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

Abstract

The invention relates to the technical field of biology, in particular to a single nucleotide polymorphism site marker composition for predicting postoperative recurrence and metastasis risks of lung squamous cell carcinoma patients and application of the single nucleotide polymorphism site marker composition. The risk of recurrence and metastasis of lung squamous cell carcinoma patients after radical treatment (operation and radiofrequency ablation) can be accurately predicted, and a more positive and effective treatment scheme is formulated for patients with poor prognosis, so that individualized treatment of the patients is realized, and the survival rate is increased. The invention further provides a kit for predicting the postoperative recurrence and metastasis risk of the lung squamous cell carcinoma patient, and the kit provided by the invention can be used for assisting and guiding individualized treatment and improving prognosis of the lung squamous cell carcinoma patient by detecting SNP sites on peripheral blood DNA or lung squamous cell carcinoma tissue DNA of the lung squamous cell carcinoma patient.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma and its application. Background Art

[0002] Lung squamous cell carcinoma (LUSC), also known as LUSC, can be treated with bronchoendoscopy in the early stage, but surgical resection is still one of the main treatments for LUSC. However, even after radical surgical resection, LUSC still has a high recurrence rate of about 30-50%.

[0003] Current studies have shown that there is a subgroup of patients with more aggressive tumors among patients with squamous cell lung carcinoma. These patients may benefit from adjuvant targeted therapy after radical resection. Therefore, accurate assessment of the risk of postoperative recurrence and metastasis of squamous cell lung carcinoma 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, squamous cell carcinoma antigen level (SCC), cytokeratin fragment 19 level (CYFRA 21-1), neuron-specific enolase level (NSE), etc. are usually used to assess the risk of tumor recurrence and metastasis in patients with squamous cell lung carcinoma after surgery.

[0004] However, patients with the same stratification results may have very different prognoses, which suggests that the current prediction methods for the postoperative recurrence assessment and staging system for squamous cell lung carcinoma need 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 squamous cell lung carcinoma. However, to date, there is still no stable and reliable molecular marker used in clinical practice to predict the risk of postoperative recurrence and metastasis of squamous cell lung carcinoma. Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by the variation of a single nucleotide at the genome 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, the research on single nucleotide polymorphisms related to disease outcomes, including cancer, has become more and more in-depth. As the 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, so it has broad application prospects. At present, no SNP sites that can accurately and effectively judge tumor prognosis have been applied to tumor prognosis prediction. 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

[0005] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a single nucleotide polymorphism site marker composition and its application for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer. The single nucleotide polymorphism site marker composition provided above can be used to assist in guiding individualized treatment and improving the prognosis of patients with squamous cell lung cancer.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer, the single nucleotide polymorphism site marker composition comprising rs12337910, rs1805794, rs734784, rs2303720, rs5998267, rs11662595, rs763780, rs2228210, rs386556287, rs16921260, rs180749, rs at least one of rs1801058, rs2229466, rs12213148, rs2617160, rs11084425, rs10181310, rs4792416, rs9350450, rs2779154, rs3132306, rs17570453, rs8071417, rs7764, rs934397, rs378528, rs7602685 and rs11166827.

[0008] After extensive research and experiments, the inventors of the present application found that by detecting the genotype of the above-mentioned SNP loci in patients with squamous cell lung cancer, the risk of recurrence and metastasis of patients with squamous cell lung cancer after radical treatment (surgery, radiofrequency ablation) can be predicted, and more active and effective treatment plans can be formulated for patients with poor prognosis, thereby achieving individualized treatment of patients and improving survival rates.

[0009] The present application 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 ID Nos: 1 to 56.

[0010] As a preferred embodiment of the primer set described in the present application, the primer sequence for detecting the rs12337910 site is shown in SEQ ID Nos: 1 to 2; the primer sequence for detecting the rs1805794 site is shown in SEQ ID Nos: 3 to 4; the primer sequence for detecting the rs734784 site is shown in SEQ ID Nos: 5 to 6; the primer sequence for detecting the rs2303720 site is shown in SEQ ID Nos: 7 to 8; the primer sequence for detecting the rs5998267 site is shown in SEQ ID Nos: 9 to 10; the primer sequence for detecting the rs11662595 site is shown in SEQ ID Nos: 11 to 12; the primer sequence for detecting the rs763780 site is shown in SEQ ID Nos: 13 to 14; the primer sequence for detecting the rs2228210 site is shown in SEQ ID Nos: 15 to 16; the primer sequence for detecting the rs386556287 site is shown in SEQ ID No: 17-18; the primer sequence for detecting the rs16921260 site is shown in SEQ ID No: 19-20; the primer sequence for detecting the rs180749 site is shown in SEQ ID No: 21-22; the primer sequence for detecting the rs1801058 site is shown in SEQ ID No: 23-24; the primer sequence for detecting the rs2229466 site is shown in SEQ ID No: 25-26; the primer sequence for detecting the rs12213148 site is shown in SEQ ID No: 27-28; the primer sequence for detecting the rs2617160 site is shown in SEQ ID No: 29-30; the primer sequence for detecting the rs11084425 site is shown in SEQ ID No: 31-32; the primer sequence for detecting the rs10181310 site is shown in SEQ ID No: 33-34; the primer sequence for detecting the rs4792416 site is shown in SEQ ID No: 35-36; the primer sequence for detecting the rs9350450 site is shown in SEQ ID No: 37-38; the primer sequence for detecting the rs2779154 site is shown in SEQ ID No: 39-40; the primer sequence for detecting the rs3132306 site is shown in SEQ ID No: 41-42; the primer sequence for detecting the rs17570453 site is shown in SEQ ID No: 43-44; the primer sequence for detecting the rs8071417 site is shown in SEQ ID No: 45-46; the primer sequence for detecting the rs7764 site is shown in SEQ ID No: 47-48; the primer sequence for detecting the rs934397 site is shown in SEQ ID No: 49-50;The primer sequences for detecting the rs378528 site are shown in SEQ ID Nos: 51-52; the primer sequences for detecting the rs7602685 site are shown in SEQ ID Nos: 53-54; and the primer sequences for detecting the rs11166827 site are shown in SEQ ID Nos: 55-56. ;

[0011] The present application adopts the above-mentioned primer set to better detect the single nucleotide polymorphism site marker composition with good specificity and high sensitivity.

[0012] The present application 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 squamous cell carcinoma.

[0013] The present application 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 squamous cell carcinoma.

[0014] The present application 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 squamous cell carcinoma.

[0015] 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;

[0016] The recurrence risk score value = rs12337910×(-0.22199754)+rs1805794×(-0.21434354)+rs734784×(-0.25266573)+rs2303720×(-0.72753082)+rs5998267×(-0.55449395)+rs11662595×(0.2210625)+rs763780×(0.6 2683182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.2 7224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.07 437168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834);

[0017] Among them, the subjects whose recurrence risk score is greater than 2.61 are classified as having a high risk of recurrence and metastasis, i.e., the high-risk group, and the subjects whose recurrence risk score is less than or equal to 2.61 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

[0018] The above recurrence risk score was used to guide the postoperative treatment selection of lung squamous cell carcinoma. Patients in the low-risk group had a longer progression-free survival (HR = 0.049, 95% CI = 0.026-0.092, P = 4.09 × 10 -21 ), these patients do not need to be given adjuvant chemotherapy after radical surgery; high-risk patients should be given adjuvant therapy to prevent recurrence.

[0019] This application obtains the genotype information of each SNP site by sequencing the DNA amplification product of the above-mentioned SNP site, and obtains scores according to the genotypes of different SNP sites. For example, when the genotype of the SNP site rs12337910 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, and then the scores of each SNP site are brought into the above-mentioned recurrence risk score formula to finally obtain the recurrence risk score. The above-mentioned recurrence risk score can be used to determine the risk of postoperative recurrence and metastasis of patients with squamous cell lung cancer, so as to guide the selection of postoperative treatment for squamous cell lung cancer.

[0020] The present application also provides a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma, wherein the kit comprises the primer set.

[0021] The kit provided in the present application for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer can be used to assist in guiding individualized treatment and improving the prognosis of patients with squamous cell lung cancer.

[0022] The kit provided in the present application can accurately predict the postoperative prognosis of patients with squamous cell lung cancer by detecting SNP sites on the peripheral blood DNA or squamous cell lung cancer tissue DNA of patients; after the kit of the present application is applied to clinical testing, the patient's prognosis can be evaluated before treatment, and a more active and effective treatment plan can be formulated for patients with poor prognosis, thereby achieving individualized treatment of patients and improving survival rate; in addition, the kit can be used for sampling and testing of peripheral blood or squamous cell lung cancer tissue, and has the characteristics of convenient sampling, simple operation, and high timeliness.

[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 squamous cell carcinoma uses the following recurrence risk score value for evaluation;

[0024] The recurrence risk score value = rs12337910×(-0.22199754)+rs1805794×(-0.21434354)+rs734784×(-0.25266573)+rs2303720×(-0.72753082)+rs5998267×(-0.55449395)+rs11662595×(0.2210625)+rs763780×(0.6 2683182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.2 7224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.07 437168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834);

[0025] Among them, the subjects whose recurrence risk score is greater than 2.61 are classified as having a high risk of recurrence and metastasis, i.e., the high-risk group, and the subjects whose recurrence risk score is less than or equal to 2.61 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

[0026] Combining the results of gene SNP analysis with the follow-up data of included lung squamous cell carcinoma patients, the correlation analysis showed that the recurrence-free survival of low-risk patients was significantly longer than that of high-risk patients, with total recurrence-free survival HR = 0.049, 95% CI = 0.026-0.092, P = 4.09×10 -21 .

[0027] Preferably, the kit further comprises PCR buffer, dNTP, magnesium chloride, DNA polymerase and deionized water.

[0028] The kit of the present application also includes reagents commonly used in the art.

[0029] The present application also provides the use of the above-mentioned kit in the preparation of a product for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung carcinoma.

[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 of patients with squamous cell lung cancer and its application. The present application also provides a kit for predicting the risk of postoperative recurrence and metastasis of patients with squamous cell lung cancer, which can be used to assist in guiding individualized treatment and improving the prognosis of patients with squamous cell lung cancer; the kit provided by the present application can accurately predict the postoperative prognosis of patients with squamous cell lung cancer by detecting SNP sites on the peripheral blood DNA or squamous cell lung cancer tissue DNA of patients with squamous cell lung cancer; after the kit of the present application is applied to clinical testing, the prognosis of patients can be evaluated before treatment, and more active and effective treatment plans 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 squamous cell lung cancer 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 out SNP sites that predict the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer

[0036] (1) Selection of research samples:

[0037] The samples included in this application are all patients with squamous cell lung cancer who have been pathologically confirmed 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 squamous cell carcinoma 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 squamous cell carcinoma 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] rs12337910, rs1805794, rs734784, rs2303720, rs5998267, rs11662595, rs763780, rs2228210, rs386556287, r s16921260, rs180749, rs1801058, rs2229466, rs12213148, rs2617160, rs11084425, rs10181310, rs4792416, rs 9350450, rs2779154, rs3132306, rs17570453, rs8071417, rs7764, rs934397, rs378528, rs7602685 and rs11166827; PCR amplification also included PCR buffer, dNTP, magnesium chloride, polymerase, deionized water, and the PCR reaction program was set as: 94°C, 2min; 98°C, 10s, 56°C, 30s, 68°C, 40s, 33cycles; 4°C, ∞;.

[0043] (4) SNP locus typing:

[0044] The SNP sites included rs12337910, rs1805794, rs734784, rs2303720, rs5998267, rs11662595, rs763780, rs2228210, rs386556287, rs16921260, rs180749, rs1801058, rs2229466, rs12213148, rs2617 The DNA amplification products of rs160, rs11084425, rs10181310, rs4792416, rs9350450, rs2779154, rs3132306, rs17570453, rs8071417, rs7764, rs934397, rs378528, rs7602685, and rs11166827 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, TNM stage, pathological grade, tumor size, vascular invasion, lymphatic invasion, perineural invasion, pleural invasion, whether the surgical margin is negative, STAS, lymph node metastasis, PD-L1 expression status, EGFR, ROS1, ALK mutation status, SCC level, CYFRA21-1 level, NSE level, whether synchronous chemoradiotherapy was used, whether postoperative adjuvant chemotherapy was used, whether neoadjuvant chemotherapy was used, etc.) and the prognosis of lung squamous cell carcinoma;

[0047] Further, factors significantly associated with tumor prognosis (including gender, age, TNM stage, pathological grade, tumor size, vascular invasion, lymphatic invasion, perineural invasion, pleural invasion, whether surgical margin is negative, STAS, lymph node metastasis, PD-L1 expression status, EGFR, ROS1, ALK mutation status, SCC level, CYFRA 21-1 level, NSE level, whether synchronous chemoradiotherapy is used, whether postoperative adjuvant chemotherapy is used, whether neoadjuvant chemotherapy is used, etc.) were used as covariates to calculate the hazard ratio (HR), 95% confidence interval (95% CI) and P value of SNP genotype for tumor prognosis. 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 tests.

[0048] (6) Result analysis:

[0049] By detecting the genotype of the above SNP loci and using the prediction formula to obtain the recurrence risk score index of the subject, the formula is as follows:

[0050] Recurrence risk score = rs12337910 × (-0.22199754) + rs1805794 × (-0.21434354) + rs734784 × (-0.25266573) + rs2303720 × (-0.72753082) + rs5998267 × (-0.55449395) + rs11662595 × (0.2210625) + rs763780 × (0.626 83182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.27 224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.074 37168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834);

[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 rs12337910 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 2.61 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 2.61 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 squamous cell carcinoma. Patients in the low-risk group had a longer progression-free survival (HR = 0.049, 95% CI = 0.026-0.092, P = 4.09 × 10 -21 ), 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

[0054]

[0055]

[0056] Example 2: Application of a marker combination in a kit for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer

[0057] This embodiment provides the use of the above-mentioned marker combination in a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma.

[0058] The SNP marker composition obtained in Example 1 includes: rs12337910, rs1805794, rs734784, rs2303720, rs5998267, rs11662595, rs763780, rs2228210, rs386556287, rs16921260, rs180749, rs1801058, rs2229466, rs12213148, rs2617160, rs1108442 5. rs10181310, rs4792416, rs9350450, rs2779154, rs3132306, rs17570453, rs8071417, rs7764, rs934397, rs378528, rs7602685 and rs11166827. The above markers were used to construct a scoring model formula for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer using a proportional hazard regression model to calculate the recurrence risk score value. The specific model formula is:

[0059] Recurrence risk score = rs12337910 × (-0.22199754) + rs1805794 × (-0.21434354) + rs734784 × (-0.25266573) + rs2303720 × (-0.72753082) + rs5998267 × (-0.55449395) + rs11662595 × (0.2210625) + rs763780 × (0.626 83182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.27 224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.074 37168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834);

[0060] The recurrence risk score value was calculated using the above model formula, and subjects with a score greater than 2.61 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 2.61 were classified as having a low risk of recurrence and metastasis, i.e., a low-risk group.

[0061] Example 3: A kit for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer

[0062] This embodiment provides a kit for predicting the risk of postoperative recurrence and metastasis in patients with squamous cell lung cancer. 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] How to use the kit: Take peripheral blood or lung squamous cell carcinoma tissue samples from patients with lung squamous cell carcinoma and process them according to the standard phenol-chloroform method 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 PCR amplification is performed using specific amplification primers (see Table 1). The PCR 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] 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, ∞.

[0068] The risk score is calculated using the recurrence risk scoring formula of this application, and subjects with a score greater than 2.61 are classified as having a high risk of recurrence and metastasis, i.e., the high-risk group; subjects with a score less than or equal to 2.61 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group; patients in the low-risk group do not need to be given adjuvant chemotherapy or other treatments after radical surgery; patients at high risk should be given adjuvant treatment to prevent recurrence.

[0069] Combining the results of gene SNP analysis with the follow-up data of included lung squamous cell carcinoma patients, the correlation analysis showed that the recurrence-free survival of low-risk patients was significantly longer than that of high-risk patients, with total recurrence-free survival HR = 0.049, 95% CI = 0.026-0.092, P = 4.09×10 -21 ,like Figure 1 shown).

[0070] 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 squamous cell carcinoma, characterized in that: The single nucleotide polymorphism site marker composition includes rs12337910, rs1805794, rs734784, rs2303720, rs5998267, rs11662595, rs763780, rs2228210, rs386556287, rs16921260, rs180749, rs1801058, rs2229466, rs at least one of rs12213148, rs2617160, rs11084425, rs10181310, rs4792416, rs9350450, rs2779154, rs3132306, rs17570453, rs8071417, rs7764, rs934397, rs378528, rs7602685 and rs11166827.

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 ID Nos: 1-56.

3. The primer set according to claim 2, characterized in that The primer sequences for detecting the rs12337910 site are shown in SEQ ID Nos: 1 to 2; the primer sequences for detecting the rs1805794 site are shown in SEQ ID Nos: 3 to 4; the primer sequences for detecting the rs734784 site are shown in SEQ ID Nos: 5 to 6; the primer sequences for detecting the rs2303720 site are shown in SEQ ID Nos: 7 to 8; the primer sequences for detecting the rs5998267 site are shown in SEQ ID Nos: 9 to 10; the primer sequences for detecting the rs11662595 site are shown in SEQ ID Nos: 11 to 12; the primer sequences for detecting the rs763780 site are shown in SEQ ID Nos: 13 to 14; the primer sequences for detecting the rs2228210 site are shown in SEQ ID Nos: 15 to 16; the primer sequences for detecting the rs386556287 site are shown in SEQ ID No: 17-18; the primer sequence for detecting the rs16921260 site is shown in SEQ ID No: 19-20; the primer sequence for detecting the rs180749 site is shown in SEQ ID No: 21-22; the primer sequence for detecting the rs1801058 site is shown in SEQ ID No: 23-24; the primer sequence for detecting the rs2229466 site is shown in SEQ ID No: 25-26; the primer sequence for detecting the rs12213148 site is shown in SEQ ID No: 27-28; the primer sequence for detecting the rs2617160 site is shown in SEQ ID No: 29-30; the primer sequence for detecting the rs11084425 site is shown in SEQ ID No: 31-32; the primer sequence for detecting the rs10181310 site is shown in SEQ ID No: 33-34; the primer sequence for detecting the rs4792416 site is shown in SEQ ID No: 35-36; the primer sequence for detecting the rs9350450 site is shown in SEQ ID No: 37-38; the primer sequence for detecting the rs2779154 site is shown in SEQ ID No: 39-40; the primer sequence for detecting the rs3132306 site is shown in SEQ ID No: 41-42; the primer sequence for detecting the rs17570453 site is shown in SEQ ID No: 43-44; the primer sequence for detecting the rs8071417 site is shown in SEQ ID No: 45-46; the primer sequence for detecting the rs7764 site is shown in SEQ ID No: 47-48; the primer sequence for detecting the rs934397 site is shown in SEQ ID No: 49-50;The primer sequences for detecting the rs378528 site are shown in SEQ ID Nos: 51-52; the primer sequences for detecting the rs7602685 site are shown in SEQ ID Nos: 53-54; and the primer sequences for detecting the rs11166827 site are shown in SEQ ID Nos: 55-56. ; 4. Use of the single nucleotide polymorphism site marker composition as described in claim 1 in the preparation of a kit for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma.

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 squamous cell carcinoma.

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 squamous cell carcinoma.

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 = rs12337910×(-0.22199754)+rs1805794×(-0.21434354)+rs734784×(-0.25266573)+rs2303720×(-0.72753082)+rs5998267×(-0.55449395)+rs11662595×(0.2210625)+rs763780×(0.6 2683182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.2 7224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.07 437168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834); Among them, the subjects whose recurrence risk score is greater than 2.61 are classified as having a high risk of recurrence and metastasis, i.e., the high-risk group, and the subjects whose recurrence risk score is less than or equal to 2.61 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

8. A kit for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma, 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 squamous cell lung carcinoma uses the following recurrence risk score values ​​for evaluation; The recurrence risk score value = rs12337910×(-0.22199754)+rs1805794×(-0.21434354)+rs734784×(-0.25266573)+rs2303720×(-0.72753082)+rs5998267×(-0.55449395)+rs11662595×(0.2210625)+rs763780×(0.6 2683182)+rs2228210×(-0.12347335)+rs386556287×(0.35318945)+rs16921260×(-0.40881666)+rs180749×(0.39101058)+rs1801058×(1.43027606)+rs2229466×(0.05553823)+rs12213148×(-0.2 7224861)+rs2617160×(-0.41250523)+rs11084425×(-0.08406672)+rs10181310×(0.30866848)+rs4792416×(0.5033008)+rs9350450×(0.36062993)+rs2779154×(-0.53839182)+rs3132306×(0.07 437168)+rs17570453×(0.55850984)+rs8071417×(1.20080923)+rs7764×(0.42011595)+rs934397×(-0.33953016)+rs378528×(0.27871644)+rs7602685×(0.44222808)+rs11166827×(1.01420834); Among them, the subjects whose recurrence risk score is greater than 2.61 are classified as having a high risk of recurrence and metastasis, i.e., the high-risk group, and the subjects whose recurrence risk score is less than or equal to 2.61 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

10. Use of the kit according to claim 8 or 9 in the preparation of a product for predicting the risk of postoperative recurrence and metastasis in patients with lung squamous cell carcinoma.

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