Application of FAT3 and LRP1B co-mutations in predicting sensitivity to immunotherapy in patients with endometrial cancer

Abstract

The present application relates to the field of molecular diagnosis technology, and specifically to an application of FAT3 / LRP1B co-mutation in the activation of anti-tumor immune response in endometrial cancer and the prognostic classification of endometrial cancer.

Description

Technical Field

[0001] The present application relates to the field of molecular diagnosis, and specifically to the application of FAT3 and LRP1B co-mutations in predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy. Background Art

[0002] Endometrial cancer is the sixth most common female cancer in the world and the most common gynecological malignancy in developed countries, with approximately 417,000 new cases and 97,000 deaths worldwide in 2020. Approximately 70% of endometrial cancers are confined to the uterus at diagnosis and have a good prognosis, while patients with metastatic and high-risk histological subtypes have a poor prognosis. Platinum-based chemotherapy is the standard first-line treatment for advanced and recurrent endometrial cancer, but there is still no standard second-line treatment option for patients who are insensitive to platinum. As a result, the 5-year overall survival rate is only 20% for patients with metastatic advanced disease.

[0003] In recent years, the emergence of immune checkpoint inhibitors based on the PD-1 / PD-L1 pathway has ignited hope for patients with endometrial cancer. For patients with endometrial cancer with high microsatellite instability (MSI-H) / mismatch repair deficiency (dMMR) or high tumor mutation burden (TMB-H), multiple studies have shown that immunotherapy has unprecedented durable responses.

[0004] However, the factors that affect the efficacy of immunotherapy have not been fully studied. Predictive immunotherapy biomarkers that have been approved by regulatory authorities include PD-L1 expression, MSI-H / dMMR, and TMB. However, the response to immunotherapy is not completely consistent with any of these existing biomarkers. They may occur in people with low PD-L1 expression but are absent in patients with MSI-H and TMB-H, which makes it often necessary to test all three biomarkers simultaneously in clinical practice to avoid missing any opportunity that may benefit from immunotherapy. On the other hand, the detection methods of these biomarkers are diverse, the threshold evaluation criteria are complicated, and a unified gold standard has not yet been formed. In addition, the difficulty in obtaining tissue samples and the high cost of large panel or whole exome sequencing limit the widespread application of these biomarkers in treatment selection. With the development of research, some new immunotherapy biomarkers have been proposed, such as aneuploidy scores, tumor microenvironment, and POLE / POLD1 gene exonuclease domain mutations. In summary, it is necessary to clearly identify new biomarkers of immunosuppressive therapy response, especially when such biomarkers can simultaneously combine the characteristics of multiple existing biomarkers and overcome all the above-mentioned shortcomings.

[0005] FAT3 and LRP1B are two tumor suppressor genes with high mutation frequencies in various cancer types. Previous studies have confirmed the predictive value of these two genes in prognosis and inducing host immune activation. However, the role and impact of FAT3 and LRP1B in endometrial cancer is still unclear.

[0006] In view of this, this application is hereby filed. Summary of the invention

[0007] To solve the above technical problems, this application, through a systematic analysis of the endometrial cancer population, discovered for the first time that co-mutations of FAT3 and LRP1B define a specific cluster associated with anti-tumor immune activation and prognosis, and that co-mutations can not only activate more powerful anti-tumor immune responses, but can also be used to improve prognostic classification.

[0008] Based on this, this application proposes the following specific technical solutions:

[0009] The present application first provides an application of FAT3 and LRP1B co-mutation as a marker in predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy.

[0010] The present application also provides a use of a detection agent for FAT3 and LRP1B co-mutation in the preparation of a kit for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy.

[0011] The present application also provides an application of FAT3 and LRP1B co-mutation as a marker in evaluating the benefit of immunotherapy in patients with endometrial cancer.

[0012] The present application also provides a use of a FAT3 and LRP1B co-mutation detection agent in the preparation of a kit for evaluating the benefits of immunotherapy for endometrial cancer.

[0013] The present application also provides an application of FAT3 and LRP1B co-mutation as a marker in the prognostic classification of endometrial cancer.

[0014] The present application also provides an application of a FAT3 and LRP1B co-mutation detection agent in the preparation of an endometrial cancer prognosis classification product.

[0015] Furthermore, the presence of the above-mentioned FAT3 and LRP1B co-mutation is an indication that the endometrial cancer patient is sensitive to immunotherapy (such as immunosuppressive therapy).

[0016] Further, the mutation is a non-synonymous mutation;

[0017] Further preferably, the non-synonymous mutation is a non-synonymous mutation in a gene coding region;

[0018] More preferably, the mutation includes but is not limited to single nucleotide polymorphism, base substitution / insertion / deletion, silent mutation, missense mutation, nonsense mutation or splicing mutation.

[0019] Furthermore, the detection agent performs detection at the nucleic acid level.

[0020] Further preferably, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

[0021] Furthermore, the detection agent is used for detection at the protein level.

[0022] Further preferably, the detection agent is used to perform any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for mutation sites.

[0023] Furthermore, the kit also includes reagents for detecting other gene mutations.

[0024] Further preferably, the other genes include but are not limited to one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

[0025] Furthermore, the kit also includes a sample processing reagent, and the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

[0026] Furthermore, the sample is selected from at least one of plasma, tissue, pleural effusion, ascites or cerebrospinal fluid of the endometrial cancer patient.

[0027] The present application also provides a kit for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy, wherein the kit comprises reagents for detecting FAT3 and LRP1B gene mutations.

[0028] The present application also provides an endometrial cancer prognosis classification product, which contains reagents for detecting FAT3 and LRP1B gene mutations.

[0029] Furthermore, the kit or product also includes mutation detection reagents for other endometrial cancer immunotherapy sensitivity genes, and the other genes include but are not limited to one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

[0030] Furthermore, the kit does not contain mutation detection reagents for other endometrial cancer immunotherapy sensitivity genes, and the co-mutation of FAT3 and LRP1B can be used as the only independent sensitivity marker.

[0031] The present application also provides a method for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy, comprising the step of detecting the co-mutation of FAT3 and LRP1B.

[0032] The present application also provides a method for evaluating the benefit of immunotherapy for patients with endometrial cancer, comprising the step of detecting the co-mutation of FAT3 and LRP1B.

[0033] The present application also provides a method for evaluating the prognosis of immunotherapy for patients with endometrial cancer, comprising the step of detecting the co-mutation of FAT3 and LRP1B.

[0034] In addition, the present application also provides any of the following uses of FAT3 and LRP1B co-mutation as markers:

[0035] Regulation of cytotoxicity in endometrial cancer;

[0036] Improved immune microenvironment in endometrial cancer;

[0037] Altered composition and abundance of lymphocytes in the microenvironment in endometrial cancer;

[0038] Inducing anti-tumor immune responses in endometrial cancer;

[0039] Promotes tumor microenvironment adaptive immune resistance in endometrial cancer;

[0040] pathway enrichment in endometrial cancer;

[0041] Evaluation of the benefit of immunotherapy in endometrial cancer;

[0042] Evaluation of sensitivity to immunotherapy in patients with endometrial cancer.

[0043] Beneficial technical effects of this application:

[0044] 1) This application proposes for the first time that FAT3 / LRP1B co-mutation is more valuable in endometrial cancer research than single mutation of FAT3 or LRP1B. Co-mutation can not only activate a stronger anti-tumor immune response, but also can be used to predict the prognosis of endometrial cancer patients.

[0045] 2) This application proposes for the first time that endometrial cancer patients with FAT3 and LRP1B co-mutations have high PD-L1 expression, TMB-H, MSI-H, low aneuploidy score, and high PD-L1 + / CD8A + These characteristics have been confirmed to be associated with better immune efficacy.

[0046] 3) All non-synonymous mutations (such as indels, missense mutations, nonsense mutations, and splicing mutations) in the coding regions of all FAT3 and LRP1B genes in this application were taken into account, with clear positive judgment criteria.

[0047] 4) Based on the co-mutation of FAT3 and LRP1B as a means of prediction, evaluation or screening, not only tumor tissue can be used for testing, but also liquid biopsy methods can be used, which can be convenient for patients who cannot obtain tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0049] Figure 1 , FAT3, and LRP1B co-mutations can affect TMB, aneuploidy score, and MSI level in endometrial cancer. Among them, (a) compared with endometrial cancer samples with only FAT3 or LRP1B mutations, the TMB level of the FAT3 and LRP1B co-mutation subgroup was significantly higher. (b) FAT3 and LRP1B co-mutations predicted TMB-H with high specificity (>10 mutations / Mb). FAT3 + / LRP1B + The prediction performance of the group for TMB-H is better than that of FAT3 + and LRP1B + The AUC of the FAT3 group was 0.664, 95% CI = 0.608-0.719, p < 0.001. (c) + / LRP1B + (d) In the entire endometrial cancer cohort, the aneuploidy score was significantly positively correlated with TMB. (e) The MSI-H proportion of the FAT3 and LRP1B co-mutated subgroup was significantly higher than that of other samples. (f) FAT3 + / LRP1B +The proportion of POLE mutation subtypes was the highest in the group. The Mann-Whitney test (a, c) and Fisher's exact test (e, f) were used for statistical comparison among the groups, and the Pearson correlation coefficient (d) was used to analyze the correlation between the variables, **, p<0.01, ***, p<0.001.

[0050] Figure 2 Co-mutation of FAT3 and LRP1B triggers anti-tumor immune responses and promotes adaptive immune resistance in the tumor microenvironment. (a) FAT3 + / LRP1B + The expression levels of cytotoxic T cell markers (CD8A, IFNG, GZMA, GZMB, and GNLY), T cell exhaustion immune checkpoints (PD-L1, CTLA-4, LAG3, TIM3, and TIGIT), and effector cytokines (CXCL9 and CXCL10) were significantly upregulated in the subgroups. (b) Compared with the WT group, only FAT3 + / LRP1B + The expression level of PD-L1 in the FAT3 group was significantly increased. + / LRP1B + Group PD-L1 and CD8A double positive (PD-L1 + / CD8A + ) ratio was significantly higher than that of other groups. p values ​​represent comparisons among different groups, using Mann-Whitney test (a, b) and Fisher's exact test (c), **, p<0.01, ***, p<0.001.

[0051] Figure 3 , FAT3 and LRP1B co-mutations can change the composition and abundance of lymphocytes in the microenvironment. (a) The stacked bar chart shows the composition of 22 immune cells in a total of 502 samples in the endometrial cancer dataset. Different cells are represented by different colors. (b) Different co-mutation states lead to differentiated immune cell abundances. (c) Pearson correlation analysis shows that there is a feedback relationship between immune cells. 0-1 is positive correlation, (-1)-0 is negative correlation, and the darker the color, the stronger the correlation. The p value is FAT3 + / LRP1B + With non-FAT3 + / LRP1B + Mann-Whitney test comparison between samples. **, p < 0.01, ***, p < 0.001.

[0052] Figure 4, FAT3 and LRP1B co-mutation pathway enrichment analysis in endometrial cancer. GSEA analysis showed that FAT3 + / LRP1B + Subgroups were significantly enriched for (a) positive regulation of DNA replication, (b) homologous recombination, (c) nucleotide excision repair, (d) processing and presentation of peptides or polysaccharides by MHC class II antigens, (e) T cell chemotaxis, (f) T cell receptor signaling, and (g) regulation of tumor cell responses. NES = normalized enrichment score.

[0053] Figure 5 , Comparison of progression-free survival of each group according to FAT3 and LRP1B mutation status. (a)-(b) PFS of endometrial cancer with FAT3 or LRP1B mutation was significantly prolonged compared with wild-type samples. (c) Among the four groups, FAT3 + / LRP1B + The PFS of the subgroup was the longest. (d)-(e) When only FAT3 or LRP1B mutations were present, the PFS was not significantly different from the other samples. (f) Only patients with both FAT3 and LRP1B mutations had better PFS. Survival curves were drawn using the Kaplan-Meier method and compared using the log-rank test.

[0054] Figure 6 , The overall survival of each group was compared according to the FAT3 and LRP1B mutation status. (a) Although there was no statistical difference, the FAT3 mutated endometrial cancer subgroup showed a potential trend of prolonged OS. (b) The LRP1B mutated samples had significantly prolonged OS compared with wild-type samples. (c) Among the four groups, FAT3 + / LRP1B + The subgroup with the longest OS. (d)-(e) When only FAT3 or LRP1B mutations were present, OS was not significantly different from other samples. (f) Only patients with both FAT3 and LRP1B mutations had better OS. p values ​​represent the results of intergroup comparisons using the uncorrected log-rank test.

[0055] Figure 7 , Analysis results of validation samples with co-mutation, among which, (a) TMB levels of endometrial cancer samples with FAT3 and LRP1B co-mutation were significantly increased. (b) Co-mutation samples had a high proportion of POLE mutation subtypes. (c) Compared with wild-type samples, FAT3 + / LRP1B +The PD-L1 expression level in the 24-hour group was significantly upregulated. Statistical analysis was performed using Mann-Whitney test (a, c) and Fisher's exact test (b), *, p<0.05, ***, p<0.001. DETAILED DESCRIPTION

[0056] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0057] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0058] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0059] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0060] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0061] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0062] The term "nucleic acid" or "nucleic acid sequence" in this application refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid or analog units thereof. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid of one strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.

[0063] The term "complementary" as used herein refers to hydrogen bond base pairing between the nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences are annealed to each other, A pairs with T, G pairs with C in DNA, and G pairs with C, and A pairs with U in RNA.

[0064] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0065] The present application relates to the use of a detection agent for co-mutation of FAT3 and LRP1B in the preparation of a kit for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy. Preferably, the presence of co-mutation of FAT3 and LRP1B is an indication that the endometrial cancer patients are sensitive to immunosuppressant therapy.

[0066] The present application relates to the use of FAT3 and LRP1B co-mutation detection agents in the preparation of endometrial cancer prognosis classification products.

[0067] It is understandable that the present application provides a new marker for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressive therapy: FAT3+LRP1B. After analysis, it was confirmed that among patients with endometrial cancer, patients with co-mutation of genes and those without gene mutations or only with FAT3 or LRP1B mutations, the characteristics of PD-L1 expression, TMB-H, MSI-H, low aneuploidy score, high PD-L1+ / CD8A+ ratio, POLE exonuclease region mutation, etc. were statistically different. And patients with co-mutation of FAT3 and LRP1B had significantly better prognosis after immunotherapy than patients with wild-type FAT3 and LRP1B and mutation groups with only FAT3 or LRP1B.

[0068] As used herein, the term "immune checkpoint" refers to some inhibitory signaling pathways present in the immune system. Under normal circumstances, the body can maintain immune tolerance by regulating the intensity of the autoimmune response. However, when the body is invaded by tumors, the activation of immune checkpoints will inhibit autoimmunity, which is conducive to the growth and escape of tumor cells. By using immunosuppressants, the body's normal anti-tumor immune response can be restored, thereby controlling and clearing tumors. The "immune checkpoints" described in this application include but are not limited to programmed death receptor 1 (PD-1), PD-L1, cytotoxic T immune cell-associated antigen 4 (CTLA-4); also include some newly discovered immune checkpoints such as immune cell activation gene 3 (LAG3), T-cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin-3 (TIM-3), V domain immunoglobulin inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR) sialic acid binding immunoglobulin-like lectin 7 / 9, etc. In some embodiments, the immunosuppressant of the present application is preferably a PD-1 inhibitor and / or a PD-L1 inhibitor. The PD-1 inhibitor can further be selected as one or more of Nivolumab (Opdivo; BMS-936558), Pembrolizumab (Keytruda; MK-3475), lambrolizumab (MK-3475), Pidilizumab (CT-011), Teplizumab (JS001), Sindilimab (IBI308), Carrelizumab (Erica) and Tislelizumab (Baizean). The PD-L1 inhibitor can further be selected as one or more of Atezolizumab (Tecentriq; MPDL3280A), JS003, Durvalumab (Imfinzi), Avelumab (Bavencio), BMS-936559, MEDI4736 and MSB0010718C.

[0069] The terms "mutational burden", "mutation burden", and "mutationalburden" are used interchangeably herein. In the context of tumors, mutational burden is also referred to herein as "tumor mutational burden", "tumor mutational burden" or "TMB".

[0070] The terms "MSI", "microsatellite instability" and "microsatellite instability" are used interchangeably herein and refer to changes in the microsatellites of tumor tissues relative to normal tissues due to insertion or deletion of repeating units resulting in any length of microsatellites.

[0071] The "typing" mentioned in the present invention refers to "molecular typing", which is generally to evaluate the DNA, RNA and / or protein of tumor patients by cells obtained from tumor tissue or by capturing circulating tumor cells in the blood. This article particularly relates to the prognostic typing of endometrial cancer patients after immunotherapy.

[0072] In this application, "immune cell infiltration" refers to immune cells that leave the bloodstream and migrate into the tumor. These immune cells include T cells and B cells, as well as natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc., which can be present in the tumor in different proportions. "The level of immune cell infiltration in the tumor" refers to the relative content of tumor-infiltrating immune cells. The tumor-infiltrating immune cells of this application are composed of tumor-infiltrating lymphocytes (TIL), and more preferably, CD8 + Composition of tumor-infiltrating T cells.

[0073] In this application, "co-mutation" refers to a common mutation in two genes.

[0074] The form of "mutation" in the present application is not limited, and all non-synonymous mutations of FAT3 and LRP1B (preferably gene coding regions) are taken into account, and all have clear positive judgment criteria.

[0075] In some embodiments, the mutation may be a point mutation, such as, but not limited to, a single nucleotide polymorphism, a base substitution / insertion / deletion, a silent mutation, a missense mutation, a nonsense mutation, or a splicing mutation.

[0076] In some embodiments, the mutation includes whether there is a mutation in the coding region, such as a frameshift mutation.

[0077] In some embodiments, after determining the presence of mutations in the coding regions of FAT3 and LRP1B genes that truncate the FAT3 and LRP1B proteins, the expression of the FAT3 and LRP1B genes, such as the protein expression levels of the FAT3 and LRP1B genes, is assessed.

[0078] In some embodiments, the kit further comprises detection agents for other gene mutations; preferably, the genes include but are not limited to: one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

[0079] Since FAT3 and LRP1B genes are proteins encoding genes, their mutations are usually manifested at the transcription level and reaction level. Those skilled in the art can detect their mutations at the RNA and protein levels to indirectly reflect whether gene mutations have occurred, which all fall within the scope of protection of this application.

[0080] In some embodiments, the detection agent detects at the nucleic acid level.

[0081] The detection agent at the nucleic acid level (DNA or RNA level) can be selected from reagents known to those skilled in the art, such as nucleic acids (usually probes or primers) that can hybridize with the DNA or RNA and are labeled with fluorescent markers, etc. It is also easy for those skilled in the art to detect the cDNA after reverse transcription of mRNA into cDNA, and the conventional replacement of these technical means does not exceed the protection scope of this application.

[0082] In some embodiments, the detection agent is used to perform any of the following methods:

[0083] Polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method. In some embodiments, the polymerase chain reaction is selected from restriction fragment length polymorphism method, single strand conformation polymorphism method, Taqman probe method, competitive allele-specific PCR and allele-specific PCR.

[0084] In some embodiments, the biological mass spectrometry is selected from flight mass spectrometer detection, such as Massarray detection.

[0085] In some embodiments, the nucleic acid sequencing method is selected from the Snapshot method.

[0086] In some embodiments of the present application, the nucleic acid sequencing method can be transcriptome sequencing or genome sequencing. In some other embodiments of the present application, the nucleic acid sequencing method is high-throughput sequencing, also known as second-generation sequencing ("NGS"). NGS is different from "Sanger sequencing" (first-generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary change to traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. The sequencing platform of the available NGS of the present application is commercially available, including but not limited to Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Applied Biosystems SOLID system, etc. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a certain species specific cell or tissue in a certain state through a second-generation sequencing platform, which can be used to study gene expression, gene function, structure, alternative splicing and new transcript prediction, etc. In some other embodiments of the present application, the nucleic acid sequencing method can be single-molecule real-time sequencing. Single-molecule DNA sequencing technology is a new generation of sequencing technology developed in the past 10 years, also known as the third-generation sequencing technology, including single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing and other technologies.

[0087] In some embodiments, the detection agent detects at the protein level.

[0088] In some embodiments, the detection agent is used to perform any of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for mutation sites. The method of detecting using antibodies specifically designed for mutation sites can further be immunoprecipitation, co-immunoprecipitation, immunohistochemistry, ELISA, and Western Blot, etc.

[0089] In some embodiments, the kit further includes a sample processing reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

[0090] In some embodiments, the sample is selected from at least one of plasma, tissue, pleural effusion, ascites, or cerebrospinal fluid of the endometrial cancer patient.

[0091] In some embodiments, the tissue is endometrial cancer tissue or paracancerous tissue.

[0092] According to another aspect of the present application, the present application also provides a method for predicting or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy, the method comprising: using the detection agent as described above to detect the presence or absence of FAT3 and LRP1B co-mutation. In some embodiments, the method is used for the prognosis assessment of endometrial cancer patients after immunosuppressant therapy.

[0093] According to another aspect of the present application, the present application also provides a kit for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy, wherein the kit comprises reagents for detecting co-mutations of FAT3 and LRP1B; in some preferred embodiments, reagents for detecting other gene mutations are also included, wherein the genes include but are not limited to one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

[0094] According to another aspect of the present application, the present application also provides a kit for predicting, evaluating or screening the degree of tumor mutation load in patients with endometrial cancer, characterized in that the kit contains reagents for detecting co-mutations of FAT3 and LRP1B; in some preferred embodiments, reagents for detecting other gene mutations are also included, and the genes include but are not limited to one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

[0095] The implementation scheme of the present application is described in detail below with reference to examples.

[0096] Example

[0097] 1. Dataset

[0098] A total of 502 endometrial cancer samples were retrieved and screened from TCGA (http: / / cancergenome.nih.gov) to form the tumor population for this study. The cohort included endometrioid, serous, and mixed endometrial adenocarcinomas. All samples also had whole exome sequencing, RNA-seq expression profiling, and somatic copy number alteration data, which provided information for immunotherapy biomarker analysis. Prognostic evaluation was performed on 500 samples with progression-free survival and overall survival results. In addition, this application also used 95 samples from the Dou cohort for validation. FAT3 and LRP1B mutations in all analyses were defined as all mutations in the coding sequence except synonymous and intronic mutations, including indels, missense mutations, nonsense mutations, and splicing mutations.

[0099] 2. Exploration of biomarkers of anti-tumor immune response

[0100] The ability of co-mutations to activate anti-tumor immune responses was comprehensively evaluated by analyzing TMB, MSI, PD-L1 expression, aneuploidy score, tumor microenvironment classification, and immune-related molecule levels in patients with endometrial cancer co-mutated with FAT3 and LRP1B. TMB was calculated as all non-synonymous somatic mutations in protein-coding regions excluding exome size, including all single nucleotide variants (SNVs) and indels. The MSI status and aneuploidy score of the cohort samples were previously determined by standard techniques. The expression levels of cytotoxic T cell markers (CD8A, IFNG, GZMA, GZMB, and GNLY), T cell exhaustion immune checkpoints (PD-L1, CTLA-4, LAG3, TIM3, and TIGIT), and effector cytokines (CXCL9 and CXCL10) were evaluated by RNASeqV2 RSEM format transcriptome data. Based on the PD-L1 mRNA expression level and the degree of lymphocyte infiltration, the tumor immune microenvironment was divided into four categories, among which CD8A expression was used to represent the presence or absence of tumor-infiltrating lymphocytes in the microenvironment, and PD-L1 and CD8A levels above the median were defined as positive. Combining the levels and characteristics of these biomarkers in samples with FAT3 and LRP1B co-mutations, the ability of co-mutations to stimulate host anti-tumor immune responses was comprehensively judged, and whether co-mutations could be used as a new biomarker to predict the efficacy of immunotherapy in endometrial cancer was explored.

[0101] 3. Gene set enrichment analysis

[0102] In order to further analyze the potential mechanism of FAT3 and LRP1B co-mutation in endometrial cancer tumorigenesis, development and treatment selection, this application uses gene set enrichment analysis (GSEA) to evaluate the effect of co-mutation on the classic signaling pathways in vivo. Based on the javaGSEA application (version 4.1.0) downloaded from http: / / software.broadinstitute.org / gsea / index.jsp, this application divides the entire population into two groups: FAT3 and LRP1B co-mutation and wild type (FAT3 + / LRP1B + vs. WT), and GSEA analysis was performed using RNA-seq data. The normalized enrichment score (NES) is a value normalized by gene size, which reflects the degree of enrichment of a gene set at the top or bottom of the entire sorted list and is the main statistic for evaluating enrichment results. Pathways with a p-value less than 0.05 were considered to be significantly enriched pathways.

[0103] 4. Distribution of tumor infiltrating lymphocytes

[0104] According to the mutation status of FAT3 and LRP1B genes, this application divided 502 endometrial cancer samples into two groups: co-mutation and wild-type (FAT3 + / LRP1B + vs. WT). Then the CIBERSORT algorithm was used to obtain the relative abundance of 22 immune cells in the microenvironment of these samples. Based on the sample grouping, this application compared the effects of co-mutations on the distribution of tumor-infiltrating immune cells and used the corrplot package and Pearson correlation coefficient to visualize the correlation between immune cells.

[0105] 5. Statistical analysis methods

[0106] Statistical analyses were performed using GraphPad Prism (version 8.0), SPSS (version 25.0), and R (version 3.6.1). Demographic, clinicopathological, and tumor molecular characteristics were treated as continuous variables (e.g., age, TMB, mRNA expression) or categorical variables (e.g., tumor type, MSI status, tumor microenvironment classification) and compared using the nonparametric Mann-Whitney test or Fisher's exact test, respectively. Survival curves were plotted using the Kaplan-Meier method, and the log-rank test was used to compare and analyze the effects of FAT3 and LRP1B co-mutations on PFS and OS. All statistical analyses were performed using two-sided tests, and p < 0.05 was considered significant.

[0107] Example 1 Demographic and clinicopathological characteristics of patients with endometrial cancer harboring FAT3 and LRP1B mutations

[0108] In 502 patients with endometrial cancer from the TCGA database, the mutation frequencies of FAT3 and LRP1B genes were 19.52% (98 / 502) and 19.32% (97 / 502), respectively, of which the co-mutation frequency of FAT3 and LRP1B was 11.16% (56 / 502). In the FAT3 mutation subgroup, at least half of the samples also had LRP1B gene mutations, and the same situation existed in the LRP1B mutation subgroup. Similarly, further detailed analysis found that FAT3 and LRP1B mutations tended to occur simultaneously in endometrial cancer (Fisher's exact test, p<0.001).

[0109] Subsequently, this example divided the entire cohort into four groups: FAT3 and LRP1B co-mutation group (FAT3 + / LRP1B + ), only the FAT3 mutation group (FAT3 + ) or LRP1B mutation group (LRP1B+ ), and FAT3 and LRP1B double wild-type group (WT), to evaluate the effect of the mutation status of the target gene on the clinical characteristics of the samples ( Table 1 ).

[0110] The analysis showed that compared with wild-type patients, FAT3 + / LRP1B + The patients in the FAT3 group had earlier onset of cancer (median age 56 vs. 64 years, p < 0.001), lighter body weight (median weight 71 kg vs. 85 kg, p < 0.001), and fewer recurrence / metastasis events after initial treatment (85.71% vs. 71.35%, p = 0.017). + / LRP1B + (94.64% vs. 70.25%, p<0.001) and FAT3 + The proportion of endometrial adenocarcinoma in the subgroup samples was higher (92.86% vs. 70.25%, p = 0.003). There was no significant difference between the three mutation data sets and wild-type samples in terms of race, stage, tumor residual, radiotherapy ratio, and tumor status.

[0111] Table 1 Comparison of demographic and clinicopathological characteristics of the endometrial cancer cohort according to FAT3 and LRP1B mutation status.

[0112]

[0113] Data are n (%) or median (range). a The data were analyzed by the nonparametric Mann-Whitney test, and other statistical comparisons between groups were performed using the Fisher exact test. * p<0.05.

[0114] Example 2 Endometrial cancer population with co-mutations of FAT3 and LRP1B shows unique molecular characteristics

[0115] TMB, MSI and aneuploidy scores are all indicators of tumor genomic stability, and their status is often consistent with immunogenicity and non-self neoantigen levels, so they can be used as biomarkers to predict the efficacy of immune checkpoint inhibitors.

[0116] In endometrial cancer, this example found that although FAT3 + and LRP1B + The TMB level of the FAT3 group was significantly higher than that of the WT group (all p values ​​were less than 0.001), but the co-mutation group had the highest TMB level, which was even significantly higher than that of the FAT3 + ( Figure 1a, 194.8 vs. 11.47, p < 0.001) and LRP1B + Group( Figure 1 a, 194.8 vs 17.68, p<0.001). FAT3 + LRP1B + There was no statistically significant difference in TMB levels between the two groups (p = 0.29). Subsequently, in order to evaluate the accuracy of FAT3 and LRP1B co-mutation in predicting TMB in endometrial cancer, this application used 10 mutations / Mb as the TMB threshold and performed ROC curve analysis. + / LRP1B + Among the patients, the proportion of TMB-H (>10 mutations / Mb) reached 96.43% (54 / 56), while the positive rate of TMB-H in the remaining samples was only 24.22% (108 / 446). In addition, the area under the ROC curve FAT3 + / LRP1B + The 0.664 of the group is also higher than that of FAT3 + (AUC = 0.543) and LRP1B + group (AUC = 0.563), proving that the co-mutation group had the best TMB level prediction performance (sensitivity of 33.33%, specificity of 99.41%, Figure 1 b, p<0.001). In addition, the present application found that FAT3 + / LRP1B + The aneuploidy score of the subgroup was significantly lower than that of FAT3 + ( Figure 1 c, p = 0.0029) and LRP1B + Group( Figure 1 c, p = 0.0026). Further analysis revealed a significant positive correlation between the aneuploidy score and TMB level in the entire endometrial cancer cohort ( Figure 1 d, r=-0.23, p<0.001), which is consistent with the conclusion that the co-mutation group had the highest TMB level and the lowest aneuploidy score.

[0117] Previous studies have demonstrated that MSI-H defines an important subtype of endometrial cancer with unique prognoses and treatment approaches. Here, we found that co-mutations were also associated with MSI status. + / LRP1B + The proportion of MSI-H in the group was significantly higher ( Figure 1e, 50% vs. 29.47%, p<0.001). Current research progress believes that MSI-H is a subset of TMB-H, and most MSI-H patients are also TMB-H, but in addition to MSI-H, there are many other possible causes of TMB-H. Another important possible source of TMB-H in endometrial cancer is the correction function defect caused by mutations in the endonuclease region of the POLE gene. Similarly, as most previous results have shown, this application found that POLE correction mutations and MSI-H rarely occur at the same time. In the POLE subtype of endometrial cancer, only 8 patients had MSI-H at the same time, and the proportion was only 16.67% (8 / 48). Therefore, this application wants to know if FAT3 + / LRP1B + Is the high level of TMB in the subgroup caused by MSI-H and POLE mutations? Surprisingly, the results showed that among the 48 samples with POLE correction defects, three-quarters of the samples had co-mutations of FAT3 and LRP1B (36 / 48), and there was also a co-occurrence relationship between POLE and FAT3 / LRP1B co-mutations in endometrial cancer (Fisher's exact test, p<0.001). Further exploration of the relationship between co-mutations and molecular classification of endometrial cancer also showed that there was a correlation between co-mutations and POLE mutations, among which FAT3 + / LRP1B + The proportion of POLE mutation subtypes in the group was significantly higher, and there was no high / low copy number subtype ( Figure 1 f, p<0.001).

[0118] Example 3 Co-mutation of FAT3 and LRP1B can stimulate more potent cytotoxicity and affect the immune microenvironment

[0119] Tumors with more mutations tend to be more immunogenic and can therefore activate the immune system to produce an anti-tumor immune response. In order to find out whether FAT3 and LRP1B co-mutations affect cytotoxicity, this application analyzed the levels of some markers related to T cell killing function in the immune microenvironment. The results showed that despite the high expression levels of multiple T cell exhaustion immune checkpoints (PD-L1, CTLA-4, LAG3, TIM3, and TIGIT), endometrial cancer samples with FAT3 and LRP1B co-mutations still showed significant upregulation of cytotoxic T cell markers (CD8A, IFNG, GZMA, GZMB, and GNLY) and effector cytokines (CXCL9 and CXCL10) ( Figure 2a) In addition, since PD-L1 expression is a predictive biomarker for the efficacy of several currently approved immunotherapy monoclonal antibodies, this application conducted a more in-depth analysis. This application found that when the entire endometrial cancer population was divided into four groups based on FAT3 and LRP1B mutation status, only FAT3 + / LRP1B + The PD-L1 expression level in the subgroup was significantly increased, while FAT3 + and LRP1B + This phenomenon did not occur in the Figure 2 b). Meanwhile, the subgroup with co-mutation of FAT3 and LRP1B was PD-L1 and CD8A double positive (PD-L1 + / CD8A + ) was significantly increased, indicating that this type of sample has a type I tumor immune microenvironment with high expression of PD-L1 and CD8A ( Figure 2 c) Based on the above analysis of T cell killing-related markers, this acquired immune resistance tumor microenvironment is not sufficient to completely inhibit cytotoxicity.

[0120] Example 4 FAT3 and LRP1B co-mutations affect the infiltration of immune cells in the microenvironment

[0121] A successful anti-tumor immune response requires the presence, activation, and co-stimulation of all lymphocyte components of the immune system, including various immune cells. In particular, the quality and quantity of tumor-infiltrating lymphocytes are closely related to immunotherapy outcomes and prognosis. Here, this application uses the CIBERSORT algorithm to specifically analyze the composition and proportion of 22 immune cells in the tumor microenvironment of the entire endometrial cancer cohort ( Figure 3 a) Based on whether there are co-mutations of FAT3 and LRP1B, this application divides the cohort into two groups. + / LRP1B + Activated CD4 + Memory T cells, CD8 + T cells, follicular helper T cells, resting NK cells, and M1 macrophages increased significantly, while plasma cells, activated NK cells, and activated dendritic cells decreased significantly ( Figure 3 b) Correlation analysis between immune cells showed that activated CD4 + Memory T cells and CD8 + T cells had the strongest positive correlation, while M1 macrophages and plasma cells had the strongest negative correlation ( Figure 3 c). This result reveals a feedback relationship between the abundance of immune cells.

[0122] Example 5 Effects of co-mutation of FAT3 and LRP1B on signaling pathways

[0123] The above study systematically analyzed the correlation between FAT3 and LRP1B co-mutations and genomic changes, cytotoxicity, immune microenvironment, and lymphocyte infiltration. This example intends to further explore the mechanism by which co-mutations affect tumor development. In order to evaluate the significance of co-mutations, this application used GSEA to perform pathway enrichment analysis on the endometrial cancer cohort. The analysis results showed that gene sets such as positive regulation of DNA replication, homologous recombination, nucleotide excision repair, processing and presentation of peptides or polysaccharides through MHC class II antigens, T cell chemotaxis, T cell receptor signaling pathways, and regulation of tumor cell responses were significantly enriched in samples with FAT3 and LRP1B co-mutations ( Figure 4 ag). The enriched gene sets revealed the potential mechanisms of co-mutation in endometrial cancer.

[0124] Example 6 Clinical outcomes of patients with endometrial cancer co-mutated with FAT3 and LRP1B

[0125] Considering that the endometrial cancer subgroup with FAT3 and LRP1B co-mutations has a reduced recurrence and metastasis rate and is entirely composed of POLE mutations and MSI-H subtypes, this application believes that co-mutations can also be used for risk stratification. Preliminary analysis showed that FAT3 ( Figure 5 a, p = 0.0035) or LRP1B ( Figure 5 b, p = 0.0013) patients with mutations had significantly longer PFS. However, when the entire cohort was further subdivided into four groups, there was a significant difference in PFS between the co-mutation subgroup and the subgroup with only FAT3 or LRP1B mutations, among which FAT3 + / LRP1B + The group with the longest PFS ( Figure 5 c, p=0.0038). Further analysis showed that when only FAT3 ( Figure 5 d, p = 0.89) or LRP1B ( Figure 5 e, p = 0.72) mutation, their PFS was not significantly different from other samples, only patients with FAT3 and LRP1B co-mutations had significantly prolonged PFS ( Figure 5 f, HR=0.16, 95%CI=0.096-0.28, p=0.0004). As with PFS, OS also had the same results ( Figure 6 ae), only FAT3 + / LRP1B + The OS of the subgroup was significantly better than that of other patients ( Figure 6 f, HR=0.15, 95%CI=0.082-0.29, p=0.0024). In conclusion, as expected, endometrial cancer patients with co-mutations of FAT3 and LRP1B have a better prognosis.

[0126] Example 7 Validation of a new cohort of patients with endometrial cancer co-mutated with FAT3 and LRP1B

[0127] This example uses a new cohort of 95 endometrial cancer samples to verify some of the results. When the entire cohort is divided into two groups: co-mutation and wild-type, this application found that the co-mutation FAT3 + / LRP1B + The TMB level of the group was significantly higher than that of the wild-type group ( Figure 7 a, 109.6 vs. 1.667, p<0.001). In addition, this group had a high proportion of POLE mutation subtypes (6 / 7) and no copy number high / low subtypes ( Figure 7 b, p<0.001). In addition, the co-mutation group had significantly higher PD-L1 expression levels compared with other samples ( Figure 7 c, p=0.030). These results obtained in the validation group are consistent with the previous conclusions of this application, which fully proves the value of FAT3 and LRP1B co-mutation in the classification and prognosis of endometrial cancer.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a FAT3 and LRP1B co-mutation detection agent in the preparation of a kit for predicting, evaluating or screening the sensitivity of endometrial cancer patients to immunosuppressant therapy, wherein the presence of the FAT3 and LRP1B co-mutation is an indication that the endometrial cancer patients are sensitive to immunosuppressant therapy.

2. Application of FAT3 and LRP1B co-mutation detector in the preparation of endometrial cancer prognosis classification products.

3. The application according to any one of claims 1-2, characterized in that: The mutation is a non-synonymous mutation in the gene coding region.

4. The use according to claim 3, characterized in that: The non-synonymous mutations include: single nucleotide polymorphism, base substitution / insertion / deletion, silent mutation, missense mutation, nonsense mutation or splicing mutation.

5. The use according to any one of claims 1 to 2, characterized in that: The detection agent performs detection at the nucleic acid level; the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

6. The use according to any one of claims 1-2, characterized in that: The detection agent is used to detect at the protein level, and is used to perform any one of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and detection using antibodies specifically designed for mutation sites.

7. The use according to any one of claims 1-2, characterized in that: The kit also includes reagents for detecting other gene mutations, and the other genes include: one or more of POLE, POLD1, TP53, PTEN, PIK3CA, ARID1A, PIK3R1, FBXW7, KRAS, and PPP2R1A genes.

8. The use according to claim 7, characterized in that: The kit also includes a sample processing reagent, which includes at least one of a sample lysis reagent, a sample purification reagent and a sample nucleic acid extraction reagent; the sample is selected from at least one of the plasma, tissue, pleural effusion, ascites and cerebrospinal fluid of the endometrial cancer patient.

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