Biomarker for prognostic typing of AML (acute myelogenous leukemia) and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a biomarker for AML prognosis typing and application of the biomarker. Wherein the biomarker for the prognostic typing of the AML is a combination of a somatic mutation gene and an AML susceptible site genotype, the somatic mutation gene is selected from DNMT3A, and the AML susceptible site is selected from rs12459419. The combination of somatic mutation genes and AML susceptible site genotypes can further refine AML prognosis risk classification and improve the recognition ability of high-risk patients; meanwhile, prognosis typing of the AML is refined, research and development of specific drug targets for patients with different genotypes are facilitated, and then accurate treatment of the AML is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to biomarkers for AML prognostic classification and their applications. Background Technology

[0002] Acute myeloid leukemia (AML) is a type of hematologic malignancy originating from hematopoietic stem cells in the bone marrow. Its core characteristic is the excessive proliferation and differentiation arrest of myeloid blast cells, ultimately leading to impaired normal hematopoietic function. Although there have been some breakthroughs in the treatment of AML in recent years, relapse and drug resistance remain prominent issues, resulting in generally poor long-term prognosis for patients.

[0003] The genomes of AML patients exhibit high heterogeneity, a key factor influencing relapse and drug resistance. This heterogeneity manifests not only in the diversity of somatic gene mutations and gene fusion types but also in a large number of germline genetic variations, particularly single nucleotide polymorphisms (SNPs). In recent years, researchers have identified at least 94 SNP loci associated with AML risk, treatment response, or prognosis based on genome-wide association studies (GWAS). These AML susceptibility loci can regulate gene expression through interactions with target genes, thereby affecting disease occurrence, diagnosis, and patient prognosis.

[0004] Furthermore, studies have found that many cancer patients carry both germline-derived gene mutations and acquired somatic mutations. Research on rare variants, common variants, ancestor-related genes, and SNP loci has confirmed that the regulation of the tumor epigenome is influenced by multiple factors, including somatic mutations. Different types of genetic variants can be used for patient subtyping, thereby helping researchers identify specific drug targets for patients with different genotypes.

[0005] Current GWAS studies targeting AML primarily identify the nearest upstream and downstream genes to the variant site in the two-dimensional linear genome as transcriptional regulatory targets. While this approach provides relatively rich information, it fails to cover all transcriptional effects induced by variant alleles, and in particular, it fails to consider the impact of interactions between germline and somatic mutations on the pathogenesis of various diseases.

[0006] For the reasons mentioned above, there are currently no typical diagnostic biomarkers for AML prognosis. In-depth analysis of the interaction between germline mutations and somatic mutations, and screening for effective biomarkers, will help provide more accurate prognostic diagnostic information for AML patients. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, a first objective of this invention is to provide a biomarker for AML prognostic typing; a second objective of this invention is to provide applications of the biomarker.

[0008] To achieve the first objective, the technical solution adopted by this invention is as follows: Biomarkers for AML prognostic classification, wherein the biomarkers are a combination of somatic mutant genes and AML susceptibility loci genotypes; Among them, the somatic cell mutation gene was selected from DNMT3A, and the AML susceptibility site was selected from rs12459419; AML stands for Acute Myeloid Leukemia.

[0009] Furthermore, the biomarkers are obtained through screening using the following steps: S100. Collect information on AML susceptibility sites and obtain AML patient cohort data from the genomic data sharing platform data portal to identify the genotype of AML susceptibility sites; S200. Divide the AML patient cohort into four subgroups, namely Group I, Group II, Group III and Group IV. Among them, patients in group I carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AA genotype; patients in group II carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AB or BB genotype; patients in group III do not carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AA genotype; and patients in group IV do not carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AB or BB genotype. A is the reference allele, B is the alternative allele, AA genotype is the homozygous reference allele, AB genotype is the heterozygous allele, and BB genotype is the homozygous alternative allele. S400. The log-rank test was used to compare the overall survival rates among the four subgroups, with the statistical significance level set at 1. P <0.05, biomarkers for AML prognostic classification were obtained through screening.

[0010] Furthermore, in step S100, the AML patient cohort data includes somatic mutation information, overall survival time, and clinical indicators of survival status.

[0011] Furthermore, in step S400, before performing overall survival analysis on the four subgroups of patients, AML patients with missing somatic mutation information, missing germline SNP genotype information, and missing or equal to 0 days of overall survival are excluded.

[0012] Further, in step S400, the Kaplan-Meier analysis method is used to analyze the overall survival rate of patients in each subgroup.

[0013] To achieve the second objective, the technical solution adopted by this invention is as follows: Application of biomarkers: using any of the above-described biomarkers for AML prognostic classification for AML prognostic grading.

[0014] Furthermore, the AML prognostic grading includes four levels, namely, Level 1, Level 2, Level 3, and Level 4. Among them, patients with a prognostic grade of AML of level 1: carrying somatic mutation genes and having the AML susceptibility site rs12459419 of the AA genotype, have a 30-month survival rate of 0 and are considered high-risk. Patients with a prognostic grade of AML level 2: carrying somatic mutation genes and having the AML susceptibility site rs12459419 with the AB or BB genotype, have a survival rate of ≤11.1% for 0 < 30 months, which is considered intermediate to high risk; Patients with a prognostic grade III for AML: those who do not carry somatic mutation genes and whose AML susceptibility site rs12459419 is of the AA genotype, have a survival rate of 11.1% < 30 months ≤ 18.5%, and are classified as low to medium risk; Patients with a prognostic grade of 4 for AML: those who do not carry somatic mutation genes and whose AML susceptibility site rs12459419 is AB or BB genotype, have a survival rate of ≤32.4% for 30 months (18.5%), and are considered low-risk.

[0015] Furthermore, the applications include using the biomarker as a targeting marker to prepare in vitro diagnostic products and / or AML prognostic treatments.

[0016] Furthermore, the in vitro diagnostic products include diagnostic kits and / or diagnostic reagents.

[0017] Furthermore, the drug includes pharmaceutically acceptable excipients, carriers, or excipients.

[0018] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides biomarkers for AML prognostic classification and their applications. The biomarker for AML prognostic classification is a combination of a somatic mutant gene and a susceptibility AML locus genotype. The somatic mutant gene is selected from DNMT3A, and the AML susceptibility locus is selected from rs12459419. This combination of the somatic mutant gene and the AML susceptibility genotype can further refine the AML prognostic risk stratification and improve the identification of high-risk patients. Simultaneously, refining the AML prognostic classification facilitates the development of specific drug targets for patients with different genotypes, thereby achieving precision treatment of AML.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. Attached Figure Description

[0020] Figure 1 This is a Kaplan-Meier survival curve of the overall survival of AML patients carrying the DNMT3A somatic mutation gene and those not carrying the DNMT3A somatic mutation gene in the discovery cohort (86 cases) provided in Embodiment 1 of the present invention.

[0021] Figure 2 This is a Kaplan-Meier survival curve of the overall survival of AML patients in the discovery cohort (86 cases) provided in Embodiment 1 of the present invention, who were jointly grouped with AML patients carrying the DNMT3A somatic mutation gene, those who did not carry the DNMT3A somatic mutation gene, and those with the AML susceptibility site rs12459419 genotype.

[0022] Figure 3 This is a Kaplan-Meier survival curve of the overall survival of AML patients carrying the DNMT3A somatic mutation gene and those not carrying the DNMT3A somatic mutation gene in the validation cohort (35 cases) provided in Embodiment 1 of the present invention.

[0023] Figure 4 This is a Kaplan-Meier survival curve of AML patients in the validation cohort (35 cases) provided in Embodiment 1 of the present invention, who were jointly grouped with AML patients carrying the DNMT3A somatic mutation gene, those who did not carry the DNMT3A somatic mutation gene, and those with the AML susceptibility site rs12459419 genotype. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0025] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0026] Example 1 The process for screening biomarkers for AML prognostic subtyping is as follows: I. We collected 94 published AML susceptibility loci. These AML susceptibility loci were all derived from literature reports and identified through genome-wide association analysis.

[0027] First, genotypic data of patients in the Cancer Genome Atlas - Acute Myeloid Leukemia Cohort (TCGA-LAML) were downloaded from the Genomic Data Commons (GDC) data portal. The patient cohort data were then randomly divided into two groups at a 7:3 ratio: a discovery cohort and a validation cohort. The discovery cohort included 86 patient samples from the TCGA-LAML cohort, while the validation cohort included 35 patient samples from the TCGA-LAML cohort, independent of the discovery cohort. All patient DNA samples were derived from normal tissue (primarily skin) and were genotyped using the Affymetrix Human SNP 6.0 microarray platform. Furthermore, somatic mutation information of patients in the cohort was downloaded and compiled, covering mutation status of common AML driver genes such as DNMT3A, along with accompanying clinical follow-up data, including key clinical indicators such as overall survival (time from initial diagnosis to death or resolution) and survival status (whether the patient died), for subsequent Kaplan-Meier survival analysis.

[0028] To obtain the specific genotype information of the aforementioned 94 AML susceptibility loci, the raw genotype data (.CEL format) from the Affymetrix Human SNP 6.0 microarray were further processed as follows: First, the raw CEL data files were converted and standardized using Affymetrix Power Tools (APT) and accompanying annotation files provided by Affymetrix, resulting in VCF format genotype data files. Then, a mainstream genotype imputation workflow was employed to fill in missing or undetected loci. This included using SHAPEIT software for haplotype reconstruction (i.e., pre-genotyping) and combining it with IMPUTE2 software and the 1000 Genomes Project Phase 3 reference panel for imputation, improving data coverage and accuracy. After imputation, genotype information for 94 AML susceptibility loci was extracted.

[0029] II. Grouping based on whether they carry somatic mutations and AML susceptibility loci genotypes, as detailed below: In the discovery cohort, AML patients were grouped according to whether they carried the DNMT3A somatic mutation gene, and survival analysis was performed using the Kaplan-Meier method. The difference in overall survival between the two groups was assessed based on the log-rank test, and a statistical significance level was set at 1. P <0.05, the result is as follows Figure 1 As shown: the survival curves of patients without the DNMT3A somatic mutation genome decreased more slowly, suggesting a longer overall survival in this group; while the survival curves of patients with the DNMT3A somatic mutation genome decreased more rapidly, indicating a shorter overall survival in this group. The long-term overall survival rate of patients with the DNMT3A somatic mutation genome was significantly lower than that of patients without the DNMT3A somatic mutation genome. P = 0.0069).

[0030] The patient survival time statistics are shown in the table below: However, assessing the prognostic risk of AML solely based on DNMT3A somatic mutations is insufficient to comprehensively and accurately predict the clinical outcomes of AML patients.

[0031] To further refine the risk stratification of AML, in addition to considering the somatic mutation gene DNMT3A, information on the AML susceptibility site rs12459419 genotype was introduced, and patients in the discovery cohort were grouped by the combination of the DNMT3A mutation gene and the AML susceptibility site rs12459419 genotype.

[0032] All AML susceptibility loci are biallelic loci, containing two alleles: a reference allele (A) and a substitute allele (B). Based on the allele type carried by each individual, patients are classified into three genotypes: homozygous reference allele (AA), heterozygous reference allele and substitute allele (AB), and homozygous substitute allele (BB). Subsequently, based on whether patients carry specific somatic mutations and whether the AML susceptibility locus genotype is a substitute allele, patients are further divided into four subgroups: Somatic mutation-SNP genotype AA group: Patients carry somatic mutation genes and have the AA genotype at the AML susceptibility locus. Somatic mutation-SNP genotype AB or BB group: The patient carries a somatic mutation gene and the AML susceptibility locus genotype is AB or BB. Wild-type somatic cell-SNP genotype AA group: Patients do not carry somatic cell mutation genes and the AML susceptibility locus genotype is AA; Wild-type somatic SNP genotype AB or BB group: The patient does not carry the somatic mutation gene and the AML susceptibility locus genotype is AB or BB.

[0033] III. Before performing survival analysis on the above four groups of patients, AML patients with missing somatic mutation information, missing germline SNP genotype information, and missing or equal to 0 days of total survival were excluded.

[0034] Subsequently, Kaplan-Meier survival curves were plotted for the four groups of patients included in the analysis within the discovery cohort; the log-rank test was used to compare the overall survival rates among the four subgroups, with a statistical significance level set at 1. P <0.05, the result is as follows Figure 2As shown, the DNMT3A somatic mutation and the homozygous reference allele (AA) of the AML susceptibility site rs12459419 exhibit a synergistic poor prognostic effect in AML patients. Specifically, the DNMT3A somatic mutation-rs12459419 genotype AA group had the lowest long-term overall survival, making it the worst-prognostic subgroup among the four groups. Among patients carrying the same DNMT3A somatic mutation, those with the rs12459419 genotype AB or BB had higher long-term overall survival. In wild-type somatic cells (without the DNMT3A somatic mutation), the overall survival rate was generally higher than that of those carrying the mutation, and the rs12459419 genotype still showed a survival trend difference, with AB or BB genotype patients having relatively higher long-term overall survival, and AA genotype patients having slightly lower long-term overall survival. Overall, considering both the DNMT3A mutation and the rs12459419 genotype, AML patients can be clearly divided into four subgroups, and the log-rank test results showed that the differences in overall survival between the groups were statistically significant. P = 0.036); The patient survival time statistics are shown in the table below: The data provided in the table above show that the 30-month survival rates for the four subgroups are as follows: For those carrying the somatic mutation gene and having the AA genotype at the AML susceptibility site, the 30-month survival rate is 0, indicating high risk and a prognosis level of 1; for those carrying the somatic mutation gene and having the AB or BB genotype at the AML susceptibility site, the 30-month survival rate is 11.1%, indicating medium to high risk and a prognosis level of 2; for those not carrying the somatic mutation gene and having the AA genotype at the AML susceptibility site, the 30-month survival rate is 18.5%, indicating low to medium risk and a prognosis level of 3; and for those not carrying the somatic mutation gene and having the AB or BB genotype at the AML susceptibility site, the 30-month survival rate is 32.4%, indicating low risk and a prognosis level of 4.

[0035] Therefore, the combination of the DNMT3A somatic mutation gene and the AML susceptibility site rs12459419 genotype can be used as a biomarker for AML prognostic typing.

[0036] IV. Verification queue analysis, the process is as follows: To verify the reliability and stability of the conclusions in the discovery cohort, 35 AML patients in the validation cohort were grouped according to whether they carried the DNMT3A somatic mutation gene. Kaplan-Meier survival analysis was performed, and the difference in overall survival between the two groups was assessed using the log-rank test. A statistical significance level was set at [value missing].P <0.05, the result is as follows Figure 3 As shown: Patients without the DNMT3A somatic cell mutation gene maintained better overall survival and had a relatively better prognosis, while patients with the DNMT3A somatic cell mutation gene had a faster decline in overall survival and a shorter survival time, with a significant difference in overall survival between the two groups. P = 0.023), which is basically consistent with the results of the discovery queue.

[0037] The patient survival time statistics are shown in the table below: Subsequently, using the same analytical method as the aforementioned cohort, based on whether patients carried the DNMT3A somatic mutation gene and whether the AML susceptibility site rs12459419 carried a substitute allele, the patients in the validation cohort were divided into four groups: the DNMT3A mutation-rs12459419 genotype AA group (carrying the somatic mutation gene and having the AML susceptibility site genotype AA), the DNMT3A mutation-rs12459419 genotype AB or BB group (carrying the somatic mutation gene and having the AML susceptibility site genotype AB or BB), the DNMT3A wild-type-rs12459419 genotype AA group (not carrying the somatic mutation gene and having the AML susceptibility site genotype AA), and the DNMT3A wild-type-rs12459419 genotype AB or BB group (not carrying the somatic mutation gene and having the AML susceptibility site genotype AB or BB).

[0038] Kaplan-Meier survival analysis was performed on the four groups of patients mentioned above, and the log-rank test was used to compare the differences in overall survival among the groups. The results are as follows: Figure 4 As shown: This result is largely consistent with the findings of the cohort. Overall, considering both the DNMT3A mutation gene and the rs12459419 genotype, AML patients can be clearly divided into four subgroups, which can be used to classify the prognosis of AML patients. The log-rank test results show that the difference in overall survival between the groups is statistically significant. P =0.00021), which is basically consistent with the results of the discovery queue.

[0039] The patient survival time statistics are shown in the table below: Therefore, the combination of somatic mutant genes and AML susceptibility genotypes can further refine the prognostic risk stratification of AML and improve the ability to identify high-risk patients; at the same time, refining the prognostic subtyping of AML will help develop specific drug targets for patients with different genotypes, thereby achieving precision treatment of AML.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker for AML prognostic typing, characterized in that, The biomarker is a combination of somatic cell mutant genes and AML susceptibility locus genotypes; Among them, the somatic cell mutation gene was selected from DNMT3A, and the AML susceptibility site was selected from rs12459419; AML stands for Acute Myeloid Leukemia.

2. The biomarker for AML prognostic typing as described in claim 1, characterized in that, The biomarkers were obtained through screening using the following steps: S100. Collect information on AML susceptibility sites and obtain AML patient cohort data from the genomic data sharing platform data portal to identify the genotype of AML susceptibility sites; S200. Divide the AML patient cohort into four subgroups, namely Group I, Group II, Group III and Group IV. Among them, patients in group I carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AA genotype; patients in group II carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AB or BB genotype; patients in group III do not carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AA genotype; and patients in group IV do not carry somatic cell mutation genes and have the AML susceptibility site rs12459419 with the AB or BB genotype. A is the reference allele, B is the alternative allele, AA genotype is the homozygous reference allele, AB genotype is the heterozygous allele, and BB genotype is the homozygous alternative allele. S400. The log-rank test was used to compare the overall survival rates among the four subgroups, with the statistical significance level set at 1. P <0.05, biomarkers for AML prognostic classification were obtained through screening.

3. The biomarker for AML prognostic typing as described in claim 2, characterized in that, In step S100, the AML patient cohort data includes somatic mutation information, overall survival time, and clinical indicators of survival status.

4. The biomarker for AML prognostic typing as described in claim 2, characterized in that, In step S400, before performing overall survival analysis on the four subgroups of patients, AML patients with missing somatic mutation information, missing germline SNP genotype information, and missing or equal to 0 days of overall survival are excluded.

5. The biomarker for AML prognostic typing as described in claim 2, characterized in that, In step S400, the Kaplan-Meier analysis method is used to analyze the overall survival rate of patients in each subgroup.

6. The application of biomarkers, characterized in that, AML prognostic classification is performed using biomarkers for AML prognostic typing as described in any one of claims 1 to 5.

7. The application of the biomarker as described in claim 6, characterized in that, The AML prognostic grading includes four levels, namely level one, level two, level three, and level four. Among them, patients with a prognostic grade of AML of level 1: carrying somatic mutation genes and having the AML susceptibility site rs12459419 of the AA genotype, have a 30-month survival rate of 0 and are considered high-risk. Patients with a prognostic grade of AML level 2: carrying somatic mutation genes and having the AML susceptibility site rs12459419 with the AB or BB genotype, have a survival rate of ≤11.1% for 0 < 30 months, which is considered intermediate to high risk; Patients with a prognostic grade III for AML: those who do not carry somatic mutation genes and whose AML susceptibility site rs12459419 is of the AA genotype, have a survival rate of 11.1% < 30 months ≤ 18.5%, and are classified as low to medium risk; Patients with a prognostic grade of 4 for AML: those who do not carry somatic mutation genes and whose AML susceptibility site rs12459419 is AB or BB genotype, have a survival rate of ≤32.4% for 30 months (18.5%), and are considered low-risk.

8. The application of the biomarker as described in claim 6, characterized in that, The applications include using the biomarkers as target markers to prepare in vitro diagnostic products and / or AML prognostic treatments.

9. The application of the biomarker as described in claim 8, characterized in that, The in vitro diagnostic products include diagnostic kits and / or diagnostic reagents.

10. The application of the biomarker as described in claim 8, characterized in that, The drug includes pharmaceutically acceptable excipients, carriers, or excipients.

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