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

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker composition for predicting postoperative recurrence and metastasis risks of a liver cancer patient and application of the SNP marker composition. The composition comprises any one or more of rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs3213869, rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs11955631, rs17827807, rs7640235, rs2472692, rs7506696 or The SNP marker composition provided by the invention can accurately predict the prognosis of a liver cancer patient, can evaluate the prognosis of the patient before treatment, and formulates a more positive and effective treatment scheme for the patient with poor prognosis, thereby realizing individualized treatment of the patient.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and tumor medicine, and in particular to a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis of liver cancer patients and its application. Background Art

[0002] Globally, approximately 865,000 people are diagnosed with primary liver cancer each year, and approximately 758,000 people die from liver cancer each year. The main histological subtype of liver cancer is hepatocellular carcinoma (HCC), which is referred to as liver cancer for the sake of convenience. Surgical resection and radiofrequency ablation are currently one of the main treatments for liver cancer. About 50-70% of liver cancer patients relapse after surgical resection and radiofrequency ablation. There is a subgroup of patients with more aggressive tumors in liver cancer 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 liver cancer is the key to determining which patients need adjuvant therapy. At present, TNM staging, pathological grade, tumor size, and the presence of vascular invasion are commonly used to assess the risk of tumor recurrence and metastasis in patients with liver cancer after surgery. However, patients with the same TNM stage, pathological grade, tumor size, and vascular invasion may have very different prognoses. Therefore, the current prediction method of the postoperative recurrence assessment and staging system for liver cancer 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 liver cancer. However, to date, there is still no stable and reliable molecular marker used in clinical practice to predict the risk of recurrence and metastasis of liver cancer after surgery. 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 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, 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

[0003] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of single nucleotide polymorphism variation site molecular markers in predicting the risk of recurrence and metastasis of liver cancer after surgery, and to provide a group of SNP molecular markers for predicting the risk of recurrence and metastasis of liver cancer after surgery, so as to assist in guiding individualized treatment and improving the prognosis of liver cancer patients.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In the first aspect, the present invention provides a single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis of liver cancer patients. The marker composition of the present invention is a molecular marker composition of single nucleotide polymorphism (SNP) sites, and the SNP sites include: any one or more of rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs3213869, rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs11955631, rs17827807, rs7640235, rs2472692, rs7506696 or rs17254718. The present invention can predict the risk of recurrence and metastasis of liver cancer patients after radical treatment (surgery, radiofrequency ablation) by detecting the genotype of the above SNP sites in liver cancer patients.

[0006] In a second aspect, the present invention provides the use of the single nucleotide polymorphism site marker composition described in the first aspect in a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients.

[0007] In a third aspect, the present invention provides an application of the single nucleotide polymorphism site marker composition described in the first aspect in constructing a scoring model for predicting the risk of postoperative recurrence and metastasis in patients with liver cancer.

[0008] As a preferred implementation of the third aspect, 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 of the first aspect in DNA;

[0009] The recurrence risk score value = rs4925 × (0.16587136) + rs3809875 ×

[0010] (-0.07779507)+rs1201559×(0.10928937)+rs2729835×(0.17100035)+rs3764795×(-0.07437999)+rs3213837×(-0.29805032)+rs17563×(-0.01873964)+rs3213869×(0. 52569608)+rs3750050×(-0.25104883)+rs2066853×(-0.17367663)+rs10983347×(-0.11852972)+rs17319721×(-0.12864303)+rs7815456×(-0.34279256)+rs12001327×

[0011] (0.81795195)+rs11955631×(0.45868298)+rs17827807×(0.41434224)+rs7640235×(-0.93421262)+rs2472692×(0.33566485)+rs7506696×(0.33566485)+rs17254718×(-0.45879418);

[0012] Among them, the subjects whose recurrence risk score is greater than or equal to 0.64 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 0.64 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

[0013] In a fourth aspect, the present invention provides the use of the recurrence risk scoring formula described in the third aspect in a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients.

[0014] In a fifth aspect, the present invention provides primers used to detect the single nucleotide polymorphism site markers of the first aspect, wherein the primer set includes nucleotide sequences as shown in SEQ Nos: 1 to 40.

[0015] As a preferred implementation of the fifth aspect, the primer sequence for detecting the rs4925 site is as shown in SEQ No: 1-2; the primer sequence for detecting the rs3809875 site is as shown in SEQ No: 3-4; the primer sequence for detecting the rs1201559 site is as shown in SEQ No: 5-6; the primer sequence for detecting the rs2729835 site is as shown in SEQ No: 7-8; the primer sequence for detecting the rs3764795 site is as shown in SEQ No: 9-10; the primer sequence for detecting the rs3213837 site is as shown in SEQ No: 11-12; the primer sequence for detecting the rs17563 site is as shown in SEQ No: 13-14; the primer sequence for detecting the rs3213869 site is as shown in SEQ No: 15-16; the primer sequence for detecting the rs3750050 site is as shown in SEQ No: No: 17-18; the primer sequence for detecting the rs2066853 site is shown in SEQ No: 19-20; the primer sequence for detecting the rs10983347 site is shown in SEQ No: 21-22; the primer sequence for detecting the rs17319721 site is shown in SEQ No: 23-24; the primer sequence for detecting the rs7815456 site is shown in SEQ No: 25-26; the primer sequence for detecting the rs12001327 site is shown in SEQ No: 27-28; the primer sequence for detecting the rs11955631 site is shown in SEQ No: 29-30; the primer sequence for detecting the rs17827807 site is shown in SEQ No: 31-32; the primer sequence for detecting the rs7640235 site is shown in SEQ No: 33-34; the primer sequence for detecting the rs2472692 site is shown in SEQ No: Nos: 35-36; the primer sequence for detecting the rs7506696 site is shown in SEQ Nos: 37-38; the primer sequence for detecting the rs17254718 site is shown in SEQ Nos: 39-40.

[0016] In a sixth aspect, the present invention provides the use of the primers described in the fifth aspect in a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients.

[0017] In a seventh aspect, the present invention provides a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients, wherein the kit comprises the primer set described in the fifth aspect.

[0018] As a preferred embodiment of the seventh aspect, the kit further comprises PCR buffer, dNTP, magnesium chloride, DNA polymerase and deionized water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides single nucleotide polymorphism (SNP) markers associated with the risk of liver cancer recurrence and metastasis and their applications, as well as a detection kit that can be used to predict liver cancer recurrence and metastasis, to assist in guiding individualized treatment and improving the prognosis of liver cancer patients. The detection kit provided by the present invention can accurately predict the prognosis of liver cancer patients by detecting SNP sites on the peripheral blood DNA or liver cancer tissue DNA of liver cancer patients; after the kit of the present invention 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 achieving individualized treatment of patients and providing survival rate; in addition, the kit only needs to detect peripheral blood, and has the characteristics of convenient sampling, simple operation, and high timeliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the overall progression-free survival curve for high-risk patients and low-risk patients. DETAILED DESCRIPTION

[0022] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Example 1 Screening out SNP sites that predict the risk of postoperative recurrence and metastasis in patients with liver cancer

[0024] (1) Selection of research samples:

[0025] The samples included in this invention are all liver cancer patients 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 tumor, and have not received tumor-related radiotherapy, chemotherapy and other anti-tumor treatments before admission; patients are all voluntary participants in the study and signed informed consent. Patients included in the study were followed up regularly, every three months within two years after the end of treatment, and every six months thereafter.

[0026] (2) Extraction of genomic DNA from peripheral blood or liver cancer tissue:

[0027] 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 liver cancer 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.

[0028] (3) Amplification of SNP sites:

[0029] Use specific primers to amplify the following SNP sites. The primer sequences used are shown in Table 1:

[0030] rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs321386 9. rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs119556 31, rs17827807, rs7640235, rs2472692, rs7506696, rs17254718; PCR amplification also includes PCR buffer, dNTP, magnesium chloride, polymerase, deionized water, and the PCR reaction program is set as: 94℃, 2min; 98℃, 10s, 56℃, 30s, 68℃, 40s, 33cycles; 4℃, ∞;.

[0031] (4) SNP locus typing:

[0032] The DNA amplification products containing SNP sites rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs3213869, rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs11955631, rs17827807, rs7640235, rs2472692, rs7506696, and rs17254718 were sequenced to obtain the genotype information of the above sites.

[0033] (5) Statistical analysis methods:

[0034] The proportional hazards model (Cox model) was used to analyze the correlation between demographic and clinical factors (including gender, age, clinical stage, alpha-fetoprotein level, serum total bilirubin level, serum albumin level, pathological differentiation grade, presence or absence of vascular invasion, tumor size, presence or absence of satellite nodules, whether concurrent chemoradiotherapy was used, whether postoperative adjuvant chemotherapy was used, whether neoadjuvant chemotherapy was used, etc.) and the prognosis of liver cancer; further, the factors significantly associated with tumor prognosis (including gender, age, clinical stage, alpha-fetoprotein level, serum total bilirubin level, serum albumin level, pathological differentiation grade, presence or absence of vascular invasion, tumor size, presence or absence of satellite nodules, 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 (HR), 95% confidence interval (95% CI) and P value of SNP genotype for tumor prognosis. Statistical analysis was performed using R software, with a statistical significance level of P < 0.05, and all tests were two-sided tests.

[0035] (6) Result analysis:

[0036] 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:

[0037] Recurrence risk score = rs4925 × (0.16587136) + rs3809875 × (-0.07779507) + rs1201559 × (0.10928937) + rs2729835 × (0.17100035) + rs3764795 × (-0.07437999) + rs3213837 × (-0.29805032) + rs17563 × (-0.01873964) + rs3213869 × (0.52569608) + rs3750050 × (-0.25104883) + rs2066853 × (-0.17367663) + r s10983347×(-0.11852972)+rs17319721×(-0.12864303)+rs7815456×(-0.34279256)+rs12001327×(0.81795195)+rs11955631×(0.45868298)+rs17827807×(0.41434224)+rs7640235×(-0.93421262)+rs2472692×(0.33566485)+rs7506696×(0.33566485)+rs17254718×(-0.45879418);

[0038] 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 rs4925 is CC, the score of the genotype is 0 points, when the genotype of the site is CA (or AC), the score of the genotype is 1 point, and when the genotype of the site is AA, 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.

[0039] Those with a score greater than or equal to 0.64 were classified as having a high risk of recurrence and metastasis, i.e., the high-risk group, and those with a score less than 0.64 were classified as having a low risk of recurrence and metastasis, i.e., the low-risk group. Patients in the low-risk group had a longer progression-free survival (HR = 0.23, 95% CI = 0.16-0.34, P = 1.79 × 10-14), and these patients did not need to receive adjuvant chemotherapy after radical surgery; patients in the high-risk group should be given adjuvant therapy to prevent recurrence, such as sorafenib, bevacizumab plus tislelizumab, or transhepatic artery infusion chemotherapy.

[0040] Table 1: SNP site scores and amplification primers for different genotypes

[0041]

[0042]

[0043] Example 2

[0044] 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 liver cancer. The SNP marker composition obtained in Example 1 includes: rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs3213869, rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs11955631, rs17827807, rs7640235, rs2472692, rs7506696, rs17254718. The above markers are used to calculate the recurrence risk score value by constructing a scoring model formula for predicting the risk of postoperative recurrence and metastasis of liver cancer patients using a proportional hazard regression model. The model formula is specifically as follows:

[0045] Recurrence risk score = rs4925 × (0.16587136) + rs3809875 × (-0.07779507) + rs1201559 × (0.10928937) + rs2729835 × (0.17100035) + rs3764795 × (-0.07437999) + rs3213837 × (-0.29805032) + rs17563 × (-0.01873964) + rs3213869 × (0.52569608) + rs3750050 × (-0.25104883) + rs2066853 × (-0.17367663) + r s10983347×(-0.11852972)+rs17319721×(-0.12864303)+rs7815456×(-0.34279256)+rs12001327×(0.81795195)+rs11955631×(0.45868298)+rs17827807×(0.41434224)+rs7640235×(-0.93421262)+rs2472692×(0.33566485)+rs7506696×(0.33566485)+rs17254718×(-0.45879418);

[0046] The recurrence risk score value was calculated by the above model formula, and the subjects with a recurrence risk score value greater than or equal to 0.64 were classified as having a high risk of recurrence and metastasis, i.e., a high-risk group, and the subjects with a recurrence risk score value less than 0.64 were classified as having a low risk of recurrence and metastasis, i.e., a low-risk group.

[0047] Example 3

[0048] This embodiment provides a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients 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.

[0049] Kit usage: Take peripheral blood or liver cancer tissue samples from liver cancer patients 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.

[0050] After the PCR reaction was completed, DNA electrophoresis (1% agarose gel, voltage: 120V, time: 30 minutes) was performed to detect the PCR products, and the PCR products with the expected fragment size (673bp), single band and moderate brightness were selected for sequencing using a sequencer.

[0051] Table 2: PCR reaction system

[0052]

[0053]

[0054] 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, ∞.

[0055] The risk score is calculated by the recurrence risk scoring formula of the present invention, and the subjects whose recurrence risk score value is greater than or equal to 0.64 are classified as having a high risk of recurrence and metastasis, i.e., a high-risk group, and the subjects whose recurrence risk score value is less than 0.64 are classified as having a low risk of recurrence and metastasis, i.e., a low-risk group; for patients in the low-risk group, no adjuvant chemotherapy or the like may be given after radical surgery; for patients in the high-risk group, adjuvant treatment for preventing recurrence should be given, such as sorafenib, bevacizumab plus tislelizumab, or transhepatic artery infusion chemotherapy.

[0056] Combining the results of gene SNP analysis with the follow-up data of included liver cancer patients, the correlation analysis 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 = 0.23, 95% CI = 0.16-0.34, P = 1.79 × 10-14, such as Figure 1 shown).

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A single nucleotide polymorphism site marker composition for predicting the risk of postoperative recurrence and metastasis in patients with liver cancer, characterized in that: The composition includes: any one or more of rs4925, rs3809875, rs1201559, rs2729835, rs3764795, rs3213837, rs17563, rs3213869, rs3750050, rs2066853, rs10983347, rs17319721, rs7815456, rs12001327, rs11955631, rs17827807, rs7640235, rs2472692, rs7506696 or rs17254718.

2. Use of the single nucleotide polymorphism site marker composition as described in claim 1 in a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients.

3. 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 liver cancer.

4. The scoring model according to claim 3, characterized in that: 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 according to claim 1 in DNA; The recurrence risk score value = rs4925×(0.16587136)+rs3809875×(-0.07779507)+rs1201559×(0.10928937)+rs2729835×(0.17100035)+rs3764795×(-0.07437999)+rs3213837×(-0.29805032)+rs17563×(-0.01873964)+rs3213869×(0.52569608)+rs3750050×(-0.25104883)+rs2066853×(-0.17367663)+rs rs10983347×(-0.11852972)+rs17319721×(-0.12864303)+rs7815456×(-0.34279256)+rs12001327×(0.81795195)+rs11955631×(0.45868298)+rs17827807×(0.41434224)+rs7640235×(-0.93421262)+rs2472692×(0.33566485)+rs7506696×(0.33566485)+rs17254718×(-0.45879418); Among them, the subjects whose recurrence risk score is greater than or equal to 0.64 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 0.64 are classified as having a low risk of recurrence and metastasis, i.e., the low-risk group.

5. Use of the recurrence risk scoring formula as described in claim 3 in a kit for predicting the risk of postoperative recurrence and metastasis in patients with liver cancer.

6. A primer set used for detecting the single nucleotide polymorphism site marker composition as claimed in claim 1, characterized in that: The primer set includes nucleotide sequences as shown in SEQ Nos: 1-40.

7. The primer set according to claim 6, characterized in that The primer sequences for detecting the rs4925 site are shown in SEQ Nos: 1 to 2; the primer sequences for detecting the rs3809875 site are shown in SEQ Nos: 3 to 4; the primer sequences for detecting the rs1201559 site are shown in SEQ Nos: 5 to 6; the primer sequences for detecting the rs2729835 site are shown in SEQ Nos: 7 to 8; the primer sequences for detecting the rs3764795 site are shown in SEQ Nos: 9 to 10; the primer sequences for detecting the rs3213837 site are shown in SEQ Nos: 11 to 12; the primer sequences for detecting the rs17563 site are shown in SEQ Nos: 13 to 14; the primer sequences for detecting the rs3213869 site are shown in SEQ Nos: 15 to 16; the primer sequences for detecting the rs3750050 site are shown in SEQ Nos: No: 17-18; the primer sequence for detecting the rs2066853 site is shown in SEQ No: 19-20; the primer sequence for detecting the rs10983347 site is shown in SEQ No: 21-22; the primer sequence for detecting the rs17319721 site is shown in SEQ No: 23-24; the primer sequence for detecting the rs7815456 site is shown in SEQ No: 25-26; the primer sequence for detecting the rs12001327 site is shown in SEQ No: 27-28; the primer sequence for detecting the rs11955631 site is shown in SEQ No: 29-30; the primer sequence for detecting the rs17827807 site is shown in SEQ No: 31-32; the primer sequence for detecting the rs7640235 site is shown in SEQ No: 33-34; the primer sequence for detecting the rs2472692 site is shown in SEQ No: Nos: 35-36; the primer sequence for detecting the rs7506696 site is shown in SEQ Nos: 37-38; the primer sequence for detecting the rs17254718 site is shown in SEQ Nos: 39-40.

8. Use of the primer set as claimed in claim 6 in a kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients.

9. A kit for predicting the risk of postoperative recurrence and metastasis of liver cancer patients, characterized in that: The kit comprises the primer set as claimed in claim 6.

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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