Single nucleotide polymorphism site marker composition for predicting postoperative recurrence and metastasis risk of lung adenocarcinoma patient and application of single nucleotide polymorphism site marker composition

Abstract

The invention relates to the technical field of genetic engineering and tumor medicine, and particularly discloses a single nucleotide polymorphism site marker composition for predicting postoperative recurrence and metastasis risks of lung adenocarcinoma patients and application of the single nucleotide polymorphism site marker composition. The single nucleotide polymorphism site marker composition provided by the invention can accurately predict the prognosis of a lung adenocarcinoma patient, can evaluate the prognosis of the patient before treatment, and formulates a more positive and effective treatment scheme for a patient with poor prognosis, thereby realizing individualized treatment of the patient.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering and tumor medicine technology, and in particular to a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma and its application. Background Art

[0002] At present, surgical resection is still one of the main treatments for lung adenocarcinoma, but early lung adenocarcinoma still has a high recurrence rate of about 30-50% even after radical surgical resection. Current studies have shown that there is a subgroup of patients with more aggressive tumors among lung adenocarcinoma patients, and these patients can benefit from adjuvant targeted therapy after radical surgery. Therefore, accurate assessment of the risk of postoperative recurrence and metastasis of lung adenocarcinoma is the key to determining which patients need adjuvant therapy. At present, TNM staging, pathological grade, tumor size, vascular invasion, lymphatic invasion, perineural invasion, pleural invasion, whether there is tumor tissue at the surgical margin, intracavitary dissemination (STAS), lymph node metastasis, PD-L1 expression status, EGFR, ROS1, ALK mutation status, etc. are usually used to assess the risk of tumor recurrence and metastasis in patients with lung adenocarcinoma after surgery.

[0003] However, patients with the same stratification results may have very different prognoses, which suggests that the current predictive means of the postoperative recurrence assessment and staging system for lung adenocarcinoma needs to be improved urgently, which is expected to be achieved by using verified specific tumor biomolecular markers. Several multi-gene classifiers have been reported to predict the risk of recurrence and metastasis of lung adenocarcinoma. However, to date, there are still no stable and reliable molecular markers used in clinical practice to predict the risk of postoperative recurrence and metastasis of lung adenocarcinoma.

[0004] Single-nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. It is the most common type of heritable variation in humans, accounting for more than 80% of all known polymorphisms. With the continuous development of whole genome technology, research on single nucleotide polymorphisms related to disease outcomes, including cancer, has become increasingly in-depth. As a third-generation genetic marker, it has been widely used in the diagnosis and prognosis prediction of major diseases such as malignant tumors in recent years, and has shown the characteristics of rapidity, sensitivity, and accuracy, and therefore has broad application prospects. At present, no SNP site that can accurately and effectively judge tumor prognosis has been applied to tumor prognosis prediction.

[0005] However, some existing reports have explored the correlation between mutations in related genes and tumor prognosis, involving DNA repair genes (ERCC1, XRCC1), chemokines (CXCL12, MCP-1), matrix metalloproteinases (MMP-9, MMP-1), interleukins (IL-8), tumor suppressor genes (TP53), cell cycle-related genes (CCNE1), key genes for prostaglandin synthesis (PTGS2), ubiquitin-protein ligase (MDM2), etc.; however, since these studies involve relatively small sample sizes and are greatly affected by confounding factors such as population structure, the results obtained are still controversial, and their potential value is not enough to be further developed and applied. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a single nucleotide polymorphism site marker composition and its application for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma. The single nucleotide polymorphism site marker composition provided in the present application is used to assist in guiding individualized treatment and improving the prognosis of patients with lung adenocarcinoma.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] The present application provides a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma, wherein the single nucleotide polymorphism site marker composition includes rs6697897, rs2235937, rs197427, rs4656197, rs2288358, rs2273697, rs17116456, rs7307331, rs1898199, rs7975, rs3743738, rs7404202, rs4239389, rs16978552, rs17816861, rs7245949, rs29 at least one of rs02317, rs8192709, rs7570807, rs276827, rs3747517, rs2231495, rs17172438, rs1358357, rs12629133, rs2289247, rs12487066, rs1049296, rs6859236, rs2073724, rs86567, rs4707569, rs3808782, rs177698, rs7034065, rs11932330, rs2253427 and rs6089219.

[0009] After extensive research and experiments, the inventors of the present application found that by detecting the above-mentioned single nucleotide polymorphism site (SNP) marker combination in patients with lung adenocarcinoma, the risk of recurrence and metastasis of patients with lung adenocarcinoma after radical treatment (surgery, radiofrequency ablation) can be predicted.

[0010] The present application also provides a primer set for detecting the single nucleotide polymorphism site marker composition, wherein the primer set includes primers having nucleotide sequences as shown in SEQ Nos: 1 to 76.

[0011] The primer set with the above sequence (shown in SEQ No: 1-76) can be used to detect the above single nucleotide polymorphism site marker composition and perform specific amplification on it.

[0012] As a preferred embodiment of the primer set described in the present application, the primer sequence for detecting the rs2235937 site is shown in SEQ Nos: 1 to 2; the primer sequence for detecting the rs197427 site is shown in SEQ Nos: 3 to 4; the primer sequence for detecting the rs4656197 site is shown in SEQ Nos: 5 to 6; the primer sequence for detecting the rs2273697 site is shown in SEQ Nos: 7 to 8; the primer sequence for detecting the rs7307331 site is shown in SEQ Nos: 9 to 10; the primer sequence for detecting the rs7975 site is shown in SEQ Nos: 11 to 12; the primer sequence for detecting the rs3743738 site is shown in SEQ Nos: 13 to 14; the primer sequence for detecting the rs7245949 site is shown in SEQ Nos: 15 to 16; the primer sequence for detecting the rs8192709 site is shown in SEQ Nos: 17 to 18; the primer sequence for detecting the rs3747517 site is shown in SEQ Nos: No: 19-20; the primer sequence for detecting the rs2231495 site is shown in SEQ No: 21-22; the primer sequence for detecting the rs12629133 site is shown in SEQ No: 23-24; the primer sequence for detecting the rs2289247 site is shown in SEQ No: 25-26; the primer sequence for detecting the rs1049296 site is shown in SEQ No: 27-28; the primer sequence for detecting the rs2073724 site is shown in SEQ No: 29-30; the primer sequence for detecting the rs4707569 site is shown in SEQ No: 31-32; the primer sequence for detecting the rs3808782 site is shown in SEQ No: 33-34; the primer sequence for detecting the rs2253427 site is shown in SEQ No: 35-36; the primer sequence for detecting the rs6089219 site is shown in SEQ No: 37-38; the primer sequence for detecting the rs6697897 site is shown in SEQ No: 39-40; the primer sequence for detecting the rs2288358 site is shown in SEQ No: 41-42; the primer sequence for detecting the rs17116456 site is shown in SEQ No: 43-44; the primer sequence for detecting the rs1898199 site is shown in SEQ No: 45-46; the primer sequence for detecting the rs7404202 site is shown in SEQ No: 47-48; the primer sequence for detecting the rs4239389 site is shown in SEQ No: 49-50; the primer sequence for detecting the rs16978552 site is shown in SEQ No: No: 51-52; the primer sequence for detecting the rs17816861 site is shown in SEQ No: 53-54; the primer sequence for detecting the rs2902317 site is shown in SEQ No: 55-56; the primer sequence for detecting the rs7570807 site is shown in SEQ No: 57-58;The primer sequences for detecting the rs276827 site are shown in SEQ Nos: 59-60; the primer sequences for detecting the rs17172438 site are shown in SEQ Nos: 61-62; the primer sequences for detecting the rs1358357 site are shown in SEQ Nos: 63-64; the primer sequences for detecting the rs12487066 site are shown in SEQ Nos: 65-66; the primer sequences for detecting the rs6859236 site are shown in SEQ Nos: 67-68; the primer sequences for detecting the rs86567 site are shown in SEQ Nos: 69-70; the primer sequences for detecting the rs177698 site are shown in SEQ Nos: 71-72; the primer sequences for detecting the rs7034065 site are shown in SEQ Nos: 73-74; the primer sequences for detecting the rs11932330 site are shown in SEQ Nos: 75-76. ;

[0013] The present application also provides the use of the above-mentioned single nucleotide polymorphism site marker composition in the preparation of a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

[0014] The present application also provides the use of the above primer set in the preparation of a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

[0015] The present application also provides the use of the above-mentioned single nucleotide polymorphism site marker combination in constructing a scoring model for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

[0016] As a preferred embodiment of the application described in the present application, the scoring model is to calculate the recurrence risk score value by using the recurrence risk scoring formula after detecting the single nucleotide polymorphism site marker composition described in the DNA;

[0017] The recurrence risk score value = rs6697897×(-0.403589)+rs2235937×(0.04457824)+rs197427×(-0.3139403)+rs4656197×(0.1026962)+rs2288358×(-0.2821615)+rs2273697×(-0.1459264)+rs17116456×(0.1917002)+rs7307331×(0.226947)+rs1898199×(0.3077543)+rs797 5×(0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.30464 5)+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0 .351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs6859 236×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981);

[0018] Among them, the subjects whose recurrence risk score value is greater than -3.68 are classified as high risk of recurrence and metastasis, that is, high-risk group, and the subjects whose recurrence risk score value is less than or equal to -3.68 are classified as low risk of recurrence and metastasis, that is, low-risk group.

[0019] The present application obtains the genotype information of each site by sequencing the DNA amplification product of the above-mentioned SNP site, and obtains a score according to the genotype of different SNP sites. For example, when the genotype of the SNP site rs2235937 is AA, the score of the genotype is 0 points, when the genotype of the site is AG (or GA), the score of the genotype is 1 point, and when the genotype of the site is GG, the score of the genotype is 2 points. Similarly, according to the different genotype scores of each SNP site, the score of each SNP site is then substituted into the above-mentioned recurrence risk score formula to finally obtain the recurrence risk score.

[0020] The above recurrence risk score can be used to determine the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma and to guide the selection of postoperative treatment for lung adenocarcinoma.

[0021] Patients with a score greater than -3.68 were classified as having a high risk of recurrence and metastasis, i.e., the high-risk group; those with a score less than or equal to -3.68 were classified as having a low risk of recurrence and metastasis, i.e., the low-risk group, to guide the selection of postoperative treatment for lung adenocarcinoma. Patients in the low-risk group had a longer progression-free survival (HR = 0.157, 95% CI = 0.114-0.216, P = 1.54 × 10 -29 ), these patients do not need to be given adjuvant chemotherapy after radical surgery; high-risk patients should be given adjuvant treatment to prevent recurrence.

[0022] The present application also provides a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma, wherein the kit comprises the primer set.

[0023] As a preferred embodiment of the kit described in the present application, the kit for predicting the risk of postoperative recurrence and metastasis of patients with lung adenocarcinoma uses the following recurrence risk score value for evaluation;

[0024] The recurrence risk score value = rs6697897×(-0.403589)+rs2235937×(0.04457824)+rs197427×(-0.3139403)+rs4656197×(0.1026962)+rs2288358×(-0.2821615)+rs2273697×(-0.1459264)+rs17116456×(0.1917002)+rs7307331×(0.226947)+rs1898199×(0.3077543)+rs797 5×(0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.30464 5)+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0 .351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs6859 236×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981);

[0025] Among them, the subjects whose recurrence risk score value is greater than -3.68 are classified as high risk of recurrence and metastasis, that is, high-risk group, and the subjects whose recurrence risk score value is less than or equal to -3.68 are classified as low risk of recurrence and metastasis, that is, low-risk group.

[0026] The kit for predicting the risk of postoperative recurrence and metastasis of patients with lung adenocarcinoma in the present application uses the above recurrence risk score value for evaluation. The detection kit can accurately predict the postoperative prognosis of patients with lung adenocarcinoma by detecting SNP sites on the peripheral blood DNA or lung adenocarcinoma tissue DNA of patients with lung adenocarcinoma.

[0027] Furthermore, after the kit of the present application is applied to clinical testing, it can evaluate the patient's prognosis before treatment and formulate more active and effective treatment plans for patients with poor prognosis, thereby achieving individualized treatment for patients and improving survival rates.

[0028] As a preferred embodiment of the kit described in the present application, the kit also includes PCR buffer, dNTP, magnesium chloride, DNA polymerase and deionized water.

[0029] The present application provides a detection kit for predicting postoperative recurrence and metastasis of lung adenocarcinoma, which can be used to assist in guiding individualized treatment and improving the prognosis of patients with lung adenocarcinoma.

[0030] Compared with the prior art, this application has the following beneficial effects:

[0031] The present application provides a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma and its application. The present application adopts the above-mentioned single nucleotide polymorphism site marker composition to assist in guiding individualized treatment and improving the prognosis of patients with lung adenocarcinoma; the detection kit provided by the present application can accurately predict the postoperative prognosis of patients with lung adenocarcinoma by detecting SNP sites on peripheral blood DNA or lung adenocarcinoma tissue DNA of patients with lung adenocarcinoma; after the kit of the present application is applied to clinical testing, the prognosis of patients can be evaluated before treatment, and a more active and effective treatment plan can be formulated for patients with poor prognosis, thereby realizing individualized treatment of patients and improving survival rate; in addition, the kit can be used for sampling and testing of peripheral blood or lung adenocarcinoma tissue, and has the characteristics of convenient sampling, simple operation, and high timeliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the overall progression-free survival curve for high-risk patients and low-risk patients. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0034] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0035] Example 1: Screening for SNP sites that predict the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma

[0036] (1) Selection of research samples:

[0037] The samples included in this application are all patients with lung adenocarcinoma diagnosed by pathology at the Sun Yat-sen University Cancer Center, with complete medical records (including tumor stage, medical history, examination results, previous treatment plans, etc.); patients must be newly diagnosed patients with no history of tumors, and have not received tumor-related radiotherapy, chemotherapy, or other anti-tumor treatments before admission; patients are all voluntary participants in the study and signed informed consent. Patients included in the study will be followed up regularly, every three months within two years after the end of treatment, and every six months thereafter.

[0038] (2) Genomic DNA extraction from peripheral blood or lung adenocarcinoma tissue:

[0039] Before the first treatment, peripheral blood was collected from patients using EDTA anticoagulant tubes or surgical resection samples were stored in a -80°C refrigerator. Genomic DNA from peripheral blood or lung adenocarcinoma tissue was extracted using the phenol-chloroform method according to routine operating procedures. The concentration of the obtained DNA samples was usually 20-50 ng / μl, and the purity (OD 260 / 280 ) is generally between 1.6-2.0.

[0040] (3) Amplification of SNP sites:

[0041] Use specific primers to amplify the following SNP sites. The primer sequences used are shown in Table 1:

[0042] rs6697897, rs2235937, rs197427, rs4656197, rs2288358, rs2273697, rs17116456, rs7307331, rs1898199, rs7975, rs3743738, rs7404202, rs4239389, rs16978552, rs17816861, rs7245949, rs2902317, rs8192709, rs7570807, rs276827, rs3747517, rs2231495, rs17172438, rs1358 357, rs12629133, rs2289247, rs12487066, rs1049296, rs6859236, rs2073724, rs86567, rs4707569, rs3808782, rs177698, rs7034065, rs11932330, rs2253427 and rs6089219; PCR amplification also included PCR buffer, dNTP, magnesium chloride, polymerase, deionized water, and the PCR reaction program was set as: 94℃, 2min; 98℃, 10s, 56℃, 30s, 68℃, 40s, 33cycles; 4℃, ∞;.

[0043] (4) SNP locus typing:

[0044] The SNP sites included rs6697897, rs2235937, rs197427, rs4656197, rs2288358, rs2273697, rs17116456, rs7307331, rs1898199, rs7975, rs3743738, rs7404202, rs4239389, rs16978552, rs17816861, rs7245949, rs2902317, rs8192709, rs7570807, rs276827, The DNA amplification products of rs3747517, rs2231495, rs17172438, rs1358357, rs12629133, rs2289247, rs12487066, rs1049296, rs6859236, rs2073724, rs86567, rs4707569, rs3808782, rs177698, rs7034065, rs11932330, rs2253427, and rs6089219 were sequenced to obtain the genotype information of the above loci.

[0045] (5) Statistical analysis methods:

[0046] The proportional hazards model (Cox model) was used to analyze the correlation between demographic and clinical factors (including gender, age, clinical stage, tumor size, vascular invasion, lymphatic invasion, perineural invasion, pleural invasion, whether there was tumor tissue at the surgical margin, intracavitary dissemination (STAS), lymph node metastasis, PD-L1 expression status, EGFR, ROS1, ALK mutation status, whether concurrent chemoradiotherapy was used, whether postoperative adjuvant chemotherapy was used, whether neoadjuvant chemotherapy was used, etc.) and the prognosis of lung adenocarcinoma; further, the factors significantly associated with tumor prognosis (gender, age, clinical stage, tumor size, vascular invasion, lymphatic invasion, perineural invasion, pleural invasion, whether there was tumor tissue at the surgical margin, intracavitary dissemination (STAS), lymph node metastasis, PD-L1 expression status, EGFR, ROS1, ALK mutation status, whether concurrent chemoradiotherapy was used, whether postoperative adjuvant chemotherapy was used, whether neoadjuvant chemotherapy was used, etc.) were used as covariates to calculate the hazard ratio (Hazard ratio) of SNP genotype for tumor prognosis. ratio (HR), 95% confidence interval (95% CI) and P value. Statistical analysis was performed using R software (Version 4.0.0), with a statistical significance level of P < 0.05, and all tests were two-sided.

[0047] (6) Result analysis:

[0048] By detecting the genotype of the above SNP loci and using the prediction formula to obtain the recurrence risk score index of the subject to be tested, the formula is as follows:

[0049] Recurrence risk score =

[0050] rs6697897×(-0.403589)+rs2235937×(0.04457824)+rs197427×(-0.3139403)+rs4656197×(0.1026962)+rs2288358×(-0.2821615)+rs2273697×(-0.1459264)+rs17116456×(0.1917002)+rs7307331×(0.226947)+rs1898199×(0.3077543)+rs7975×(0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.304645)+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0.351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs6859236×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981);

[0051] By sequencing the DNA amplification products of the above-mentioned SNP sites, the genotype information of each site is obtained (see Table 1), and the genotypes of different SNP sites are scored according to Table 1. For example, when the genotype of the SNP site rs2235937 is AA, the score of the genotype is 0 points, when the genotype of the site is AG (or GA), the score of the genotype is 1 point, and when the genotype of the site is GG, the score of the genotype is 2 points. Similarly, different genotype scores of each SNP site are obtained according to Table 1, and then the score of each SNP site is substituted into the above-mentioned recurrence risk score formula to finally obtain the recurrence risk score.

[0052] Patients with a score greater than -3.68 were classified as having a high risk of recurrence and metastasis, i.e., the high-risk group; those with a score less than or equal to -3.68 were classified as having a low risk of recurrence and metastasis, i.e., the low-risk group, to guide the selection of postoperative treatment for lung adenocarcinoma. Patients in the low-risk group had a longer progression-free survival (HR = 0.157, 95% CI = 0.114-0.216, P = 1.54 × 10 -29 ), these patients do not need to be given adjuvant chemotherapy after radical surgery; high-risk patients should be given adjuvant treatment to prevent recurrence.

[0053] Table 1 SNP site scores and amplification primers for different genotypes

[0054]

[0055]

[0056] Example 2

[0057] This example provides the use of the marker composition of Example 1 in a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

[0058] The SNP marker composition obtained in Example 1 includes: rs6697897, rs2235937, rs197427, rs4656197, rs2288358, rs2273697, rs17116456, rs7307331, rs1898199, rs7975, rs3743738, rs7404202, rs4239389, rs16978552, rs17816861, rs7245949, rs2902317, rs8192709, rs7570807, rs276827, rs3747517, rs 2231495, rs17172438, rs1358357, rs12629133, rs2289247, rs12487066, rs1049296, rs6859236, rs2073724, rs86567, rs4707569, rs3808782, rs177698, rs7034065, rs11932330, rs2253427, rs6089219. The above markers were used to construct a scoring model formula for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma using a proportional hazard regression model to calculate the recurrence risk score value. The specific model formula is:

[0059] Recurrence risk score = rs6697897 × (-0.403589) + rs2235937 × (0.04457824) + rs197427 × (-0.3139403) + rs4656197 × (0.1026962) + rs2288358 × (-0.2821615) + rs2273697 × (-0.1459264) + rs17116456 × (0.1917002) + rs7307331 × (0.226947) + rs1898199 × (0.3077543) + rs7975 × (0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.304645 )+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0. 351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs68592 36×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981);

[0060] The recurrence risk score value was calculated using the above model formula, and subjects with a score greater than -3.68 were classified as having a high risk of recurrence and metastasis, i.e., a high-risk group; subjects with a score less than or equal to -3.68 were classified as having a low risk of recurrence and metastasis, i.e., a low-risk group.

[0061] Example 3

[0062] This embodiment provides a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma and its application. The kit includes: specific primers for detecting each SNP site in Example 1 (primer sequences are shown in Table 1), PCR buffer, dNTP, magnesium chloride, DNA polymerase and deionized water.

[0063] Kit usage: Take peripheral blood or lung adenocarcinoma tissue samples from patients with lung adenocarcinoma and process them according to the phenol-chloroform method standard process to extract genomic DNA. The DNA concentration is 30-60ng / μL and the purity (OD 260 / 280 ) is between 1.6 and 2.0, and specific amplification primers (see Table 1) are used for PCR amplification. The reaction system is shown in Table 2.

[0064] After the PCR reaction was completed, DNA electrophoresis (1% agarose gel, voltage: 120 V, time: 30 minutes) was performed to detect the PCR products, and the PCR products with the expected fragment size (673 bp), single band and moderate brightness were selected for sequencing using ABI 3730 sequencer.

[0065] Table 2 PCR reaction system

[0066]

[0067]

[0068] The PCR amplification reaction program was set as: 94°C, 2 min; 98°C, 10 s, 56°C, 30 s, 68°C, 40 s, 33 cycles; 4°C, ∞.

[0069] The risk score is calculated by the recurrence risk scoring formula of the present invention, and subjects with a score greater than -3.68 are classified as having a high risk of recurrence and metastasis, i.e., a high-risk group; subjects with a score less than or equal to -3.68 are classified as having a low risk of recurrence and metastasis, i.e., a low-risk group; patients in the low-risk group may not be given adjuvant chemotherapy or other treatments after radical surgery; patients at high risk should be given adjuvant treatment to prevent recurrence.

[0070] The correlation analysis based on the results of gene SNP analysis and the follow-up data of included lung adenocarcinoma patients showed that the recurrence-free survival of low-risk patients was significantly longer than that of high-risk patients, with an overall recurrence-free survival (HR = 0.157, 95% CI = 0.114-0.216, P = 1.54 × 10 -29 ,like Figure 1 shown).

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma, characterized in that: The single nucleotide polymorphism site marker composition includes rs6697897, rs2235937, rs197427, rs4656197, rs2288358, rs2273697, rs17116456, rs7307331, rs1898199, rs7975, rs3743738, rs7404202, rs4239389, rs16978552, rs17816861, rs7245949, rs2902317, rs8192709, rs75 at least one of rs70807, rs276827, rs3747517, rs2231495, rs17172438, rs1358357, rs12629133, rs2289247, rs12487066, rs1049296, rs6859236, rs2073724, rs86567, rs4707569, rs3808782, rs177698, rs7034065, rs11932330, rs2253427 and rs6089219.

2. A primer set for detecting the single nucleotide polymorphism site marker composition according to claim 1, characterized in that: The primer set includes primers with nucleotide sequences as shown in SEQ Nos: 1-76.

3. The primer set according to claim 2, characterized in that The primer sequences for detecting the rs2235937 site are shown in SEQ Nos: 1 to 2; the primer sequences for detecting the rs197427 site are shown in SEQ Nos: 3 to 4; the primer sequences for detecting the rs4656197 site are shown in SEQ Nos: 5 to 6; the primer sequences for detecting the rs2273697 site are shown in SEQ Nos: 7 to 8; the primer sequences for detecting the rs7307331 site are shown in SEQ Nos: 9 to 10; the primer sequences for detecting the rs7975 site are shown in SEQ Nos: 11 to 12; the primer sequences for detecting the rs3743738 site are shown in SEQ Nos: 13 to 14; the primer sequences for detecting the rs7245949 site are shown in SEQ Nos: 15 to 16; the primer sequences for detecting the rs8192709 site are shown in SEQ Nos: 17 to 18; the primer sequences for detecting the rs3747517 site are shown in SEQ Nos: No: 19-20; the primer sequence for detecting the rs2231495 site is shown in SEQ No: 21-22; the primer sequence for detecting the rs12629133 site is shown in SEQ No: 23-24; the primer sequence for detecting the rs2289247 site is shown in SEQ No: 25-26; the primer sequence for detecting the rs1049296 site is shown in SEQ No: 27-28; the primer sequence for detecting the rs2073724 site is shown in SEQ No: 29-30; the primer sequence for detecting the rs4707569 site is shown in SEQ No: 31-32; the primer sequence for detecting the rs3808782 site is shown in SEQ No: 33-34; the primer sequence for detecting the rs2253427 site is shown in SEQ No: 35-36; the primer sequence for detecting the rs6089219 site is shown in SEQ No: 37-38; the primer sequence for detecting the rs6697897 site is shown in SEQ No: 39-40; the primer sequence for detecting the rs2288358 site is shown in SEQ No: 41-42; the primer sequence for detecting the rs17116456 site is shown in SEQ No: 43-44; the primer sequence for detecting the rs1898199 site is shown in SEQ No: 45-46; the primer sequence for detecting the rs7404202 site is shown in SEQ No: 47-48; the primer sequence for detecting the rs4239389 site is shown in SEQ No: 49-50; the primer sequence for detecting the rs16978552 site is shown in SEQ No: 51-52; the primer sequence for detecting the rs17816861 site is shown in SEQ No: 53-54; the primer sequence for detecting the rs2902317 site is shown in SEQ No: 55-56; the primer sequence for detecting the rs7570807 site is shown in SEQ No: 57-58;The primer sequences for detecting the rs276827 site are shown in SEQ Nos: 59-60; the primer sequences for detecting the rs17172438 site are shown in SEQ Nos: 61-62; the primer sequences for detecting the rs1358357 site are shown in SEQ Nos: 63-64; the primer sequences for detecting the rs12487066 site are shown in SEQ Nos: 65-66; the primer sequences for detecting the rs6859236 site are shown in SEQ Nos: 67-68; the primer sequences for detecting the rs86567 site are shown in SEQ Nos: 69-70; the primer sequences for detecting the rs177698 site are shown in SEQ Nos: 71-72; the primer sequences for detecting the rs7034065 site are shown in SEQ Nos: 73-74; the primer sequences for detecting the rs11932330 site are shown in SEQ Nos: 75-76. ; 4. Use of the single nucleotide polymorphism site marker composition as described in claim 1 in preparing a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

5. Use of the primer set as claimed in claim 2 or 3 in preparing a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

6. Use of the single nucleotide polymorphism site marker composition as described in claim 1 in constructing a scoring model for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma.

7. The use according to claim 6, characterized in that The scoring model is to calculate the recurrence risk score value by using a recurrence risk scoring formula after detecting the single nucleotide polymorphism site marker composition as described in claim 1 in DNA; The recurrence risk score value = rs6697897×(-0.403589)+rs2235937×(0.04457824)+rs197427×(-0.3139403)+rs4656197×(0.1026962)+rs2288358×(-0.2821615)+rs2273697×(-0.1459264)+rs17116456×(0.1917002)+rs7307331×(0.226947)+rs1898199×(0.3077543)+rs797 5×(0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.30464 5)+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0 .351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs6859 236×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981); Among them, the subjects whose recurrence risk score value is greater than -3.68 are classified as high risk of recurrence and metastasis, that is, high-risk group, and the subjects whose recurrence risk score value is less than or equal to -3.68 are classified as low risk of recurrence and metastasis, that is, low-risk group.

8. A kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma, characterized in that: The kit comprises the primer set according to claim 2 or 3.

9. The kit according to claim 8, characterized in that The kit for predicting the risk of postoperative recurrence and metastasis in patients with lung adenocarcinoma uses the following recurrence risk score values ​​for evaluation; The recurrence risk score value = rs6697897×(-0.403589)+rs2235937×(0.04457824)+rs197427×(-0.3139403)+rs4656197×(0.1026962)+rs2288358×(-0.2821615)+rs2273697×(-0.1459264)+rs17116456×(0.1917002)+rs7307331×(0.226947)+rs1898199×(0.3077543)+rs797 5×(0.03250266)+rs3743738×(0.9469271)+rs7404202×(-0.3247954)+rs4239389×(-0.1814657)+rs16978552×(0.2101912)+rs17816861×(-0.4634299)+rs7245949×(0.03088702)+rs2902317×(0.3739028)+rs8192709×(-0.1407154)+rs7570807×(-0.30464 5)+rs276827×(0.2172139)+rs3747517×(0.04860136)+rs2231495×(-0.02135492)+rs17172438×(-0.1719029)+rs1358357×(-0 .351861)+rs12629133×(-0.06124469)+rs2289247×(-0.0946711)+rs12487066×(0.4249074)+rs1049296×(0.1246213)+rs6859 236×(0.3202245)+rs2073724×(0.3202245)+rs86567×(0.2838189)+rs4707569×(0.1357111)+rs3808782×(0.1564333)+rs177698×(-0.3689062)+rs7034065×(-0.2581711)+rs11932330×(-0.6463428)+rs2253427×(0.09991192)+rs6089219×(0.08003981); Among them, the subjects whose recurrence risk score value is greater than -3.68 are classified as high risk of recurrence and metastasis, that is, high-risk group, and the subjects whose recurrence risk score value is less than or equal to -3.68 are classified as low risk of recurrence and metastasis, that is, low-risk group.

10. The kit according to claim 8, characterized in that The kit also includes PCR buffer, dNTPs, magnesium chloride, DNA polymerase and deionized water.

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