfDNA were identified and confirmed as gastric cancer-progression-time specific cfDNA, and the DNA sequence of the amplified cfDNA was analyzed and verified. In particular, A_16_P01781625 at ENTPD6, T1_e, T1_a and T2_a at T1_e was below Ct = 30 and a single melting curve was observed, which can be regarded as a specific candidate cfDNA according to the progression of gastric cancer and stomach cancer. The level of blood cfDNA obtained in the present invention and the characteristics of the extracted cfDNA are expected to be applicable to biomarkers for measuring gastric cancer progression, evaluation of treatment progress, performance of anticancer agents, and suitability evaluation.
The specific embodiments described herein are representative of preferred embodiments or examples of the present invention, and thus the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that modifications and other uses of the invention do not depart from the scope of the invention described in the claims.
<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Use of Cell-Free DNA for Diagnosing Gastric Cancer <130> MP15-0135 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer of ENTPD6 <400> 1 ctggggctct cagtagttgc 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer of ENTPD6 <400> 2 tggtgttccc aaactgttc 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer of AP_16_P01781625 <400> 3 ctgcctacaa cctcacccta a 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer of AP_16_P01781625 <400> 4 tagaacatgg tcccgggtta 20 <210> 5 <211> 116 <212> DNA <213> Homo sapiens <400> 5 agggacggag tttaaacctt tcgaaaagta aattgcctga atagaagtat aactgaggat 60 tgaaagactg aatcagatac gcgcttctgt tcccatgaac agtttgggaa caccaa 116 <210> 6 <211> 91 <212> DNA <213> Homo sapiens <400> 6 taagactcac atcgaggtaa actgcttgtt ttaagaacta acctctgtcc ttaattcagt 60 ctcctattcc taacccggga ccatgntcta a 91 <210> 7 <211> 115 <212> DNA <213> Homo sapiens <400> 7 agggacggag tttaaccttt cgaaaagtaa attgcctgaa tagaagtata actgaggatt 60 gaaagactga atcagatacg cgcttctgtt cccatgaaca gtttgggaac accaa 115 <210> 8 <211> 114 <212> DNA <213> Homo sapiens <400> 8 agggacggag tttaaccttt cgaaagtaaa ttgcctgaat agaagtataa ctgaggattg 60 aaagactgaa tcagatacgc gcttctgttc ccatgaacag tttgggaaca ccaa 114 <210> 9 <211> 115 <212> DNA <213> Homo sapiens <400> 9 agggacggag tttaaccttt cgaaaagtaa attgcctgaa tagaagtata actgaggatt 60 gaaagactga atcagatacg cgcttctgtt cccatgaaca gttwgggaac accaa 115
Claims (14)
Wherein the method further comprises the step of separating cell-free DNA from the biological sample.
A first primer set consisting of oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2;
A second primer set consisting of the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4; or
The first primer set and the second primer set;
Wherein the method further comprises the step of performing a polymerase chain reaction (PCR) using a DNA polymerase chain reaction (PCR).
Free DNA consisting of a nucleotide sequence selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOS: 6 to 9, and a cell- free DNA.
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| KR101980819B1 (en) | 2016-12-23 | 2019-05-21 | 주식회사 제놉시 | Magnetic nanostructure for detecting and isolating circulating cell-free DNA comprising conductive polymers containing magnetic nanoparticles and cationic polymers |
| JP7239973B2 (en) * | 2017-12-19 | 2023-03-15 | 国立大学法人山口大学 | Method for assisting prediction of presence or absence of precancerous lesion or cancer |
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