Biomarker and immunokit for diagnosis and treatment and prognosis evaluation of HER2 positive metastatic breast cancer and application of biomarker and immunokit

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a biomarker for diagnosis and treatment and prognosis evaluation of HER2 positive metastatic breast cancer, an immunokit and application of the biomarker and the immunokit. Serum adenosine is adopted as the HER2 positive metastatic breast cancer diagnosis and treatment and prognosis evaluation biomarker. The prognosis condition of the patient is evaluated by detecting the relative expression level of the serum adenosine. The serum adenosine level is a potential early index for predicting the treatment effect and disease progression of trastuzumab. The invention discloses the prediction capability of serum adenosine on the disease progress and targeted therapy response of HER2 positive metastatic breast cancer patients for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a biomarker, an immune kit and an application thereof for the diagnosis and treatment of HER2-positive metastatic breast cancer and prognosis evaluation. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is positive and is defined as HER2 protein overexpression, with six or more copies of the HER2 gene or a HER2 / CEP17 ratio of 2.0 or more as measured by immunohistochemistry status (IHC3+) or fluorescence in situ hybridization (FISH). HER2-positive breast cancer is highly invasive and has a poor prognosis, accounting for about 15% to 20% of breast cancer. With the continuous emergence and widespread use of anti-HER2 drugs, the prognosis of patients with HER2-positive breast cancer has been significantly improved, with a survival rate of nearly 5 years for female patients with metastatic HER2-positive breast cancer, and 75% of patients achieving pathological complete remission. Despite these achievements, the high number of deaths caused by HER2-positive breast cancer continues to grow due to resistance and heterogeneity of response to anti-HER2 treatment, as well as brain metastases in up to 40% to 50% of patients with HER2-positive advanced breast cancer. There is an urgent need to strengthen the clinical invention of new therapies and new combinations.

[0003] According to the latest cancer statistics, there were more than 2 million cases of breast cancer worldwide in 2018, accounting for one-tenth of all cancers. It is also the second leading cause of cancer death. There are four main molecular subtypes of breast cancer, of which the proportion of human epidermal growth factor receptor 2 (HER2) positive subtype accounts for about 25%-30% of all breast cancers. HER-positive tumors are highly invasive, prone to recurrence and progression, and resistant to drug treatment, with poor clinical prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 that can significantly reduce the risk of recurrence and death. A large number of inventions have confirmed that trastuzumab alone or in combination with chemotherapy has significant clinical benefits for patients with metastatic breast cancer (MBC). In the treatment of MBC patients, the effective rate of trastuzumab reached 35%, and the combination of trastuzumab and chemotherapy drugs can increase the effective rate to 57.3%. In addition, the median survival time was extended by more than 5 months.

[0004] Despite the abundance of treatment options, the clinical efficacy of HER2-targeted combined chemotherapy remains unstable because some patients do not respond to treatment. Due to the biological heterogeneity of tumors caused by gene mutations and tumor microenvironment, HER2 expression and amplification may also have intratumoral heterogeneity, resulting in different sensitivity of patients to HER2-targeted drugs. A recent invention showed that breast cancer patients with tumor heterogeneity were insensitive to HER-2 targeted therapy and did not achieve clinical remission. This has prompted the invention of trastuzumab resistance mechanisms and the identification of effective biomarkers to guide personalized treatment. Although the mechanism of action of the immune system in HER2-positive breast cancer is still unclear, the view that the immune mechanism is a key factor in regulating the action of HER2-targeted drugs has been recognized. Although the mechanism of action of the immune system in HER2-positive breast cancer is still unclear, the view that the immune mechanism is a key factor in regulating the action of HER2-targeted drugs has been recognized, promoting tumor development and response to treatment. This process plays an indispensable role in cancer biology.

[0005] Previous studies have shown that ATP metabolism is abnormal in tumor cells, and CD39 and CD73 break down ATP into the end product adenosine. Local accumulation of adenosine in the microenvironment inhibits dendritic cells and effective T cells and enhances the polarization of tumor-associated macrophages to the M2 phenotype. In addition, adenosine can effectively regulate the progression and cell growth of breast cancer. Therefore, adenosine metabolism in the breast cancer microenvironment is crucial for tumor immunity. However, the prognostic significance of adenosine in breast cancer is still unclear, and data from large cohort studies are needed to fully understand its clinical relevance. In summary, adenosine has potential value in tumor targeting and clinical translation.

[0006] Adenosine metabolism in the breast cancer microenvironment is crucial for tumor immunity. However, the prognostic significance of adenosine in breast cancer remains unclear. We aimed to dynamically monitor serum adenosine levels in patients with HER2-positive metastatic breast cancer and explore its predictive significance during trastuzumab treatment. Summary of the invention

[0007] In the present invention, a biomarker, an immunological kit and its application for the diagnosis and treatment of HER2-positive metastatic breast cancer and prognosis evaluation are provided. The present invention aims to dynamically monitor the serum adenosine level of HER2-positive MBC patients and explore its predictive value in trastuzumab treatment. The present invention is of great significance for early identification of treatment benefit groups, further promoting MBC targeted and immune synergistic treatment, and providing new ideas for MBC targeted treatment.

[0008] The technical solution of the present invention is as follows:

[0009] A biomarker for the diagnosis, treatment and prognosis evaluation of HER2-positive metastatic breast cancer, characterized in that serum adenosine is used as a biomarker for the diagnosis, treatment and prognosis evaluation of HER2-positive metastatic breast cancer.

[0010] The prognosis of the patient is evaluated by detecting the relative expression level of the serum adenosine.

[0011] The serum adenosine level is a potential early indicator for predicting the therapeutic effect of trastuzumab and disease progression.

[0012] An immunoassay kit comprising a reagent for detecting serum adenosine levels.

[0013] The serum adenosine detection reagent is used in the preparation of an immunological test kit for auxiliary diagnosis of HER2-positive metastatic breast cancer.

[0014] The beneficial effects of the present invention are as follows:

[0015] Trastuzumab is a monoclonal antibody that targets HER2-positive tumor cells through immune mechanisms such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Clinical inventions have found that the characteristics of immune infiltration are associated with the clinical benefits of trastuzumab. A large number of inventions have shown that the more tumor-infiltrating T lymphocytes (til), the greater the clinical benefit of HER2-targeted therapy and the higher the complete remission rate. In addition, the types of lymphocytes and circulating tumor cells (CTCS) are also valuable for predicting efficacy and prognosis. Some inventions screen for benefit indicators of targeted HER2 therapy through gene sequencing and other aspects. Therefore, finding a biomarker that is simple, stable and effective to screen has become an urgent problem to be solved in clinical personalized medicine. The present invention dynamically monitors the serum adenosine level of HER2-positive MBC patients during trastuzumab treatment. We have confirmed that adenosine levels may be a potential early indicator for predicting the efficacy of trastuzumab treatment and disease progression.

[0016] Like most solid tumors, breast cancer has a higher energy metabolism and extracellular ATP release, and therefore, the hydrolysis of ATP, ADP and AMP and the accumulation of extracellular adenosine. In the cancer microenvironment, adenosine binds to its ligands and triggers signal transduction such as cAMP and NF-κB, thereby inhibiting the anti-tumor function of immune cells. Atezolizumab is the only FDA-approved immunotherapy for breast cancer, which is unfortunately limited to MBC. The lack of response to current immune checkpoint inhibitors can be attributed to the upregulation of other compensatory immune checkpoints after treatment. In addition, HIF-1α, as a biomarker of the hypoxic tumor microenvironment in cancer, can serve as a potent enhancer of CD39 and CD73 ectonucleotide expression, thereby mediating extracellular adenosine accumulation and establishing immune suppression. These conclusions stimulated our interest in exploring the clinical predictive value of adenosine in HER2-positive MBC patients.

[0017] Considering the high economic cost of multi-gene testing and the limited application scope of clinical institutions or hospitals, it is reasonable to find easier alternative tests. Metabolites can assess metabolic status and related disease risks by directly analyzing the concentration and changes of individual metabolites. It is relatively simple, can directly reflect the current metabolic state, and has certain practical value. Adenosine is an endogenous purine nucleoside that mediates intracellular cAMP through four G protein-coupled receptors. Most reports have explored the relationship between adenosine deaminase 2 (ADA2), a polymorphic enzyme that regulates adenosine concentration, and various clinical outcomes. Few reports have implicated the role of peripheral adenosine levels in physiological and pathological processes. However, the predictive role of adenosine in malignant tumors has not been reported. Some reports have shown that the type and density of immune cells in the tumor microenvironment play a central role in disease progression. Immunological analysis of the tumor microenvironment has shown great promise in predicting prognosis and predicting treatment response. CD4+T cells play multiple roles in suppressing inflammation and inhibiting adaptive immune responses. NK cells are innate immune cells that have the ability to directly kill malignant cells. Treg cell-mediated suppression is an important mechanism of negative regulation of immune-mediated inflammation. CD4CD25 regulatory T lymphocytes are a class of immune regulatory cells discovered in recent years. They have two main characteristics: immunosuppression and immune energy. They also have potential application value in the treatment of autoimmune diseases, tumor immunotherapy, induction of transplant tolerance, etc. Adenosine has been shown to mediate pro-tumor activity, tumor tissue angiogenesis, and chemotherapy resistance. Adenosine regulates anti-tumor immune responses and promotes tumor immune escape. Therefore, adenosine is beneficial and indispensable in downregulating anti-tumor immune responses and tumor progression. This invention shows that serum adenosine is closely related to the progression of MBC and will become a promising biomarker for treatment response. We also constructed a prognostic model based on adenosine and verified its prognostic and predictive abilities. Single-cell derived data from existing public data revealed a transcriptome feature map of HER2-positive breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The schematic diagram of the prognostic model of the present invention is shown in FIG.

[0019] Figure 1 Zhong AB. Lasso regression model of DEGs based on adenosine signaling. Figure 1 Overview of mutations in the C breast cancer cohort. Figure 1 Middle D. Waterfall plot of the top 30 mutated genes in the TCGA breast cancer cohort. Figure 1 E. Somatic cell interaction analysis. Figure 1 Middle F. Survival curves of high-risk group and low-risk group. Figure 1 Middle G. ROC curve of adenosine-based risk signature.

[0020] Figure 2 Schematic diagram of adenosine-related immune scoring of the present invention.

[0021] Figure 2 AB. OS survival curves between adenosine groups in the subgroups of patients with high or low HRD scores. Figure 2 C. Volcano plot of deg between adenosine groups. Figure 2 D. ssGSEA enriched the top terms of the adenosine group. Figure 2 E. Heat map of infiltrate immune signature scores based on five algorithms. Figure 2 F. Box plot of immune checkpoint gene expression in two adenosine-related risk groups.

[0022] Figure 3 This is a single-cell transcriptome feature analysis map of HER2-positive breast cancer of the present invention.

[0023] Figure 3 A. UMAP visualization of HER2-positive sequencing data at the single-cell level from 5 breast cancer patients. Figure 3 B. The percentage of each cell in the sample. Figure 3 Middle C. Activated metabolic pathways in HER2-positive breast cancer patients. Figure 3 Middle D. UMAP visualization of the oxidative phosphorylation pathway. Figure 3 Middle E. Visualization of interaction weights for HER2-positive breast cancer subsets. Figure 3 Analysis of FANGPTL ligand-receptor interactions and visualization of ring clustering. Figure 3 Middle H. Heatmap visualization of re-clustered CAFs. Figure 3 Heat map of cluster marker genes in CAFs of HER2-positive breast cancer patients. Figure 3 JK. Changes in cell state and gene expression over pseudotime.

[0024] Figure 4 This is a schematic diagram of the predictive value of serum adenosine for the clinical progression of HER2-positive MBC patients according to the present invention.

[0025] Figure 4 A. Serum adenosine levels in 15 HER2-positive MBC patients. Figure 4 Middle B. One patient was assessed as stable and progressive after initial PD status. Figure 4 CD. Patient was evaluated as PD after initial SD status. Figure 4 Middle E. During the follow-up period, a representative HER2-positive MBC patient was evaluated as SD. Serum TR ( Figure 4 F) and cfDNA ( Figure 4G) levels. TR: thioredoxin reductase; cfDNA, circulating free DNA. MBC, metastatic breast cancer

[0026] Figure 5 This is a schematic diagram of the differences in immune microenvironment between the PD group and the SD group of HER2-positive MBC patients in the present invention.

[0027] Figure 5 (A) CD4+ cells, Figure 5 Middle (B) NKT, Figure 5 Middle (C) CD4+CD25+ cells, Figure 5 Middle (D)NK, Figure 5 (E) The proportion of Treg cells in HER2-positive MBC patients between the PD group and the SD group.

[0028] Figure 6 Representative CT images of the HER2-positive MBC patients in the PD and SD subgroups of the present invention ( Figure 5 Patients with elevated adenosine levels in the brain ( Figure 5 A and C), lymph nodes ( Figure 5 Middle B) and liver ( Figure 5 D) all had new lesions. They were evaluated as PD. Figure 5 Middle E) Representative brain images of MBC patients with stable adenosine levels. They were assessed as SD. MBC, metastatic breast cancer; PD, progressive disease; SD, stable disease. DETAILED DESCRIPTION

[0029] The following is combined with Figure 1-Figure 6 The present invention is further described.

[0030] A biomarker for the diagnosis, treatment and prognosis evaluation of HER2-positive metastatic breast cancer, characterized in that serum adenosine is used as a biomarker for the diagnosis, treatment and prognosis evaluation of HER2-positive metastatic breast cancer.

[0031] The prognosis of the patient is evaluated by detecting the relative expression level of the serum adenosine.

[0032] The serum adenosine level is a potential early indicator for predicting the therapeutic effect of trastuzumab and disease progression.

[0033] An immunoassay kit comprising a reagent for detecting serum adenosine levels.

[0034] The serum adenosine detection reagent is used in the preparation of an immunological test kit for auxiliary diagnosis of HER2-positive metastatic breast cancer.

[0035] Methods: Sequence and clinical data were downloaded from TCGA and GSE176078, and the “DESeq2” package was used to analyze adenosine-related differentially expressed genes. Multivariate Cox regression and lasso regression were used to construct prognostic risk markers. Risk scores were calculated based on the expression of the screened core genes. Bioinformatics analysis was performed using R and its related packages. We also recruited HER2-positive metastatic breast cancer patients from our center and divided them into different treatment outcome groups based on the clinical results evaluated by enhanced CT. Adenosine levels in these patients were dynamically detected, and differences in the immune microenvironment between different groups were evaluated by recording immune cells in our center.

[0036] Results: A total of 109 patients with HER2-positive metastatic breast cancer were included, and 22 adenosine-related genes were screened from the TCGA database. A survival model based on 15 core genes was constructed, and the risk score of each patient was used as a prognostic risk factor. Single-cell transcriptome sequencing data determined the transcriptome profile of patients with HER2-positive breast cancer. We also demonstrated the predictive value of serum adenosine in clinical progression of patients with HER2-positive metastatic breast cancer. The different immune microenvironments between subgroups supported the reliability of the predictive ability of adenosine in patients with HER2-positive metastatic breast cancer.

[0037] Conclusion: The dynamic changes of serum adenosine are predictive biomarkers for monitoring disease progression. Adenosine metabolism-based markers have potential application value in the prognosis of patients with HER2-positive metastatic breast cancer.

[0038] Patient sources and adenosine-related categories:

[0039] HER-positive breast cancer patients with a survival time of at least 30 days were downloaded from The Cancer Genome Atlas Program (TCGA). Adenosine-related genes were derived from a previous invention. Differentially expressed gene analysis was performed using the “DESeq2” package. Multivariate Cox and lasso penalized Cox regression were used to construct the best prognostic risk signature. Risk scores were calculated based on the expression of the central genes.

[0040] The present invention includes female patients with HER2-positive breast cancer. All patients received trastuzumab and docetaxel treatment at the same time. The initial dose of trastuzumab was 8 mg / kg, followed by maintenance treatment at a dose of 6 mg / kg every three weeks until disease progression or unbearable side effects. Docetaxel was administered at a dose of 75 mg / m2 every 3 weeks for a total of 8 cycles, at least 6 cycles were completed, and the treatment period was 1 year.

[0041] Somatic mutation analysis:

[0042] Mutant data were obtained and visualized using the “maftools” package. The “somatic interaction” function was used to analyze co-occurrence or exclusivity between mutant genes using paired Fisher exact tests.

[0043] Establishment of prognostic model and survival analysis:

[0044] Patients were divided into high-risk and low-risk groups using the median risk score of each cohort. Kaplan-Meier survival and receiver operating characteristic (ROC) analysis were used to analyze the association of adenosine-related risk scores with overall survival (OS), as well as the accuracy of the model. The interaction between BRCA1 / BRCA2 mutations and homologous recombination deficiency (HRD) and adenosine-related features was assessed by hierarchical analysis and interaction P values ​​using the Wald test. The telomeric allele imbalance number (NtAI) counts, large-scale state transition (LST) counts, homologous recombination deficiency (HRD) scores, and loss of heterozygosity (HRD-loh) of TCGA samples were derived from a previous pan-cancer report.

[0045] ssGESA enrichment and biological immune characteristics analysis:

[0046] The enrichment scores in patients with different adenosine signatures were calculated using the ssGSVA method implemented in the “GSVA” package to explore the molecular mechanisms. In addition, the immune infiltration signature, that is, the association between the adenosine risk group and the immune microenvironment, was calculated using the CIBERSORT, ESTIMATE, MCPcounter, TIMER, and TIDE algorithms and compared using the Wilcoxon rank sum test. The expression of immune checkpoint genes was also analyzed.

[0047] Download and process single cell sequencing data of HER2-positive breast cancer patients:

[0048] GSE176078 single cell breast cancer was downloaded from the GEO public database (https: / / www.ncbi.nlm.nih.gov). GSE176078 included 5 patients with HER2-positive breast cancer. Seurat objects were constructed with min.cells=3 and min.features=300, and the correlation between nFeatures, counts, and sequencing depth was calculated. The percentages of mitochondrial genes, ribosomal genes, and erythroid genes were calculated. Cells were filtered using nFeature_RNA>4000, mitochondrial gene percentage ≥15%, counts ≥10000, and erythroid gene percentage ≥1%. The following settings were used: normal. method = "LogNormalize", scaling. “NormalizeData” function with factor = 10,000. “FindVariableFeatures” calculated the top 3000 highly variable genes. The UMAP method was used to reduce the dimensionality of the data, and the “KNN” (k-nearest neighbor) method with a resolution of 0.2 was used to cluster the cells. Subsequently, known cell-specific markers were used for annotation.

[0049] Serum sample collection and adenosine content detection:

[0050] During treatment, serum samples were collected every two treatment cycles. Serum samples were collected every 8 weeks 24 weeks after the end of treatment. Serum samples were collected every 12 weeks 24-48 weeks after the end of treatment, or until disease progression or the end of the invention. The human adenosine assay kit (Abcam) was used to detect the patient's serum adenosine level. During treatment or follow-up after treatment, about 500 μL of serum specimens were collected for hematological examination and preservation. The present invention was approved by the Invention Ethics Committee of Jiangsu Cancer Hospital. All patients had written informed consent to participate in the present invention. The TR detection kit (No. 20110901) and QuantiDNATM DNA assay kit (No. 20110901) were used to detect the activity of thioredoxin reductase (TR) and the level of circulating free DNA (cfDNA) in plasma (No. DC-08-0092R). All samples to be tested were extracted from the remaining serum samples for clinical testing. Flow cytometry was used to analyze immune cell counts. Natural killer (NK) cells are marked with CD3-CD16+CD56+, natural killer T (NKT) cells are marked with CD3+CD16+CD56+, B cells are marked with CD19+, active T cells are marked with CD3+HLA-DR+, and regulatory T (Treg) cells are marked with CD4+CD25+CD127low.

[0051] Statistical analysis:

[0052] All statistical analyses were performed using SPSS 22.0 (SPSS, Inc. Chicago, USA) and GraphPad Prism software (version 9.0). Two-tailed t-test was used to compare the differences in immune cell counts. P values ​​less than 0.05 were considered statistically significant.

[0053] Patient screening and adenosine-related classification:

[0054] Since adenosine signaling has an immunosuppressive effect in the microenvironment, we first screened for significantly differentially expressed genes (DEGs) that intersected with adenosine genes. A total of 15 DEGs related to adenosine signaling were identified (Table 1).

[0055] Table 1. Table 1 Differential expression of ABO-related genes in normal and tumor specimens

[0056]

[0057]

[0058]

[0059] We screened and matched 109 HER2-positive breast cancer patients and the expression of 22 adenosine-related genes from the TCGA database. Considering the close relationship between adenosine signaling and the immune microenvironment, we used the lasso regression model to establish a survival model based on adenosine signaling deg ( Figure 1 AB). The formula for calculating the adenosine-related risk signature of HER2-positive breast cancer patients was: ENPP1*0.039+NME2*0.033+BST1*0.058-ADORA3*0.0744+ADORA2A*0.044. The risk score of each patient was calculated based on the expression of central adenosine genes in HER2-positive breast cancer patients. All patients were divided into high-risk or low-risk groups according to the median score value.

[0060] Mutational landscape and adenosine-related survival analysis:

[0061] The mutation database of breast cancer patients was obtained from TCGA. For all patients, Missense_Mutation, SNP, and C>T mutation were the most common terms ( Figure 1 C). In the waterfall plot using the MutSigCV algorithm, the somatic mutation status of TP53, PIK3CA, TTN, CDH1, GATA3, and MUC16 was higher ( Figure 1 D) Many pathogenic genes have the characteristics of co-occurrence or exclusivity in their mutation patterns. Correlation analysis of mutant genes showed that there was a strong exclusivity between TP53 and PIK3CA, which is consistent with literature reports ( Figure 1 E).

[0062] Patients with high adenosine risk scores had shorter OS than those with low risk scores (HR, 17; 95% CI, 5-56, P < 0.001, Figure 1 F). The AUC value further demonstrates the superiority of this feature in predicting short-term and long-term survival ( Figure 1 G). Due to the limitation of the number of patients, we further validated the reliability of adenosine-related risk score.

[0063] HRD score and biological immune characteristics of adenosine group:

[0064] Since HRD can be considered as a genomic instability score, we further validated the prognostic value of adenosine signatures based on HRD score subgroups. There were no significant differences in NtAI and LST counts, HRD-loh, and HRD scores between the high-risk and low-risk groups (Supplementary Figure 1 A). Patients were divided into two subgroups according to the median HRD score. Patients with high HRD scores had significant prognostic differences between high-risk or low-risk adenosine signature scores, as did patients with low HRD scores ( Figure 2 AB). This suggests that adenosine signature has a strong survival benefit in patients with HRD.

[0065] To further explore the genomic patterns of variation between adenosine-related risk groups, we performed DEGs analysis and revealed the molecular mechanisms by gene set enrichment analysis (GSEA). The most enriched terms were neuroactive ligand receptor interaction, maturity-onset diabetes of the young, and cytokine receptor interaction ( Figure 2 CD). In addition, five immune signature algorithms were applied to explore the correlation between adenosine scores and the immune microenvironment. The results showed that the immune signature of the low adenosine group was enriched in cancer-associated fibroblasts, NK cells, and M2 macrophages ( Figure 2 E). In contrast, the expression of checkpoint genes TNFRSF2, TNFRSF4, TNFRSF14, CD40, LAIR1, and ADORA2A was decreased in the high-risk group compared with the low-risk group ( Figure 2 F).

[0066] Single-cell transcriptomic characterization of HER2-positive breast cancer patients:

[0067] The single-cell transcriptome data of 5 HER2-positive breast cancer patients were reanalyzed and clustered, and a total of 9 clusters were identified ( Figure 3 A). The cell type ratios between samples were determined and it was found that T cells accounted for the majority percentage of cells ( Figure 3B). Other cell clusters are B cells, cancer epithelial cells, endothelial cells, perivascular-like cells, myeloid cells, plasmablasts, and cancer-associated fibroblasts (CAFs). Considering that metabolic reprogramming is an important hallmark of cancer cells and TME cells, we further demonstrated the activated metabolic pathways in HER2-positive breast cancer patients ( Figure 3 C). The most well-known classical pathways are activated, such as oxidative phosphorylation, glycolysis and gluconeogenesis, and glutathione metabolism ( Figure 3 D and supplement Figure 1 BC), indicating significant mitochondrial activity. Thanks to the advantages of scRNA-seq technology, we revealed high-resolution interactions between different subpopulations ( Figure 3 E). ANGPTL was confirmed to be a dependent mediator of CAF tumor-promoting function, and it communicated most strongly with the SDC and CDH families ( Figure 3 FG). In addition, CAFs from the HER2-positive subtype are unique compared to other types of breast cancer. We further explored the developmental trajectory of the cells using pseudo-time trajectory analysis. CAFs cells were re-aggregated and marked by MEST, CADM1, GRP, ARC, CFD, IDO1, and HIGD1B ( Figure 3 HI and supplement Figure 1 DE). We also showed the evolutionary trajectories of our proposed model genes and found that NME2 had the most significant changes ( Figure 3 JK).

[0068] Predictive value of serum adenosine in clinical progression of HER2-positive MBC patients:

[0069] Since proteins are the most direct response to biological processes and are easy to detect in clinical practice, we tested adenosine levels, which is a regulated end product of the above-mentioned characteristic genes. A total of 15 patients with HER2-positive breast cancer in our hospital were included in the present invention. The left ventricular ejection fraction of patients aged 18 to 70 years was greater than 50%. The median follow-up was 22 (5-23) months, the median age was 55 (34-75) years old, and 73% (11 / 15) patients were diagnosed with distant metastasis of varying degrees at the initial diagnosis. Among them, the primary lesion was in the left breast in 6 cases and the right breast in 8 cases, of which 1 case was diagnosed with bilateral breast cancer. At the last review, 10 cases were found to be in stable condition (SD) by imaging examination, and 5 cases were in progressive disease (PD). There were statistical differences in serum adenosine levels, which were higher in the progressive disease group ( Figure 4 A). In addition, serum adenosine levels fluctuate with clinical outcomes ( Figure 4 BD), while serum adenosine levels in SD patients were stable ( Figure 4E). In addition, TR and cfDNA were also included in the follow-up analysis. TR activity was significantly increased, indicating hyperactivity of tissue cells in vivo. cfDNA levels can be used for efficacy evaluation, tumor metastasis risk analysis, and tumor recurrence monitoring. A downward trend in cfDNA levels generally indicates a better response to treatment. Here, we observed higher TR and cfDNA levels in the PD group ( Figure 4 FG). The above results suggest that decreased adenosine levels can predict SD based on CT examination and tumor markers.

[0070] Differences in immune microenvironment and clinical outcomes between PD and SD groups in HER2-positive MBC patients:

[0071] To further explore the immune microenvironment between the PD and SD groups of HER2-positive MBC patients, we performed immune cell counts in our hospital. We found that CD4+, NKT+, and CD4+CD25+ cells were significantly increased in the PD group ( Figure 5 AC), NK+ and Treg cells were significantly reduced ( Figure 5 DE). There was no difference in CD8+, B cells and activated T cells (Figure S2A-C).

[0072] Imaging studies were used to assess clinical outcomes and confirmed the predictive role of serum adenosine in patients with HER2-positive MBC. We found that patients with elevated adenosine levels developed new lesions and were evaluated for disease progression ( Figure 6 AD). The remaining 10 patients had stable adenosine levels and stable clinical outcomes after evaluation ( Figure 6 E).

[0073] in conclusion:

[0074] The present invention reveals for the first time the predictive ability of serum adenosine for disease progression and targeted therapy response in patients with HER2-positive metastatic breast cancer.

Claims

1. A biomarker for the diagnosis, treatment and prognosis assessment of HER2-positive metastatic breast cancer. It is characterized in that Serum adenosine is used as a biomarker for the diagnosis, treatment and prognosis of HER2-positive metastatic breast cancer.

2. A use of the biomarker for diagnosis, treatment and prognosis assessment of HER2-positive metastatic breast cancer as claimed in claim 1, It is characterized in that The prognosis of the patient is evaluated by detecting the relative expression level of the serum adenosine.

3. A use of the biomarker for diagnosis, treatment and prognosis assessment of HER2-positive metastatic breast cancer as claimed in claim 2, It is characterized in that The serum adenosine level is a potential early indicator for predicting the therapeutic effect of trastuzumab and disease progression.

4. An immunoassay kit comprising a reagent for detecting serum adenosine levels.

5. Use of the serum adenosine detection reagent according to claim 1 in the preparation of an immunoassay kit for auxiliary diagnosis of HER2-positive metastatic breast cancer.

Images