Application of a mutant gene in prediction of lung cancer immunotherapy-related thrombocytopenia

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of a MAPK9-based mutant gene in predicting thrombocytopenia associated with lung cancer immunotherapy. The mutant gene corresponds to the wild-type MAPK9 gene, and the T allele at the rs17080141 site of MAPK9 is mutated to the A allele. This invention provides predictive biomarkers for thrombocytopenia associated with lung cancer immunotherapy and their applications, as well as a detection kit for predicting the efficacy of lung cancer immunotherapy. The detection kit provided by this invention can accurately predict the treatment effect in patients with inflammatory breast cancer by detecting the presence of this mutant gene in the tumor tissue of lung cancer patients undergoing immunotherapy. After applying the kit to clinical testing, it can assess the treatment effect of patients before treatment, allowing for the development of more aggressive and effective treatment plans for patients with poor treatment outcomes, thereby improving survival rates.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a mutant gene based on MAPK9 in predicting thrombocytopenia associated with lung cancer immunotherapy. Background Technology

[0002] Lung cancer has consistently ranked first in incidence and mortality among all cancers for decades, accounting for approximately 22.7% of all cancer deaths in China. Immunotherapy is a significant breakthrough in lung cancer treatment in recent years. It activates the body's own immune system to attack cancer cells, offering advantages such as high targeting and long-lasting efficacy. However, immunotherapy has limitations, including the potential for immune-related adverse events (irAEs), such as immunotherapy-associated pneumonia, immunotherapy-associated hepatitis, immunotherapy-associated thyroiditis, immune-related hematologic toxicity, and immunotherapy-associated thrombocytopenia. These adverse events can significantly impact patient treatment.

[0003] The impact of immunotherapy-related thrombocytopenia is particularly significant. Thrombocytopenia affects cancer patients throughout their treatment journey; once the peripheral blood platelet count falls below 100 × 10⁻⁶, the effect is even more pronounced. 9 Chemotherapy, radiotherapy, or surgery may be forced to be delayed or even interrupted, leading to a narrowing of the tumor control window. If continued, the dosage needs to be reduced or a regimen with less bone marrow suppression needs to be used, thus weakening the therapeutic effect. Simultaneously, thrombocytopenia directly increases the risk of bleeding, ranging from mild gum and nasal bleeding to severe gastrointestinal, urinary tract, or intracranial hemorrhage. Studies have shown that patients with solid tumors who develop thrombocytopenia have a 67% increased risk of hospitalization due to bleeding. Thrombocytopenia is significantly associated with mortality risk; those with all grades of thrombocytopenia have a 3.5-fold increased risk of death, and the more severe the grade, the worse the prognosis. The incidence of immunotherapy-related thrombocytopenia is approximately 8%, with severe thrombocytopenia occurring in approximately 4.3%. The mechanism by which tumor immunotherapy drugs cause thrombocytopenia is unclear, but it may be related to T-cell activation. Currently, there is no existing technology for predicting immunotherapy-related thrombocytopenia. Therefore, finding a predictive method for thrombocytopenia associated with lung cancer immunotherapy is of great clinical significance.

[0004] The mitogen-activated protein kinase (MAPK) cascade comprises three sequentially activated protein kinases: MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K / MEK / MKK), and MAPK. Members of the MAPK family, including ERK, JNK, and p38, are key components of a series of important signal transduction pathways, regulating processes such as proliferation, differentiation, and death in all eukaryotic cells from yeast to humans. MAPK9 belongs to the JNK subfamily of the MAPK family and is also commonly referred to as JNK2 or p54a. Different MAPKs are activated by specific MEKs: ERK1 / 2 and ERK5 are activated by MEK1 / 2 and MEK5, respectively; p38 is activated by MEK3 / 6; and JNK is activated by MEK4 / 7. However, each MEK corresponds to more than one MAP3K, and each MAP3K can respond to different stimuli, making the MAPK pathway complex and diverse. Different stimuli (growth factors, cytokines, viruses, G protein-coupled receptor ligands, transformants, and carcinogens, etc.) can activate the ERK1 / 2 pathway. After activation, small G proteins (such as rat sarcoma Ras) recruit and activate the downstream proto-oncogene Raf. Activated Raf then progressively stimulates MEK1 / 2 and activates ERK1 / 2. In the cytoplasm, ERK promotes protein phosphorylation and participates in regulating cytoskeleton formation, cell movement, transport, cell adhesion, and metabolism. It also separates from anchoring proteins and translocates to the nucleus, regulating various transcription factors (such as c-Fos, c-Myc, and ETS domain protein 1), thereby modulating gene expression. Current technology discloses the presence of MAPK in platelets. Summary of the Invention

[0005] The purpose of this invention is to provide an application of MAPK9-based mutant genes in predicting lung cancer immunotherapy-related thrombocytopenia.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The application of a mutated gene in the preparation of a reagent for predicting lung cancer immunotherapy-related thrombocytopenia, wherein the wild-type gene corresponding to the mutated gene is MAPK9, and the T allele at the rs17080141 site of MAPK9 is mutated to the A allele.

[0008] Detailed information about the mutated gene is as follows: Figure 1 As shown (from the NCBI website, https: / / www.ncbi.nlm.nih.gov / snp / ?term=rs17080141).

[0009] The sequence of MAPK9 is known (from https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000005.10?report=fasta&from=180233143&to=180292083&strand=true).

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0011] In one preferred embodiment, the immunotherapy is a PD-1 / PD-L1 immune checkpoint inhibitor therapy for lung cancer.

[0012] In one preferred embodiment, the inhibitor is one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, slulimab, camrelizumab, atezolizumab, durvalumab, sugemalimab, adebelimab, and bemosubaimub.

[0013] Based on the same inventive concept, this invention also claims protection for the use of reagents for detecting mutated genes in the preparation of reagents for predicting lung cancer immunotherapy-related thrombocytopenia, wherein the wild-type gene corresponding to the mutated gene is MAPK9, and the T allele at the rs17080141 site of MAPK9 is mutated to the A allele.

[0014] In one preferred embodiment, the reagents for detecting mutated genes include: specific primers, probes, gene chips, labeled fluorescent agents, or enzymes for detecting mutated genes.

[0015] Based on the same inventive concept, the present invention also claims the use of reagents for detecting rs17080141 in the preparation of reagents for predicting lung cancer immunotherapy-related thrombocytopenia, wherein the rs17080141 site is located on MAPK9.

[0016] In one preferred embodiment, the reagents for detecting rs17080141 include: specific primers, probes, gene chips, labeled fluorescent agents, or enzymes for detecting mutated genes.

[0017] In one preferred embodiment, the gene chip is an Illumina Infinium Chinese Genotyping Array-24.

[0018] In one preferred embodiment, the probes include TaqMan MGB probes, SYBR Green probes, and immunoblotting probes.

[0019] Based on the same inventive concept, the present invention also claims the use of a kit in the preparation of a reagent for predicting lung cancer immunotherapy-related thrombocytopenia, the kit containing a reagent for detecting a mutated gene or a reagent for detecting rs17080141, wherein the wild-type gene corresponding to the mutated gene is MAPK9, and the T allele at the rs17080141 site of the mutated MAPK9 is mutated to the A allele.

[0020] In one preferred embodiment, the kit contains one or more of the following: specific primers, probes, gene chips, labeled fluorescent agents, or enzymes for detecting mutated genes or detecting rs17080141.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention provides predictive biomarkers for thrombocytopenia associated with lung cancer immunotherapy and their applications, as well as a diagnostic kit for predicting the efficacy of lung cancer immunotherapy, to assist in guiding individualized treatment and improving the treatment outcomes of cancer patients. The diagnostic kit provided by this invention can accurately predict the treatment outcomes of inflammatory breast cancer patients by detecting the presence of the mutated gene in the tumor tissue of lung cancer patients undergoing immunotherapy. After applying the kit of this invention to clinical testing, the treatment effect of patients can be evaluated before treatment, and more proactive and effective treatment plans can be developed for patients with poor treatment outcomes, thereby achieving individualized treatment and improving survival rates. Attached Figure Description

[0023] Figure 1 This is detailed information about the rs17080141 site of the present invention.

[0024] Figure 2 This is a PCA plot showing the correlation between lung cancer immunotherapy and chemotherapy-associated thrombocytopenia.

[0025] Figure 3 This is a QQ plot showing the correlation between lung cancer immunotherapy and chemotherapy-related thrombocytopenia.

[0026] Figure 4 This is a Manhattan plot showing the correlation between lung cancer immunotherapy and chemotherapy-associated thrombocytopenia.

[0027] Figure 5 This is a PCA plot showing the correlation between chemotherapy-related thrombocytopenia and other conditions.

[0028] Figure 6 This is a QQ plot of the correlation analysis of chemotherapy-related thrombocytopenia.

[0029] Figure 7 This is a Manhattan plot of correlation analysis of chemotherapy-related thrombocytopenia.

[0030] Figure 8 This is a linkage disequilibrium analysis diagram of the rs17080141 locus.

[0031] Figure 9 This is an eQTL analysis diagram of the rs17080141 locus.

[0032] Figure 10 This is the ROC curve of the rs17080141 locus for predicting thrombocytopenia associated with immunotherapy combined with chemotherapy. Detailed Implementation

[0033] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other. Example 1

[0034] Genome-wide association analysis (GWAS) was performed on the genes of 455 lung cancer patients. The main inclusion criteria included: 1) histological diagnosis of lung cancer; 2) treatment with PD-1 / PD-L1 inhibitors; and 3) relatively complete follow-up data. Exclusion criteria included: 1) patients who had not received immune checkpoint inhibitors (ICIs); 2) patients who switched to targeted therapy based on gene testing; and 3) patients with other malignancies or serious diseases. Peripheral blood samples were collected from lung cancer patients, and genomic DNA was extracted from the samples according to the instructions of the Wizard® Genomic DNA Purification (Promega, A1120) DNA extraction kit. Relevant clinical information was also collected, including gender, age, smoking history, pathological type, clinical stage, PD-L1 expression level, whether ICIs were used as monotherapy, type of ICI, number of lines of treatment, chemotherapy, and immune toxicity / adverse reactions. The degree of thrombocytopenia was evaluated according to the CSCO guidelines for the management of immune checkpoint inhibitor-related toxicities (2023), and classified into grades 1-4. The number of patients with thrombocytopenia in the discovery cohort and the validation cohort were 90 and 64, respectively. Table 1 shows the following data: gender, age, smoking history, stage, pathological type, PD-L1 expression level, number of lines of treatment, type of ICIs, and treatment regimen.

[0035] Table 1 Information on 785 lung cancer patients

[0036]

[0037] The collected blood sample DNA was then genotyped using the Illumina Infinium Chinese Genotyping Array-24 v1.0 chip, with the genomic version being GRCh37 (hg19). This chip can detect 697,214 SNPs. Genomestudio 2.0 was used to process the genotyping data from all samples. Quality control of the raw genotyping data was performed using PLINK (version 1.9). The quality control requirements (routine requirements for GWAS analysis) were as follows: 1. Removal of loci with a detection rate <95%; 2. Removal of loci with MAF <0.01; 3. Removal of loci with a P-value <1×10⁻⁶. -4 4. Filtering sex chromosome SNP loci; 5. Removing samples with a detection rate <95%; 6. Removing samples with an abnormal heterozygosity rate exceeding 3 times the standard deviation; 7. Filtering by kinship, excluding samples with PI-HAT >0.25; 8. Population analysis quality control, using principal component analysis (PCA) to exclude samples with a detection rate greater than 4 times the standard deviation, and verifying the results using PCA with three datasets: 1000 Geomics CEU (Utah Residents with Northern and Western European Ancestry), JPT (Japanese in Tokyo), and CHB (Han Chinese in Beijing), to detect population outliers and stratification. Results are as follows... Figure 2 and Figure 3 As shown. The results showed that PCA of the collected blood sample DNA did not reveal any obvious stratification. Figure 2 For the DNA samples collected from 455 patients, genomic binary logistic regression analysis (standard analysis method) was performed in an additive mode. Data quality was assessed using QQ plots (standard analysis method), and significant deviations from the expected P-values ​​were only observed in the tail region. Figure 3 ).

[0038] 3. Genotype filling. Using 1000 Genomes Phase 3 data as a baseline, the SHAPEIT tool was applied to establish haplotypes, and the IMPUTE2 software was used to perform SNP interpolation and filling on each chromosome segment.

[0039] 4. Post-filling quality control. Filtration detection rate <95%, MAF <0.01, HWP equilibrium P value <1×10⁻⁶. -4Locator; sample detection rate needs to be >95%; abnormal heterozygosity and kinship filtering.

[0040] 5. Correlation Analysis. Statistical data were analyzed using R (version 4.3.2) statistical software. GWAS was performed using PLINK (v1.9) for binary logistic regression analysis. Baseline data were analyzed using the direct count method, and measurement data were expressed as median. Comparisons between groups were performed using the χ² method. 2 The test or Fisher's exact test was used. A two-sided p-value < 0.05 was considered statistically significant. Results are as follows... Figure 4 As shown. Figure 4 The Manhattan plot shows the results of the genome-wide association analysis, demonstrating a significant association between rs17080141 and immunotherapy-related thrombocytopenia.

[0041] 6. Continue to collect genes from another 330 lung cancer patients for site verification (Table 1). The collection and exclusion criteria for these 330 lung cancer patients are the same as above.

[0042] Linkage disequilibrium analysis was used to screen sites with p-values ​​less than 10 from 740,000 sites in the Illumina Infinium Chinese Genotyping Array-24 v1.0 chip. -4 Of the 322 sites identified, linkage disequilibrium analysis was performed on sites with MAF ≥ 0.01, and four tag SNPs were selected for further validation (Table 2). These four sites, significantly associated with thrombocytopenia, were genotyped using Massarray mass spectrometry (a standard technique) in 330 validation samples.

[0043] Table 2. Four loci significantly associated with thrombocytopenia

[0044]

[0045] The verification results are shown in Table 3. The results show that rs17080141 has the most significant correlation.

[0046] Table 3 Validation results of rs17080141

[0047]

[0048] The results show that rs17080141 had the smallest P-value, less than 0.05, indicating that it was the only site significantly associated with thrombocytopenia. While other loci met the same screening criteria, the validation results showed that their P-values ​​were greater than 0.05, indicating poorer correlation.

[0049] 7. Verify the specificity of rs17080141 for immunotherapy-related thrombocytopenia.

[0050] To rule out chemotherapy-induced thrombocytopenia, this invention further collected data from first-line chemotherapy-only lung cancer patients. All patients were pathologically diagnosed with non-small cell lung cancer and had received at least two cycles of platinum-based single-agent or combination chemotherapy. Prior to chemotherapy, they had not received surgery, targeted therapy, radiotherapy, immunotherapy, or other anti-tumor treatments and underwent comprehensive physical examinations. A total of 461 eligible samples were obtained, and DNA samples were extracted using the same method as described above. These DNA samples were genotyped using the Illumina BeadChip Array Global Screening Array-24+ v1.0 chip (Illumina Inc., San Diego, CA). This chip can detect 688,783 SNPs. Quality control was performed on these 461 samples. SNPs and samples removed were as follows: 1. 6288 loci with a detection rate <95%; 2. 277499 loci with MAF <0.01; 3. 963 loci with Hardy equilibrium P-value <1×10⁻⁴; 4. 20115 sex chromosome SNP loci; 5. 2 samples with a detection rate <95%; 6. 10 samples with an abnormal heterozygosity exceeding 3 times the standard deviation; 7. 2 kinship-related SNPs; 8. 0 outlier samples removed for population analysis quality control. After quality control, 446 samples and 383918 loci remained. After filling and quality control again, 446 samples and 4,689552 loci remained. Baseline data for patients with thrombocytopenia caused by chemotherapy alone are shown in Table 4.

[0051] Table 4 Baseline data of patients with thrombocytopenia induced by chemotherapy alone

[0052]

[0053] PCA was performed on the research cohort (using the same method as above), and the results are as follows: Figure 5 As shown in the figure, the results indicate that no obvious stratification occurred, suggesting that the sample members were not a discrete group.

[0054] Multivariate Cox regression analysis of the genome was performed on DNA samples from 446 patients in an additive pattern. Data quality was assessed using QQ plots, and the results are as follows: Figure 6 As shown in the figure. The results show that the expected P-value deviates significantly only in the tail region. A Manhattan plot was generated from the DNA samples of 446 patients, and the results are shown in the figure. Figure 7As shown in the figure. The results show the location of rs17080141 in the genome-wide association analysis. The results indicate that rs17080141 was not associated with the occurrence of thrombocytopenia in NSCLC patients receiving chemotherapy alone (P=0.081, greater than 10). -4 The results showed that rs17080141 was not significantly associated with the occurrence of chemotherapy-related thrombocytopenia alone.

[0055] 8. Linkage disequilibrium of SNPs and eQTL analysis.

[0056] PLINK and LDBlockShow-1.40 software were used to find and verify strongly linked sites of successful SNPs. R 2 The value R is used as a standard to assess the degree of linkage disequilibrium. 2 The value of is between 0 and 1, where R0 is... 2 The closer the value is to 1, the stronger the linkage between the two sites. When R 2 A value of 1 indicates complete linkage between the two loci, meaning that a variation at one locus can accurately predict the status of the other. rs17080141 is located in an intron region of the MAPK9 gene. LD analysis was performed on locus rs17080141, and LD haplotype block diagrams were generated using LDBlockShow, along with a regional plot to display its LD and haplotype information. The results are as follows: Figure 8 As shown.

[0057] Using FUMA GWAS (Functional Mapping and Annotation of Genome-Wide Association Studies) and multiple databases, we performed expression quantitative trait loci (eQTL) analysis, chromatin interaction localization, and transcription factor binding site analysis on validated SNPs to identify SNP-related enhancers and their regulated target genes. Results are as follows: Figure 9 As shown in the figure. The results show that rs17080141 interacts with chromatin regions of multiple genes (such as CNOT6, GFPT2 and C5orf45).

[0058] Example 2

[0059] ROC curve validation of the predictive efficacy of rs17080141 for lung cancer immunotherapy-related thrombocytopenia

[0060] Sixty samples from patients who developed immunotherapy-related thrombocytopenia and 60 samples from patients who did not develop immunotherapy-related thrombocytopenia were re-screened, with the same inclusion criteria. The rs170801041 genotype was obtained from these patients using the same method, and ROC curves were plotted based on the prediction results. The predictive ability of the rs170801041 locus for immunotherapy-related thrombocytopenia in these 120 samples was analyzed using ROC curves. Results are as follows: Figure 10 As shown, the area under the curve (AUC) is 0.804, which statistically confirms that this locus significantly predicts immune-related thrombocytopenia.

[0061] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of a reagent for detecting mutated genes in the preparation of products for predicting lung cancer immunotherapy-related thrombocytopenia, characterized in that, The wild-type gene corresponding to the mutated gene is MAPK9, and the T allele at the rs17080141 site of MAPK9 is mutated to the A allele; the immunotherapy is PD-1 / PD-L1 immune checkpoint inhibitor therapy for lung cancer.

2. The application according to claim 1, characterized in that, Reagents for detecting mutated genes include: specific primers, probes, gene chips, labeled fluorescent agents, or enzymes.

3. The application according to claim 1, characterized in that, The gene chip in question is an Illumina Infinium Chinese Genotyping Array-24.

4. The application according to claim 2, characterized in that, The probes include TaqMan MGB probes, SYBRGreen probes, and immunoblotting probes.

5. The use of a reagent kit in preparing reagents for predicting lung cancer immunotherapy-related thrombocytopenia, characterized in that, The kit contains reagents for detecting rs17080141.

6. The application according to claim 5, characterized in that, The kit contains one or more of the following: specific primers, probes, gene chips, labeled fluorescent agents, or enzymes for detecting rs17080141.

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