ADGRA2 mutation gene and its application as a marker for bone metastasis of lung cancer

Abstract

The present invention provides a specific human ADGRA2 mutant gene for the first time, which is formed by the g.18084_18086insGCT mutation of the wild-type ADGRA2 gene, and the specific sequence of the gene fragment where the mutation is located is shown in SEQ ID NO: 2. Lung cancer patients with the g.18084_18086insGCT mutation in the ADGRA2 gene have a high risk of bone metastasis, which can be used as an effective and reliable molecular marker for lung cancer bone metastasis; the present invention also provides a detection kit and detection method for the ADGRA2 mutant gene based on digital PCR, which can be simply and effectively applied to the analysis of bone metastasis in lung cancer patients, which is helpful for the early detection and early intervention of bone metastasis in lung cancer patients, and can also provide clues for the mechanistic analysis of lung cancer bone metastasis.

Description

Technical Field

[0001] The present invention relates to the field of medical molecular biology, and in particular to ADGRA2 mutant genes, ADGRA2 mutant gene detection and application thereof as a molecular marker in lung cancer metastasis. Background Art

[0002] Lung cancer is the most common malignant tumor of the respiratory system. It is generally believed that smoking, air pollution and heredity are closely related to the high incidence of lung cancer. However, many molecular mechanisms of the occurrence and development of lung cancer have not yet been fully clarified clinically. Lung cancer often has no obvious symptoms in the early stages, but it is highly malignant and prone to metastasis. More than half of patients are diagnosed in the late stages and are often accompanied by metastasis. In addition, after various treatment methods such as surgery, radiotherapy and chemotherapy, a large number of recurrences and metastases still occur, making it difficult to achieve complete cure. Therefore, recurrence and metastasis are the main causes of death from lung cancer (more than 90%), and its 5-year survival rate is less than 15%.

[0003] Lung cancer can metastasize to various organs in the late stage, and is prone to intrathoracic dissemination and distant metastasis, such as metastasis to the lungs, pleura, bones, brain, liver and adrenal glands. Among them, brain and bones are the most common metastatic sites of lung cancer. 20%-65% of lung cancer patients will have brain metastasis, and the average natural survival time is only 1-2 months, and the prognosis is very poor. At present, the comprehensive application of treatment methods such as surgery, radiotherapy and chemotherapy can prolong the survival of patients with brain metastasis of lung cancer to a certain extent and significantly improve their quality of life. Bone metastasis of lung cancer can cause symptoms such as bone pain, pathological fractures, vertebral compression or deformation, and spinal cord compression, which seriously reduces the quality of life of patients. The median survival time is only 6 months to 10 months. Bone metastasis of lung cancer should be treated with comprehensive treatment based on systemic treatment. On the basis of systemic treatment of the primary disease, a multidisciplinary comprehensive treatment model can be adopted for bone metastasis. An individualized comprehensive treatment plan can be formulated in a planned and reasonable manner to reduce or delay the occurrence of bone metastasis complications and bone-related events, which will help improve the quality of life of patients.

[0004] The occurrence, development and metastasis of lung cancer involve the temporal mutation of multiple genes. It is a complex disease with multiple genes, multiple pathways and multiple stages. It undergoes a series of molecular events during its occurrence, development and metastasis, among which mutations of certain important genes occur, leading to its continuous development and changes, and the continuous accumulation of mutations leads to progressive deterioration. The mutant genes at these key nodes can be considered as the driver genes of lung cancer, controlling its growth, progression, invasion and metastasis. Some current serological and imaging indicators in clinical practice have a certain lag, and abnormalities can often be detected only after obvious symptoms appear, and the sensitivity and specificity are often not high. For the metastasis of lung cancer, it is necessary to reveal its key gene mutations at the molecular level in order to provide corresponding molecular markers, and to assess the risk and location of its metastasis as early as possible, so as to intervene and treat as soon as possible, and ultimately effectively improve the patient's survival and quality of life.

[0005] However, due to the complexity of lung cancer, the molecular mechanism of its metastasis has not yet been resolved. In view of this, the present invention uses lung cancer metastasis pedigrees as the starting point for exploration. A malignant tumor pedigree refers to a family in which multiple members have one or several malignant tumors in the same anatomical position, which has the characteristics of familial onset. However, the clinical phenotype and development between family members may be different. Therefore, under a similar genetic background, the molecular nature behind it can be analyzed by the differential characterization of tumors in the family, which helps to find the key control genes. Therefore, lung cancer metastasis families are a good model for exploring lung cancer metastasis. The present invention first collects lung cancer pedigrees with coexisting lung cancer brain metastasis and bone metastasis, and classifies groups of similar molecular groups through whole exome sequencing analysis of the metastatic patient group, finds out the differential gene mutations of brain metastasis and bone metastasis groups, and then comprehensively verifies through the population distribution and expression of mutant genes, animal model simulation and clinical prediction, thereby revealing that a specific ADGRA2 mutant gene is a key determining gene for lung cancer brain metastasis and bone metastasis, and provides a detection kit and detection method based on digital PCR. The ADGRA2 mutant gene provided by the present invention has not been reported before, and its clinical significance and value for metastasis of lung cancer patients have not been revealed before. Summary of the invention

[0006] The present invention provides for the first time a specific human ADGRA2 mutant gene, which can be used as a molecular biomarker for brain and bone metastasis of lung cancer, and also provides a digital PCR-based ADGRA2 specific mutant gene detection kit and detection method, which can be applied to the risk prediction of brain and bone metastasis in lung cancer patients, and can also provide clues for the mechanistic analysis of brain and bone metastasis of lung cancer.

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0008] The object (1) of the present invention is to provide a human ADGRA2 mutant gene, wherein the sequence of the region 37796883-37844896 in the DNA fragment with NCBI accession number NC_000008.11 is a wild-type ADGRA2 gene reference sequence, and the wild-type ADGRA2 gene sequence with NCBI accession number NC_000008.11 is referred to. The ADGRA2 mutant gene is formed by the wild-type ADGRA2 gene undergoing a g.650_652insAAC mutation, with the first nucleotide of the wild-type ADGRA2 gene as the starting site (i.e., the nucleotide 37796883 in the DNA fragment with NCBI accession number NC_000008.11), that is, the wild-type ADGRA2 gene has three nucleotides AAC inserted at its 650-652 sites, and the specific sequence of the gene fragment where the mutation is located is shown in SEQ ID NO: 1, and the remaining gene sequences refer to the wild-type ADGRA2 gene sequence. Lung cancer patients with g.650_652insAAC in the ADGRA2 gene have a high risk of brain metastasis. Therefore, this mutant gene can be used as a molecular marker for brain metastasis in lung cancer patients and be used to construct detection kits and methods for analyzing brain metastasis in lung cancer patients.

[0009] It should be noted that the nucleotide at position 37796883 of the DNA fragment with NCBI accession number NC_000008.11 is the starting position nucleotide of the wild-type ADGRA2 gene, and the nucleotide at position 37844896 of the DNA fragment with NCBI accession number NC_000008.11 is the last nucleotide of the wild-type ADGRA2 gene. For the convenience of expression, the ADGRA2 gene mutation is referenced and identified with the first nucleotide of the wild-type ADGRA2 gene as the starting point (position 1). For the sake of simplicity, SEQ ID NO: 1 only shows the sequence of 500 bp (a total of 1003 bp) before and after the ADGRA2 mutant gene with the g.650_652insAAC mutation as the center. The rest of the sequence of the ADGRA2 mutant gene is consistent with the sequence of the wild-type ADGRA2 gene, and the only difference between the two is the insertion variation of the three nucleotides AAC at positions 650-652.

[0010] The object (2) of the present invention is to provide another human ADGRA2 mutant gene, wherein the sequence of the region 37796883-37844896 in the DNA fragment with NCBI accession number NC_000008.11 is a wild-type ADGRA2 gene reference sequence, and the wild-type ADGRA2 gene sequence with NCBI accession number NC_000008.11 is referred to. The ADGRA2 mutant gene is formed by the wild-type ADGRA2 gene undergoing a g.18084_18086insGCT mutation, with the first nucleotide of the wild-type ADGRA2 gene as the starting site (i.e., the nucleotide 37796883 in the DNA fragment with NCBI accession number NC_000008.11), that is, the wild-type ADGRA2 gene has three nucleotides GCT inserted at its 18084-18086 sites. The specific sequence of the gene fragment where the mutation is located is shown in SEQ ID NO: 2, and the remaining gene sequences refer to the wild-type ADGRA2 gene sequence. Lung cancer patients with the g.18084_18086insGCT mutation in the ADGRA2 gene have a high risk of bone metastasis. Therefore, this mutant gene can be used as a molecular marker for bone metastasis in lung cancer patients and be used to construct detection kits and methods for analyzing bone metastasis in lung cancer patients.

[0011] It should be noted that the nucleotide at position 37796883 of the DNA fragment with NCBI accession number NC_000008.11 is the starting position nucleotide of the wild-type ADGRA2 gene, and the nucleotide at position 37844896 of the DNA fragment with NCBI accession number NC_000008.11 is the last nucleotide of the wild-type ADGRA2 gene. For the convenience of expression, the ADGRA2 gene mutation is referenced and identified with the first nucleotide of the wild-type ADGRA2 gene as the starting point (position 1). For simplicity, SEQ ID NO: 2 only shows the sequence of 500 bp before and after the ADGRA2 mutant gene with the g.18084_18086insGCT mutation (1003 bp in total). The rest of the sequence of the ADGRA2 mutant gene is consistent with the sequence of the wild-type ADGRA2 gene, and the only difference between the two is the insertion mutation of the three nucleotides GCT at the 18084-18086 sites.

[0012] The object (3) of the present invention is to provide a detection kit for the g.650_652insAAC mutant fragment of the ADGRA2 gene based on digital PCR, the kit comprising primers and probes capable of amplifying the g.650_652insAAC mutant fragment of the ADGRA2 gene, specifically digital PCR amplification primers as shown in SEQ ID NO: 3 and SEQ ID NO: 4 and a detection probe targeting the g.650_652insAAC mutation as shown in SEQ ID NO: 7, wherein the 5' end and 3' end bases of the probe as shown in SEQ ID NO: 7 are modified with a fluorescent group and a quenching group, respectively.

[0013] The object (4) of the present invention is to provide a detection kit for the g.18084_18086insGCT mutant fragment of the ADGRA2 gene based on digital PCR, the kit comprising primers and probes capable of amplifying the g.18084_18086insGCT mutant fragment of the ADGRA2 gene, specifically digital PCR amplification primers as shown in SEQ ID NO: 5 and SEQ ID NO: 6 and a detection probe targeting the g.18084_18086insGCT mutation as shown in SEQ ID NO: 8, wherein the 5' end and 3' end bases of the probe as shown in SEQ ID NO: 8 are modified with a fluorescent group and a quenching group, respectively.

[0014] The object (5) of the present invention is to provide a method for detecting and analyzing specific ADGRA2 mutant genes in lung cancer patients based on digital PCR, the method comprising the following main steps: obtaining cancer tissue or blood samples from lung cancer patients, the cancer tissue can be samples obtained by surgery or biopsy, and the blood sample can be peripheral venous blood that is easy to obtain, and extracting genomic DNA from the cancer tissue sample or extracellular free DNA from the blood sample using a corresponding kit. Using the extracted DNA as a template, the g.650_652insAAC mutant fragment of the ADGRA2 gene is amplified using digital PCR primers as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and a detection probe targeting the g.650_652insAAC mutation as shown in SEQ ID NO: 7 is used. According to a conventional digital PCR amplification system, the amplification conditions are: 96°C pre-denaturation for 8 minutes; 95°C denaturation for 15 seconds, 58°C annealing and extension for 35 seconds, 40 cycles; 98°C final reaction for 10 minutes. For the g.18084_18086insGCT mutation fragment of the ADGRA2 gene, digital PCR primers as shown in SEQ ID NO: 5 and SEQ ID NO: 6 were used for amplification, and a detection probe targeting the g.18084_18086insGCT mutation as shown in SEQ ID NO: 8 was used. According to the conventional digital PCR amplification system, the amplification conditions were: 96°C pre-denaturation for 8 minutes; 95°C denaturation for 15 seconds, 59°C annealing and extension for 30 seconds, 40 cycles; 98°C final reaction for 10 minutes. If the g.650_652insAAC mutation of the ADGRA2 gene is detected in the sample of the lung cancer patient, it indicates that the patient has a high risk of brain metastasis; if the g.18084_18086insGCT mutation of the ADGRA2 gene is detected in the sample of the lung cancer patient, it indicates that the patient has a high risk of bone metastasis.

[0015] The human ADGRA2 mutant genes with g.650_652insAAC mutation and g.18084_18086insGCT mutation provided by the present invention can be used as effective and reliable molecular markers for brain and bone metastasis of lung cancer, respectively, and can also provide clues for the mechanistic analysis of brain and bone metastasis of lung cancer; these two ADGRA2 mutant genes and their detection methods can be simply and effectively applied to the analysis and prediction of brain and bone metastasis of lung cancer patients, which is helpful for the early detection and early intervention of brain and bone metastasis of lung cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of high-frequency gene mutation clustering of 23 lung cancer metastasis patient samples. Family #11 contains 3 lung cancer metastasis patients, including 11.1 and 11.1', two brain metastasis patients, and 11.2, a bone metastasis patient.

[0017] Figure 2 This is the Sanger sequencing local map of the ADGRA2 wild-type gene (A) and mutant gene (B) at the g.650_652insAAC mutation site. The boxed area in the figure is where the mutation occurs.

[0018] Figure 3 This is the Sanger sequencing local map of the ADGRA2 wild-type gene (A) and mutant gene (B) at the g.18084_18086insGCT mutation site. The boxed area in the figure is where the mutation occurs.

[0019] Figure 4 The ADGRA2 protein immunohistochemistry results in cancer tissue sections (S1, S7 and S12 are shown as examples). The dark-stained area on the left is the cancerous tissue, and the light-stained area on the right is the adjacent tissue. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments.

[0021] Example 1: Specific gene mutations in a family group of patients with lung cancer metastasis

[0022] Specific lung cancer metastasis pedigrees in Henan Province were collected. Each pedigree contained at least 2 patients with clinically pathologically confirmed lung cancer metastasis (including patients with metastasis at the time of diagnosis and patients without metastasis at the time of diagnosis but metastasis occurred after consultation. The clinical pathological types of lung cancer in the family should be consistent, such as non-small cell lung cancer, and without other tumors and serious diseases). Lung cancer patients in the same family must be direct relatives (such as father and daughter, mother and son, and siblings, but husband and wife are not included). Only one of the metastatic lesions, brain or bone, was found at the time of consultation and during the treatment process. The same lung cancer pedigree contained patients with brain metastasis and bone metastasis at the same time. A total of 23 patients with lung cancer metastasis from 11 specific lung cancer metastasis pedigrees (numbered #1-#11) were collected for research (of which families #1-#10 each had only two patients with lung cancer metastasis, corresponding in order, family #1 was patients 1.1 and 1.2, family #2 was patients 2.1 and 2.2…family #10 was patients 10.1 and 10.2, and family #11 contained three patients with lung cancer metastasis, 11.1, 11.1', and 11.2, where .1 or .1' represented brain metastasis and .2 represented bone metastasis). Exploring patients with different metastatic sites in the same lung cancer family is helpful to explore the genetic determinants associated with brain metastasis and bone metastasis through analytical comparison, which is more advantageous than simply collecting sporadic cases.

[0023] Under the premise of meeting clinical diagnosis and treatment requirements and informed consent of patients, pre-treatment and post-treatment samples were collected as much as possible, mainly including pre-treatment blood samples and blood samples at important points during treatment (including circulating tumor DNA, ctDNA) and tissue samples (paraffin blocks of postoperative cancer lesions or biopsy tissue samples, if any). Each blood sample was no less than 5 ml of anticoagulated venous blood, and no less than 6 paraffin sections (1 cm long × 1 cm wide × 10 um thick, with cancer tissue area exceeding 50%) were taken from each FFPE (formalin-fixed, paraffin-embedded) paraffin block sample, as well as biopsy tissue samples obtained by one puncture. Cell-free DNA (cell-free DNA, extracellular free DNA, including ctDNA) or genomic DNA were extracted from the corresponding samples of patients using free DNA and genomic extraction kits (QIAamp Circulating Nucleic Acid kit, QIAamp DNA Blood Mini Kit, QIAamp Fast DNA Tissue Kit, and QIAampDNA FFPE Tissue Kit from Qiagen, Germany) according to the instructions. Each patient's whole blood sample is first processed into plasma and paired peripheral blood mononuclear cells (PBMC) and then DNA is extracted separately. The sequencing results of the PBMC samples will be used as the individual's genomic genetic background to screen out cancer-related gene mutations. The concentration and purity of all sample genomic DNA are tested by nucleic acid analyzers such as Qubit4.0, and after passing the test, they are sent to a genetic company for whole exome sequencing (WES) analysis.

[0024] For WES, the sequencing library was constructed using Agilent's SureSelect All exon 50 MB (V6) capture kit, and high-throughput DNA sequencing was run on Illumina's HiSeq 4000 sequencer, with an average coverage depth of no less than 100× (PBMC) and 1000× (other samples). For FASTQ raw date, the adapters and reads that did not meet the quality standards were removed first, and then the clean reads obtained were aligned to the human genome reference genome sequence GRCh38 using Burrows-WheelerAligner software (BWA). Somatic DNA variations were analyzed and annotated using Genome Analysis Toolkit (GATK), ANNOVAR, and VarScan251 software (the p value for mutation identification was 0.1, and the minimum quality score was 15). Databases such as dbSNP and 1000 Genomes were then combined to exclude polymorphic sites and determine mutation site information, and high-frequency mutations were selected for Sanger sequencing verification. The sequencing results showed that for the sequencing samples of 23 patients with lung cancer metastasis, an average of 36 high-frequency gene mutations were detected (ranging from 9 to 156 genes, involving a total of 317 genes, mutation frequency >1%, sequencing reads >500), and each gene had multiple mutation types.

[0025] Furthermore, considering that lung cancer is a complex disease with multiple gene mutations and may have multiple molecular types, only by analyzing lung cancer of the same molecular group can we find the pathogenic gene mutations. The gene mutations detected in all samples of each patient were combined, and 317 mutant genes were used as the benchmark, with mutations recorded as 1 and non-mutation recorded as 0. The gene mutation information was converted into a digital matrix, and then these 23 matrices were clustered based on Euclidean distance (i.e., the sum of squared deviations method). The results are as follows: Figure 1As shown. According to the high-frequency gene mutation sequencing results of 23 lung cancer metastasis patient samples, they can be divided into 4 categories, of which 1-7 are category I, 8-14 are category II, 15-19 are category III, and 20-23 are category IV (for the convenience of display, the samples and numbers have been reorganized). Among the 11 lung cancer metastasis pedigrees #1-#11, only the 7 pedigrees #1-#7, with a total of 14 metastatic patients, are clustered in one category (including categories I and II) and show obvious regular clustering. Among them, the brain metastasis patients of these 7 families are clustered into one category (category I, including 1-7, i.e. patients 1.1-7.1), and the bone metastasis patients are also clustered into one category (category II, including 8-14, i.e. patients 1.2-7.2). However, the 9 lung cancer patients in the 4 families #8-#11 did not show regular clustering. In the Class III (including 8.1, 8.2, 10.1, 11.1' and 11.2) and Class IV (including 9.1, 9.2, 10.2 and 11.1), there were both patients with brain metastasis and patients with bone metastasis, and even related patients in the same family. Therefore, the subsequent analysis was only conducted on the 7 lung cancer metastasis families #1-#7, in order to better find out the molecular rules behind the phenotype.

[0026] For the 14 patients in the 7 lung cancer metastasis families #1-#7, the gene mutation information measured in their samples was further analyzed, and the gene mutations that met the 7 brain metastasis patients 1.1-7.1 and the 7 bone metastasis patients 1.2-7.2 were found in the high-frequency gene mutations. The results showed that the g.650_652insAAC mutation of the ADGRA2 gene was detected in the samples of the 7 brain metastasis patients (1.1-7.1) of the families #1-#7, and the g.18084_18086insGCT mutation of the ADGRA2 gene was detected in the samples of the 7 bone metastasis patients (1.2-7.2). The g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene were not present in any samples of other lung cancer metastasis patients in categories III and IV. The ADGRA2 gene mutations detected in the tissue samples and blood samples of the same patient were consistent. Furthermore, special common PCR primers were designed for amplification of the g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene. The amplification primer pair sequences of the g.650_652insAAC mutation gene fragment are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the amplification primer pair sequences of the g.18084_18086insGCT mutation gene fragment are shown in SEQ ID NO: 5 and SEQ ID NO: 6. The PCR amplification products were subjected to Sanger sequencing, and the results showed that the Sanger sequencing results of the ADGRA2 gene mutation of all successfully amplified samples were consistent with the WES sequencing results.

[0027] Combined with the WES and Sanger sequencing results, the sequence of the g.650_652insAAC mutant gene fragment is shown in SEQ ID NO: 1, the sequence of the g.18084_18086insGCT mutant gene is shown in SEQ ID NO: 2, and the Sanger sequencing maps at the mutation sites are shown in Figure 2 and Figure 3. It should be noted that the nucleotide at position 37796883 of the DNA fragment with NCBI accession number NC_000008.11 is the starting position nucleotide of the wild-type ADGRA2 gene, and the nucleotide at position 37844896 of the DNA fragment with NCBI accession number NC_000008.11 is the last nucleotide of the wild-type ADGRA2 gene. For the convenience of expression, the ADGRA2 gene mutation is referenced and identified with the first nucleotide of the wild-type ADGRA2 gene as the starting point (position 1). For the sake of simplicity, SEQ ID NO: 1 only shows the sequence of 500 bp (a total of 1003 bp) before and after the ADGRA2 mutant gene with the g.650_652insAAC mutation as the center. The rest of the sequence of the ADGRA2 mutant gene is consistent with the sequence of the wild-type ADGRA2 gene, and the only difference between the two is the insertion variation of the three nucleotides AAC at positions 650-652. SEQ ID NO: 2 only shows the sequence of 500bp before and after the g.18084_18086insGCT mutation of the ADGRA2 mutant gene (1003bp in total). The rest of the sequence of the ADGRA2 mutant gene is consistent with that of the wild-type ADGRA2 gene. The only difference between the two is the insertion mutation of the three nucleotides GCT at the 18084-18086 sites.

[0028] Based on the results of this example, it can be inferred that ADGRA2 gene mutation is closely related to the metastasis of lung cancer, and different mutation types of ADGRA2 gene (g.650_652insAAC mutation and g.18084_18086insGCT mutation) are related to different metastatic sites of lung cancer (brain metastasis and bone metastasis), which will be further verified below.

[0029] Example 2 Distribution and expression of g.650_652insAAC mutation and g.18084_18086insGCT mutation of ADGRA2 gene in lung cancer metastasis patient population

[0030] The sample was further expanded to explore the distribution of specific mutations of the ADGRA2 gene in patients with conventional lung cancer metastasis. Patients with conventional lung cancer metastasis confirmed by clinical pathology were collected, including 17 patients with brain metastasis and 12 patients with bone metastasis (the selection of these 29 patients with lung cancer metastasis was not subject to family requirements) and 30 patients with lung cancer metastasis in other parts. Informed consent was obtained from the patients and no less than 5 ml of anticoagulated venous blood was drawn during the clinical examination. Plasma was separated, and the QIAamp Circulating Nucleic Acid kit of Qiagen, Germany, was used to extract cfDNA (cell-free DNA, which contains ctDNA, i.e., circulating tumor DNA) from blood samples according to the instructions. Using the extracted cfDNA as a template, according to the primers, probes and conditions in Table 1, the reagents and instruments (ddPCR Supermix and QX200ddPCR system) of BioRad, USA were selected to prepare the system and operate the equipment according to the instructions to detect the g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene. As shown in Seq ID NO: 7 and Seq ID NO: 8, the 5' end base of the probe is modified with the fluorescent group FAM, and the 3' end base is modified with the fluorescent quenching group BHQ1. All samples were tested for g.650_652insAAC mutation and g.18084_18086insGCT mutation at the same time, and each sample was tested in parallel three times. The results are shown in Table 2. The results showed that the g.650_652insAAC mutation of the ADGRA2 gene only existed in samples of patients with brain metastasis, and the g.18084_18086insGCT mutation of the ADGRA2 gene only existed in samples of patients with bone metastasis. These two specific mutations of the ADGRA2 gene were not detected in samples of patients with other metastatic sites. Specifically, up to 70.59% (12 / 17) of patients with brain metastasis had g.650_652insAAC mutations in the ADGRA2 gene, and up to 75.00% (9 / 12) of patients with bone metastasis had g.18084_18086insGCT mutations in the ADGRA2 gene. Due to the complexity of cancer, the remaining patients with brain metastasis or bone metastasis who did not have the corresponding two specific mutations in the ADGRA2 gene may be affected by other genetic mechanisms.

[0031] Mutation type Amplified fragment sequence Amplification primers Mutation Probes Amplification conditions g.650_652insAAC mutation Seq ID No: 1 Seq ID NO: 3 and Seq ID NO: 4 Seq ID No: 7 Pre-denaturation at 96°C for 8 min; denaturation at 95°C for 15 s, annealing and extension at 58°C for 35 s, 40 cycles; final reaction at 98°C for 10 min. g.18084_18086insGCT mutation Seq ID No: 2 Seq ID NO: 5 and Seq ID NO: 6 Seq ID NO: 8 Pre-denaturation at 96°C for 8 min; denaturation at 95°C for 15 s, annealing and extension at 59°C for 30 s, 40 cycles; final reaction at 98°C for 10 min.

[0032] ADGRA2 gene mutation types Number of patients with brain metastases Average copy number (copies / ul) Proportion of patients with brain metastases Number of patients with bone metastasis Average copy number (copies / ul) Proportion of patients with bone metastasis g.650_652insAAC mutation 12 96.32±6.92 70.59%（12 / 17） 0 0 0 g.18084_18086insGCT mutation 0 0 0 9 135.7±8.31 75.00%（9 / 12）

[0033] According to the results in Table 2, 4 patients with brain metastasis (3 of them had g.650_652insAAC mutation of ADGRA2 gene, and 1 did not have g.650_652insAAC mutation of ADGRA2 gene) and 4 patients with bone metastasis (3 of them had g.18084_18086insGCT mutation of ADGRA2 gene, and 1 did not have g.18084_18086insGCT mutation of ADGRA2 gene) were selected from these 29 patients with lung cancer metastasis, and 4 patients were selected from 30 patients with metastasis in other parts. The extracted cfDNA was subjected to whole exome sequencing according to the method in Example 1, and the ADGRA2 gene mutation detection results obtained by whole exome sequencing were consistent with the detection results of digital PCR. In addition, except for g.650_652insAAC mutation and g.18084_18086insGCT mutation, whole exome sequencing did not find other high-frequency mutations (>1%) of ADGRA2 gene in the samples. The distribution of two specific mutations of the ADGRA2 gene (g.650_652insAAC mutation and g.18084_18086insGCT mutation) in patients with brain metastasis and bone metastasis of lung cancer further indicates that the ADGRA2 gene is closely related to the metastasis of lung cancer, and that different mutation types of the ADGRA2 gene (g.650_652insAAC mutation and g.18084_18086insGCT mutation) are related to different metastatic sites of lung cancer (brain metastasis and bone metastasis).

[0034] Then, immunohistochemistry was used to explore the expression of ADGRA2 gene in cancerous tissue and non-cancerous tissue at the protein distribution level. For all lung cancer metastasis patients (52 patients) in Example 1 and Example 2, 6 patients with brain metastasis (5 samples of which contained g.650_652insAAC mutation of ADGRA2 gene, and 1 did not contain ADGRA2 gene mutation) and 5 patients with bone metastasis (4 samples of which contained g.18084_18086insGCT mutation of ADGRA2 gene, and 1 did not contain ADGRA2 gene mutation) had metastasis after surgery. These 11 lung cancer patients (patient numbers S1-S11) had undergone surgical resection of the primary cancer lesion before, so tissue paraffin block samples were retained. In addition, 6 patients (patient numbers S12-S17) with paraffin block samples retained after surgical resection of the primary cancer lesion were selected from the above 30 patients with lung cancer metastasis in other parts as controls. Wax blocks of cancerous lesions (including adjacent tissues) were sliced ​​(5 μm) and placed in an oven at 65°C for 40 min. The slices were dewaxed and hydrated with xylene and gradient alcohol, and then placed in PBS buffer. Antigen retrieval was performed by microwave for 30 min. 3% H2O2 was added and incubated at room temperature for 15 min. 10% sheep serum was added and blocked at room temperature for 30 min to block nonspecific binding. Monoclonal antibodies (TEM5 Polyclonal Antibody, ADGRA2, also known as TEM5, from Thermo Company, USA) were added according to the instructions. The slices were incubated at 4°C overnight, and conventional secondary antibodies were added and incubated at room temperature for 1 h. DAB was used for color development, hematoxylin was used for counterstaining, the slices were washed with water to turn blue, gradient alcohol was used for dehydration and xylene was used for transparency, and the slices were sealed with neutral gum. The slices were observed under a microscope (by two experienced pathologists) and scanned. Under an optical microscope, the cell membrane was observed to be unstained, light yellow, dark yellow, or brownish yellow. Five different fields of view were taken for each section, and the expression of ADGRA2 protein was comprehensively evaluated according to the proportion of stained cells and the depth of staining. More than 90% of cells were stained and brownish yellow, which was evaluated as "+++++", less than 10% of cells were stained and light yellow, which was evaluated as "+", and basically no cell staining was evaluated as "-". The results are shown in Table 3 and Figure 4 shown.

[0035] Patient number ADGRA2 gene mutation test results Expression in cancer tissues Expression in adjacent tissues S1 g.650_652insAAC mutation +++++ + S2 g.650_652insAAC mutation +++++ + S3 g.650_652insAAC mutation +++++ + S4 g.650_652insAAC mutation +++++ - S5 g.650_652insAAC mutation +++++ - S6 none - - S7 g.18084_18086insGCT mutation +++++ + S8 g.18084_18086insGCT mutation +++++ + S9 g.18084_18086insGCT mutation +++++ + S10 g.18084_18086insGCT mutation +++++ + S11 none - - S12-S17 none - -

[0036] The results in Table 3 show that ADGRA2 protein is only expressed in large quantities in samples of patients with ADGRA2 gene mutations (S1-S5 and S7-S10), regardless of whether it is samples of patients with brain metastasis or bone metastasis. In addition, ADGRA2 protein is expressed in large quantities in cancer tissue cells, but is lowly expressed or not expressed in adjacent tissues (Tables 3 and Figure 4 , Figure 4The results are shown in S1, S7 and S12, and the rest are shown in S1, S7 and S12. The samples without ADGRA2 gene mutations had no ADGRA2 protein expression in either the cancer lesions or adjacent tissues. This verifies the association between specific ADGRA2 gene mutations and brain metastasis / bone metastasis in lung cancer patients at the protein level.

[0037] Example 3 Mutation analysis of brain and bone metastases of lung cancer formed by in situ injection of human lung cancer cells

[0038] The commonly used lung cancer cell line NCI-H1299 (ATCC catalog number: CRL-5803) was used. This cell line is a stable human non-small cell lung cancer (NSCLC) cell line derived from lymph nodes. The lung cancer cell line was tested to be free of g.650_652insAAC mutations and g.18084_18086insGCT mutations of the ADGRA2 gene. 60 specific pathogen-free SPF-grade BALB / c immunodeficient nude mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) were taken and intraperitoneally anesthetized with 5% chloral hydrate. A skin incision was made at the lower edge of the right scapula. The right lung was observed through the pleura by pushing aside the subcutaneous fat and muscle. 10ul of a single cell suspension of the lung cancer cell line NCI-H1299 was slowly injected into the intercostal space using a microsyringe. The cell concentration was 10×10 6 / ml, and then suture the wound for routine care and feeding. Once the mice die, they are dissected in time, and the main tissues and organs such as lungs, liver, bones, brain, and heart are taken, and pathological sections are made to observe the formation and metastasis of cancer foci, and blood samples are taken at the same time. When half of the mice die, all are euthanized, and the main tissues and organs such as lungs, liver, bones, brain, and heart are taken, and pathological sections are made to observe the formation and metastasis of cancer foci, and blood samples are taken at the same time. The results showed that 38 of the 60 mice successfully achieved lung cancer modeling, of which 23 had metastasis, including 6 brain metastases and 5 bone metastases. The remaining brain tissue and bone tissue metastases and blood of mice with brain metastasis and bone metastasis were taken out respectively, and genomic DNA and cfDNA were extracted. The g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene were detected with reference to the system and method in Example 2 (each sample detects both mutations at the same time). The results showed that the g.650_652insAAC high-frequency mutation of the ADGRA2 gene was detected in the primary lesions, metastatic lesions and blood of 4 out of 6 mice with lung cancer brain metastasis, and the g.18084_18086insGCT high-frequency mutation of the ADGRA2 gene was detected in the metastatic lesions and blood of 4 out of 5 mice with lung cancer bone metastasis. However, the g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene were not detected in the samples of mice with metastasis in other parts. This further proves that the ADGRA2 gene mutation is closely related to the metastasis of lung cancer, and lung cancer cells with the g.650_652insAAC mutation of the ADGRA2 gene are prone to brain metastasis, and lung cancer cells with the g.18084_18086insGCT mutation of the ADGRA2 gene are prone to bone metastasis. There is an association between specific mutations in the ADGRA2 gene and brain metastasis / bone metastasis of lung cancer. The presence of the ADGRA2 gene g.650_652insAAC mutation indicates a high risk of brain metastasis, and the presence of the ADGRA2 gene g.18084_18086insGCT mutation indicates a high risk of bone metastasis.

[0039] G protein-coupled receptor (GPCR) is a super membrane protein family that can recognize a variety of extracellular signaling molecules and regulate almost all known physiological processes. Its abnormality will cause many physiological process disorders and lead to various diseases and even tumors. GPR124 belongs to class B GPCR, also known as TEM5 or ADGRA2. Tumor angiogenesis is one of the hallmarks of malignant solid tumors and an important factor affecting their invasion and metastasis. ADGRA2 gene expression is closely related to angiogenesis, and its effect on angiogenesis in the central nervous system has been studied more. The present invention finds that specific mutations in the ADGRA2 gene will cause high expression of ADGRA2 protein in the cancer tissue of lung cancer patients, and are respectively related to brain metastasis and bone metastasis of lung cancer. Combined with the characteristics of the ADGRA2 gene, it is speculated that the high expression of ADGRA2 protein caused by the ADGRA2 gene mutation in the cancer lesion will promote the formation of blood vessels in the cancer lesion and lead to the formation of metastases; however, different ADGRA2 variant proteins have different promoting effects on the blood vessels required for lung cancer cells to form corresponding metastatic lesions in different organs (brain and bone), thus leading to different metastatic site preferences.

[0040] Example 4 ADGRA2 specific gene mutation can predict brain metastasis and bone metastasis in lung cancer patients

[0041] Patients with lung cancer diagnosed by clinical pathology (both those who can undergo resection and those who cannot undergo resection) were further collected. They were required to meet the requirements of first diagnosis of lung cancer and no metastasis was found at the time of diagnosis and no other tumors were combined. 5 ml of peripheral anticoagulated venous blood was drawn before admission for treatment. 5 ml of peripheral venous blood was drawn at key nodes during the diagnosis and treatment process (no less than 3 times) until the patient had metastatic lesions confirmed by clinical imaging and pathology, and patients with brain or bone metastasis were included. There were 14 effective patients in the end (patient numbers Y1-Y14), including 4 patients with brain metastasis of lung cancer, 5 patients with bone metastasis, and 5 patients with metastasis in other parts (control group). The free nucleic acid in all blood samples of these 14 patients with lung cancer metastasis was extracted according to the method in Example 1, and the extracted DNA was used as a template. The two specific mutations (g.650_652insAAC mutation and g.18084_18086insGCT mutation) of the ADGRA2 gene of all samples were detected by digital PCR according to the method in Example 2. The results are shown in Table 4.

[0042] The results showed that the g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene were only present in the blood samples of some patients with brain metastasis (Y2, Y7 and Y13) and bone metastasis (Y1, Y11 and Y14), and were not detected in the samples of patients with metastasis in other parts, which is consistent with the results in Example 1 and Example 2. In addition, before treatment, no metastasis was found in clinical blood and imaging, but the g.650_652insAAC mutation of the ADGRA2 gene was detected in the blood sample, and then brain metastasis was found clinically (Y2, Y7 and Y13); before treatment, no metastasis was found in clinical blood and imaging, but the g.18084_18086insGCT mutation of the ADGRA2 gene was detected in the blood sample, and then bone metastasis was found clinically (Y1, Y11 and Y14). In other words, in all these six lung cancer patients, molecular detection of specific mutations in the ADGRA2 gene was earlier than the discovery and diagnosis of metastases by clinical imaging and / or pathology. Compared with the discovery and diagnosis of metastases by clinical imaging and / or pathology, the earliest detection of specific mutations in the ADGRA2 gene in blood samples ranged from 37 to 106 days earlier. This further indicates that the g.650_652insAAC mutation and g.18084_18086insGCT mutation of the ADGRA2 gene can be used as molecular markers for brain metastasis and bone metastasis in lung cancer patients, respectively. Lung cancer patients with these two specific gene mutations of the ADGRA2 gene detected in the blood are at high risk of brain metastasis and bone metastasis, respectively. Molecular testing is earlier than imaging and pathology results and can better reflect the molecular nature of the tumor, especially when combined with liquid biopsy and second-generation genome sequencing technology, which will make it more advantageous for the prediction and early detection of metastasis.

[0043] Patient number ADGRA2 gene mutation in blood samples before treatment ADGRA2 gene mutation in blood samples during treatment Final metastatic site The time interval between the earliest detection of specific ADGRA2 gene mutations in blood samples and the diagnosis of metastasis by imaging / pathology (days) Y1 g.18084_18086insGCT g.18084_18086insGCT bone 64 Y2 g.650_652insAAC g.650_652insAAC brain 106 Y3 - - Other parts none Y4 - - brain none Y5 - - Other parts none Y6 - - Other parts none Y7 g.650_652insAAC g.650_652insAAC brain 49 Y8 - - bone none Y9 - - Other parts none Y10 - - bone none Y11 g.18084_18086insGCT g.18084_18086insGCT bone 37 Y12 - - Other parts none Y13 g.650_652insAAC g.650_652insAAC brain 45 Y14 g.18084_18086insGCT g.18084_18086insGCT bone 68

[0044] Note: “-” means not detected.

[0045] Although the present invention is described by preferred specific embodiments, it is obvious to those skilled in the art that the present invention may be modified and varied in various ways. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting ADGRA2 gene mutation in constructing a detection kit for risk analysis of bone metastasis in lung cancer patients, characterized in that: The ADGRA2 gene mutation is a GCT insertion mutation at positions 501-503 of the sequence shown in SEQ ID NO:

2. If a lung cancer patient has the ADGRA2 gene mutation, it means that the patient has bone metastasis or has a high risk of bone metastasis.

2. Use of a reagent for detecting ADGRA2 gene mutation according to claim 1 in constructing a detection kit for risk analysis of bone metastasis in lung cancer patients, characterized in that: The reagent for detecting ADGRA2 gene mutation includes primers and probes, wherein the primers and probes are digital PCR amplification primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 and a detection probe targeting the ADGRA2 gene mutation as shown in SEQ ID NO: 8.

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