CN109385478B

CN109385478B - Application of gene marker for detecting 19-GCS in preparation of product for diagnosing early lung adenocarcinoma prognosis

Info

Publication number: CN109385478B
Application number: CN201811562197.2A
Authority: CN
Inventors: 安广宇; 姚健楠; 葛洋; 刘健
Original assignee: Beijing Chaoyang Hospital
Current assignee: Beijing Chaoyang Hospital
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-08-03
Anticipated expiration: 2038-12-20
Also published as: CN109385478A

Abstract

The invention discloses application of a gene marker of 19-GCS in preparation of a product for diagnosing early lung adenocarcinoma prognosis. The invention claims the use of a product for obtaining a 19-GCS score for the preparation of a system for prognosis of patients with early stage lung adenocarcinoma. Subjects with a 19-GCS score below the threshold are at low risk, subjects with a 19-GCS score greater than or equal to the threshold are at high risk, and the prognosis of low risk is better than that of high risk. 19-GCS score-sum of abundance of 8 positive regulatory genes in lung cancer tissue of subject-sum of 11 negative regulatory genes in lung cancer tissue of subject. 8 positive regulatory genes: ST14, RDH14, DARS, CTSL2, C16orf88, ARL6IP1, SLC6A10P, MEX 3D. 11 negative regulatory genes: SERPINB6, FAM162B, PARK2, GPX3, HLA-DQB1, HOXA2, LOC100128288, RPL13AP17, SETDB2, SLC5A4, LRP2 BP. The invention has great application and popularization value for prognosis of early lung cancer.

Description

Application of gene marker for detecting 19-GCS in preparation of product for diagnosing early lung adenocarcinoma prognosis

Technical Field

The invention relates to application of gene markers for detecting 19-GCS in preparation of products for diagnosing early lung adenocarcinoma prognosis.

Background

Lung adenocarcinoma (lung adenocarinoma) is a type of lung cancer, and belongs to non-small cell carcinoma. Lung adenocarcinoma originates in the bronchial mucosal epithelium, a few originate in the mucus glands of large bronchi, and most of them originate in smaller bronchi, peripheral lung cancer. Early stage lung adenocarcinoma generally has no obvious clinical symptoms, often found on chest X-ray examination, and appears as a rounded or oval mass.

Surgical resection is the most main treatment means for early lung adenocarcinoma and is the only method for clinically curing lung cancer at present. Lung cancer surgery is divided into radical surgery and palliative surgery, and radical resection should be strived for to achieve optimal and complete tumor resection and reduce tumor metastasis and recurrence.

Disclosure of Invention

The invention aims to provide application of a gene marker of 19-GCS in preparing a product for diagnosing early lung adenocarcinoma prognosis.

The invention claims the application of the product A in preparing a system for prognosis of early lung adenocarcinoma patients; the product A is a product for detecting 19 specific genes;

the invention also protects the application of the product A and the carrier recorded with the method A-1 in preparing a system for prognosis of patients with early lung adenocarcinoma;

the method A-1 comprises the following steps:

(1) detecting the abundance of the 19 specific genes in the lung cancer tissue of the subject;

(2) obtaining a 19-GCS score;

(3) subjects with a 19-GCS score below the threshold are at low risk, subjects with a 19-GCS score greater than or equal to the threshold are at high risk, and the prognosis of low risk is better than that of high risk.

The invention also protects the application of the product A and the carrier recorded with the method A-2 in preparing a system for prognosis of patients with early lung adenocarcinoma;

the method A-2 comprises the following steps:

(2) obtaining a 19-GCS score; 19-GCS score-value a minus value B; the value A is the sum of the abundances of 8 positive regulatory genes in the lung cancer tissue of the subject, and the value B is the sum of 11 negative regulatory genes in the lung cancer tissue of the subject;

(3) subjects with the 19-GCS score smaller than the threshold value are low in risk, subjects with the 19-GCS score larger than or equal to the threshold value are high in risk, the prognosis of the high risk is poor, and the prognosis of the low risk is good.

The invention also claims the application of the product B in the preparation of a system for prognosis of patients with early lung adenocarcinoma; the product B is a product for detecting the abundance of 19 specific genes in lung cancer tissues of a subject.

The invention also protects the application of the product B and the carrier recording the method B-1 in preparing a system for prognosis of patients with early lung adenocarcinoma;

the method B-1 comprises the following steps:

(2) obtaining a 19-GCS score;

The invention also protects the application of the product B and the carrier recording the method B-2 in preparing a system for prognosis of patients with early lung adenocarcinoma;

the method B-2 comprises the following steps:

The invention also claims the application of the product C in preparing a system for prognosis of patients with early lung adenocarcinoma; the product C is a product for obtaining a 19-GCS score.

The invention also protects the application of the product C and the carrier which is recorded with the judgment standard-1 in the preparation of a system for prognosis of patients with early lung adenocarcinoma; the judgment standard-1 is as follows: subjects with a 19-GCS score below the threshold are at low risk, subjects with a 19-GCS score greater than or equal to the threshold are at high risk, and the prognosis of low risk is better than that of high risk.

The invention also protects the application of the product C and the carrier which is recorded with the judgment standard-2 in the preparation of a system for prognosis of patients with early lung adenocarcinoma; the judgment standard-2 is as follows: subjects with the 19-GCS score smaller than the threshold value are low in risk, subjects with the 19-GCS score larger than or equal to the threshold value are high in risk, the prognosis of the high risk is poor, and the prognosis of the low risk is good.

The invention also protects a system for use in prognosing patients with early stage lung adenocarcinoma; the system is a system A, a system B or a system C.

The system A comprises the product A. The system A further comprises a vector loaded with the method A-1 or the method A-2.

The system B comprises the product B. The system B also comprises a carrier loaded with the method B-1 or the method B-2.

The system C comprises the product C. The system also comprises a carrier loaded with the judgment standard-1 or the judgment standard-2.

19-GCS score-value a minus value B; the value A is the sum of the abundances of 8 positive regulatory genes in the lung cancer tissue of the subject, and the value B is the sum of 11 negative regulatory genes in the lung cancer tissue of the subject.

The 19 specific genes are the following 19 genes: ST14 gene, RDH14 gene, DARS gene, CTSL2 gene, C16orf88 gene, ARL6IP1 gene, SLC6A10P gene, MEX3D gene, SERPINB6 gene, FAM162B gene, PARK2 gene, GPX3 gene, HLA-DQB1 gene, HOXA2 gene, LOC100128288 gene, RPL13AP17 gene, SETDB2 gene, SLC5A4 gene, LRP2BP gene.

The 8 positive regulatory genes are the following 8 genes: ST14 gene, RDH14 gene, DARS gene, CTSL2 gene, C16orf88 gene, ARL6IP1 gene, SLC6A10P gene, MEX3D gene.

The 11 negative regulatory genes are the following 11 genes: SERPINB6 gene, FAM162B gene, PARK2 gene, GPX3 gene, HLA-DQB1 gene, HOXA2 gene, LOC100128288 gene, RPL13AP17 gene, SETDB2 gene, SLC5A4 gene, and LRP2BP gene.

Any of the above systems is a kit or a detection platform.

Any of the above subjects are patients with early stage lung adenocarcinoma.

The early stage lung adenocarcinoma is stage I lung adenocarcinoma.

Any of the above thresholds is 53.41.

The abundance of 19 specific genes in any one of the lung cancer tissues can be specifically as follows: abundance of 19 specific genes in lung cancer tissue RNA.

Any of the above abundances may specifically be: taking the RNA of lung cancer tissues of a subject as a sample, and carrying out high-throughput sequencing by an illuminatHiseq platform and an NGS technology to obtain a normalized value of an original value by using matched software.

The prognosis is (a) and/or (b):

(a) the postoperative survival rate of low risk patients is higher than that of high risk patients;

(b) patients at low risk have a lower risk of new tumors after surgery than patients at high risk.

The low risk prognosis is better than the high risk by (a) and/or (b):

The prognosis is preferably (a) and/or (b):

(a) the survival rate after operation is high;

(b) the risk of new tumors after operation is low.

The prognosis difference is represented by (a) and/or (b):

(a) the survival rate after operation is low;

(b) the risk of new tumors after operation is high.

The survival rate is 1-year survival rate after operation or 2-year survival rate after operation or 3-year survival rate after operation or 5-year survival rate after operation.

The invention has great application and popularization values for early lung adenocarcinoma prognosis.

Drawings

FIG. 1 is a Kaplan-Meier curve of recurrence-free survival in each training set for the high-risk group and the low-risk group of 19-GCS (groups 1 to 6).

FIG. 2 is a Kaplan-Meier curve of recurrence-free survival in each training set for the high-risk group and the low-risk group of 19-GCS (groups 7 to 10).

FIG. 3 is a Kaplan-Meier curve of recurrence-free survival in each test set for the high-risk group and the low-risk group of 19-GCS (groups 1 to 6).

FIG. 4 is a Kaplan-Meier curve of recurrence-free survival in each test set for the high-risk group and the low-risk group of 19-GCS (groups 7 to 10).

FIG. 5 is a Kaplan-Meier curve of recurrence-free survival in the total case set for the high risk group and the low risk group of 19-GCS.

FIG. 6 is a Kaplan-Meier curve of overall survival in the total case set for the high risk group and the low risk group of 19-GCS.

FIG. 7 is a Kaplan-Meier plot of recurrence-free survival and overall survival for the high risk group and low risk group of 19-GCS in the disease case library collected by Okayama.

FIG. 8 is a Kaplan-Meier curve of progression free survival for high risk and low risk groups of 19-GCS in the disease case library collected by Sheden.

FIG. 9 is a Kaplan-Meier plot of overall survival for the high risk group and the low risk group of 19-GCS in the disease case library collected by Tang.

Figure 10 compares 19-GCS scores in groups based on primary treatment effect.

FIG. 11 is a comparison of 19-GCS scores based on whether there were new tumor groupings after initial treatment.

FIG. 12 is a comparison of 19-GCS scores based on new emerging tumor type groupings.

Figure 13 is a 19-GCS score compared against follow-up treatment effect groups.

FIG. 14 is the statistics of the ratio of the 5-year follow-up of new tumors in example 2.

FIG. 15 is the results of the follow-up 5-year overall survival statistics in example 2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1, 19-establishment of a method for diagnosing early stage Lung adenocarcinoma

Early lung adenocarcinoma-related gene data in the american tumor database (TCGA database), early lung adenocarcinoma-related gene data in published literature, and clinically discovered early lung adenocarcinoma-related gene data were collected. 17334 early lung adenocarcinoma related genes are obtained in total.

The method comprises the steps of randomly extracting 35% of sample information of 275 patients with stage I lung adenocarcinoma in a TCGA database as a test set (test sets, n is 97), 65% of sample information as a training set (training sets, n is 178), randomly allocating 10 different groups of test sets and training sets, dividing the expression of each gene in each group into high expression (top 25% expression) and low expression (bottom 25% expression), calculating the P value of each gene in each group, which is relevant to RFS prognosis, by Cox analysis, taking the genes which are relevant to RFS with statistical significance, namely relevant to RFS, obtaining at least 541 and at most 1696 meaningful genes in the 10 groups of training sets, wherein 19 genes coexist in the 10 groups of meaningful genes.

The obtained combination of 19 genes was named as 19-GCS (19gene consensus signature) gene marker.

The number of positive control genes is 8, the species is Homo sapiens (human), and the specific information is shown in Table 1. The number of the negative control genes is 11, the species is Homo sapiens (human), and the specific information is shown in Table 2.

TABLE 1

TABLE 2

Adding the abundances of 8 positive control genes (values obtained by carrying out high-throughput sequencing on an original value by an illuminaHiseq platform by an NGS technology and standardizing the original value by matched software) to obtain a value A. Adding the abundances of 11 negative control genes (values obtained by carrying out high-throughput sequencing on an original value by an illuminatHiseq platform by an NGS technology and standardizing the original value by matched software) to obtain a value B. The value B was subtracted from the value A to give a 19-GCS score.

The Kaplan-Meier curves for the recurrence-free survival of the 19-GCS scores in each training set are shown in FIGS. 1 and 2, and the relevant data are shown in Table 1. The Kaplan-Meier curves for recurrence-free survival for the 19-GCS score in each set of tests are shown in FIGS. 3 and 4, and the data for correlation is shown in Table 2. In fig. 1, 2, 3 and 4, the solid line bar represents the low risk group classified based on the 19-GCS score, and the dotted line bar represents the high risk group classified based on the 19-GCS score. The results show that the 19-GCS high-risk and low-risk groups have statistical differences in the Kaplan-Meier curves of recurrence-free survival rates in each training set and test set.

TABLE 1

TABLE 2

Scores for the 19-GCS gene markers were calculated and the heatmap showed that as the scores increased, the risk increased with the score, 235 out of 275 patients with stage I lung adenocarcinoma were low risk cases and 40 were high risk cases, but the recurrence rate in the high risk case area was significantly higher than in the low risk area. The Kaplan-Meier curves for recurrence-free survival (RFS) for the high-risk and low-risk groups of 19-GCS are shown in FIG. 5. The Kaplan-Meier curves of Overall Survival (OS) for the high risk group and the low risk group of 19-GCS are shown in FIG. 6. The high-risk group and the low-risk group of 19-GCS have differences in OS and RFS in patients with lung adenocarcinoma at the whole stage I, are respectively applicable to the stage IA and the stage IB of the lung adenocarcinoma, and have obviously increased 19-GCS scores of tumor tissues compared with normal tissues.

Multifactorial analysis suggests that 19-GCS is an independent prognostic factor for OS and RFS.

The utility of 19-GCS was verified by obtaining individual gene information and sample information from several databases of NCBI GEO, see FIGS. 7, 8 and 9. In FIGS. 7, 8 and 9, Low-Risk represents the Low Risk group and High-Risk represents the High Risk group. 19-GCS also had prognostic value, and the Kaplan-Meier curve showed significant differences in both OS and RFS for the high-risk and low-risk groups.

Wilcox test and the FDR correction are used for comparing the genes with high risk and low risk related differences, FDR <0.01 is selected as a standard, 777 genes are screened out in total, and through KEGG, WikiPathways and Reactome pathway enrichment analysis, 7 specific related pathways are prompted and respectively: the complete and coaggation cassettes pathway (KEGG; reaction), the muscle concentration pathway and the cardiac concentration pathway (reaction), the gateway pathway (WikiPathway), and the cell cycle/promotion pathway, the animal surveillance/response pathway and the DNA damage/repay pathway are commonly contained in KEGG, reaction and WikiPathway. The Proliferation pathway is one of the most important pathways in the pathways related to tumor formation, cell cycle and Proliferation, the Hallmark-50G2/M pathway gene expression analysis shows that the 19-GCS risk score is related to the low-risk and high-risk components of normal tissues and tumor tissues, the expression of Cyclin D (checkpoint at the G1/S phase) is gradually reduced, the expression of Cyclin E/A/B (promoting the cells to transform to the S phase and the G2/M phase) is gradually increased from the normal tissues to the low-risk and high-risk groups of tumors, and the 19-GCS score is positively related to the expression of Proliferation indexes Ki-67 and PCNA in clinical application. Immune-mediated killing of tumor cells generally directs infiltration by antigen presentation and subsequent specific immune cells, CD4+ helper T cells and CD8+ cytotoxic T cells. CD4+ T cells are activated by detection of Major Histocompatibility Complex (MHC) class II presented antigen, whereas CD8+ T cells initiate cell destruction upon detection of MHC class I presented antigen. With an increasing 19-GCS risk score, there is a gradual decrease in MHC subunit gene expression and an almost complete lack of expression in high-risk tumors. Compared with normal tissues and low-risk tumors, the antigen presentation is obviously absent in high-risk tumors. Expression of T cells, CD8+ T cells and other types of tumor infiltrating immune cells is significantly reduced in high risk tumors compared to normal tissue, low risk tumors. Immune-related and cell proliferation-related gene analysis and pathway analysis suggest that the 19-GCS high-risk group accelerates cell proliferation and promotes immune escape.

PD: the sum of the maximum diameters of target focuses is at least increased by more than or equal to 20 percent or new focuses appear in the disease progression (progressive disease). SD: stable disease (stable disease), the sum of the maximal diameters of the target lesions either diminished to short PR or increased to short PD. PR: partial response, the sum of the maximum diameters of the target lesions decreased by more than or equal to 30% for at least 4 weeks. CR: complete remission (complete response), disappearance of all target lesions, no new ones appearing, and normal tumor markers for at least 4 weeks. The 19-GCS score is adopted to predict the curative effect of the first and subsequent treatments of the patient with stage I lung adenocarcinoma, the 19-GCS high risk is related to SD and PD in the first and subsequent treatments, the 19-GCS score of the patient with PD is higher than that of the patient with CR/PR/SD, the 19-GCS score of the patient with relapse is higher, and the risk score of distant metastasis is higher than that of local relapse. The results are shown in FIGS. 10 to 13.

Example 2 prognosis of early stage Lung adenocarcinoma based on 19-GCS score

95 patients with stage I lung adenocarcinoma (confirmed in hospital) were all volunteers with informed consent.

1. Taking lung cancer tissues obtained by the operation of a patient, and immediately freezing and storing in liquid nitrogen at-80 ℃ after the operation.

2. Extracting RNA of tissues, and detecting the abundance of 19 genes related to the genes in the table 1 and the table 2 in the RNA (the original value obtained by high-throughput sequencing by an NGS technology on the illuminathHiseq platform is standardized by matched software for scoring).

The abundances of the 8 positive regulatory genes were added to obtain a value a. And adding the abundances of the 11 negative control genes to obtain a value B. The value B was subtracted from the value A to give a 19-GCS score.

Based on the 19-GCS score, 53.41 was used as a threshold, and a group with a High Risk (High-Risk) was classified as one having a threshold value of not less than 53.41, and a group with a Low Risk (Low-Risk) was classified as one having a threshold value of not less than 53.41. 95 patients with stage I lung adenocarcinoma, 72 patients classified into the low risk group, and 23 patients classified into the high risk group.

Each patient was followed up for a long period of time after surgery.

Statistics of the rate of new tumors within 5 years of surgery are shown in figure 14. Within 1 year after surgery, the rate of new tumors in the high risk group patients after surgery was 25.5%, and the rate of new tumors in the low risk group patients after surgery was 4.6%. Within 2 years after surgery, the rate of new tumors in the high risk group patients after surgery was 67.0%, and the rate of new tumors in the low risk group patients after surgery was 23.6%.

The results of the overall survival statistics within 5 years after surgery are shown in fig. 15. The overall survival rate for the high risk group patients was 11.1% and for the low risk group patients was 45.0% 5 years post-surgery.

Claims

1. The product B is applied to the preparation of a system for prognosis of patients with early lung adenocarcinoma;

the product B is a product for detecting the abundance of 19 specific genes in lung cancer tissues of a subject;

the subject is an early stage lung adenocarcinoma patient;

2. The product C is applied to the preparation of a system for prognosis of patients with early lung adenocarcinoma;

the product C is a product for obtaining a 19-GCS score; 19-GCS score-value a minus value B; the value A is the sum of the abundances of 8 positive regulatory genes in the lung cancer tissue of the subject, and the value B is the sum of the abundances of 11 negative regulatory genes in the lung cancer tissue of the subject;

the subject is an early stage lung adenocarcinoma patient;

the 8 positive regulatory genes are the following 8 genes: ST14 gene, RDH14 gene, DARS gene, CTSL2 gene, C16orf88 gene, ARL6IP1 gene, SLC6A10P gene, MEX3D gene;

3. Use of a product b as claimed in claim 1 and a vector describing the method b-1 for the preparation of a system for prognosis of patients with early stage lung adenocarcinoma;

the method B-1 comprises the following steps:

(2) obtaining a 19-GCS score; 19-GCS score-value a minus value B; the value A is the sum of the abundances of 8 positive regulatory genes in the lung cancer tissue of the subject, and the value B is the sum of the abundances of 11 negative regulatory genes in the lung cancer tissue of the subject;

(3) subjects with the 19-GCS score smaller than the threshold are at low risk, subjects with the 19-GCS score larger than or equal to the threshold are at high risk, and the prognosis of the low risk is better than that of the high risk; the threshold is 53.41;

4. Use of the product of claim 2 and a vector bearing criteria-1 for the preparation of a system for prognosis of patients with early stage lung adenocarcinoma;

the judgment standard-1 is as follows: subjects with the 19-GCS score smaller than the threshold are at low risk, subjects with the 19-GCS score larger than or equal to the threshold are at high risk, and the prognosis of the low risk is better than that of the high risk; the threshold is 53.41.

5. Use of a product b as claimed in claim 2 and a vector describing method b-2 for the preparation of a system for prognosis of patients with early stage lung adenocarcinoma;

the method B-2 comprises the following steps:

(3) subjects with the 19-GCS score smaller than the threshold are low-risk subjects, subjects with the 19-GCS score larger than or equal to the threshold are high-risk subjects, the prognosis of the high-risk subjects is poor, and the prognosis of the low-risk subjects is excellent; the threshold is 53.41;

6. Use of the product of claim 2 and a vector bearing criteria-2 for the preparation of a system for prognosis of patients with early stage lung adenocarcinoma;

the judgment standard-2 is as follows: subjects with the 19-GCS score smaller than the threshold are low-risk subjects, subjects with the 19-GCS score larger than or equal to the threshold are high-risk subjects, the prognosis of the high-risk subjects is poor, and the prognosis of the low-risk subjects is excellent; the threshold is 53.41.