Application of PIK3CA and ESR1 co-mutation in predicting sensitivity of breast cancer patient to pilosilide and fulvestrant combined treatment

Abstract

The invention discloses an application of PIK3CA and ESR1 gene co-mutation in predicting the sensitivity of a breast cancer patient to combined treatment of piloxil and fulvestrant. Through verification, it is found that PIK3CA and ESR1 gene co-mutation has high accuracy, sensitivity and specificity, can be used for accurate prediction of the sensitivity of breast cancer to pilosilil and fulvestrant combined treatment, can assist clinicians in judging the sensitivity of breast cancer patients to a pilosilil and fulvestrant combined treatment scheme, and can be used for predicting the sensitivity of the breast cancer patients to the pilosilil and fulvestrant combined treatment scheme. And good balance and maximum benefit among curative effect, adverse reaction and medication cost are achieved.

Description

Technical Field

[0001] This invention belongs to the biomedical field and relates to the application of PIK3CA / ESR1 co-mutation in predicting the sensitivity of breast cancer patients to combination therapy with piroxicillin and fulvestrant. Background Technology

[0002] The successful development and approval of cyclin-dependent kinase 4 / 6 inhibitors (CDK4 / 6 inhibitors) has transformed the treatment landscape for hormone receptor-positive (HR+) breast cancer. Currently, the four most commonly used CDK4 / 6 inhibitors in clinical practice include palbociclib, reboxiclib, abeciclib, and dalcilib. The median progression-free survival for these drugs as second-line treatment after progression on first-line endocrine therapy in HR+ advanced breast cancer is 11.2, 14.6, 16.4, and 15.7 months, respectively, with hazard ratios ranging from 0.42 to 0.57. Pireociclib (trade name: Xuan Yue Ning) is a novel CDK4 / 6 inhibitor independently developed by the Chinese pharmaceutical company Xuanzhu Biotechnology. Its structure is similar to abeciclib. Studies have demonstrated that pirociclib monotherapy or in combination with endocrine therapy has good efficacy and safety in HR+, HER2-negative advanced breast cancer patients, especially in patients with primary endocrine resistance, liver metastases, and other refractory conditions. Based on this evidence-based medicine, the China National Medical Products Administration approved piroxicillin on May 13, 2025, for use as monotherapy in adult patients with HR+ / HER2- advanced or metastatic breast cancer who have experienced disease progression after prior metastatic treatment with two or more endocrine therapies and one chemotherapy regimen, and in combination with fulvestrant for patients who have experienced disease progression after prior endocrine therapy. Based on these research results and drug approval, piroxicillin has joined the ranks of CDK4 / 6 inhibitors in clinical application in my country, providing a new treatment option for HR+ / HER2- breast cancer patients.

[0003] It is worth noting that although various CDK4 / 6 inhibitors offer more effective treatment options for HR+ / HER2- breast cancer patients, the large HR+ patient population (accounting for approximately 70% of all breast cancer patients) leads to significant differences in the efficacy of CDK4 / 6 inhibitors combined with endocrine therapy among different patients in clinical practice. Some patients achieve long-term remission or disease stabilization, while others develop resistance and disease progression in a short period. Therefore, accurately identifying treatment-sensitive or resistant patient groups and selecting individualized treatment regimens is crucial. Currently, relevant research mainly focuses on the mechanisms of resistance, with key mechanisms including RB gene deletion or mutation, RTK / RAS pathway gene activation mutations, AURKA amplification, high expression of cyclin E, and excessive activation of CDK2 and CDK6. No readily available and reliable biomarkers have yet been identified for screening and predicting treatment-sensitive populations.

[0004] Recently, the results of the INAVO-120 study were published, a groundbreaking study that further optimized first-line treatment options for HR+ advanced breast cancer. This study enrolled breast cancer patients with PIK3CA mutations, who received a three-drug combination regimen of phosphatidylinositol 3-kinase (PI3K) inhibitor, CDK4 / 6 inhibitor, and endocrine inhibitor as first-line treatment. The results showed that the triple regimen of enoxaparin, palbociclib, and fulvestrant significantly prolonged progression-free survival (PFS) and overall survival (OS) compared to palbociclib or fulvestrant alone. The differential analysis in this study suggested that the significant benefit of the triple regimen may be related to the simultaneous blockade of the estrogen receptor (ER), cell cycle, and PI3K pathways, with significant interactions among these pathways. However, the broad inhibition of these key pathways by the triple regimen also affected the physiological functions of normal tissues, including hyperglycemia, stomatitis, rash, and diarrhea. Therefore, among all HR+ advanced breast cancer patients with PIK3CA mutations, selecting patients who are highly sensitive to CDK4 / 6 inhibitors for step-down therapy while considering efficacy and the incidence of adverse reactions, and extending survival while ensuring patients' quality of life, has become an urgent problem to be solved. Summary of the Invention

[0005] To address the aforementioned technical issues, this application, through a systematic analysis of HR+ / HER2- advanced breast cancer patients, has for the first time discovered that co-mutations of PIK3CA and ESR1 define a specific cluster associated with the prognosis of piroxicillin and fulvestrant in the treatment of breast cancer. These co-mutations can be used to guide clinical medication regimens.

[0006] Based on this, this application proposes the following specific technical solutions: In a first aspect, the present invention provides the use of a reagent for detecting co-mutations of PIK3CA and ESR1 in a sample in the preparation of a product for predicting the sensitivity of breast cancer patients to combination therapy with piroxicillin and fulvestrant.

[0007] Furthermore, the sample includes any one of peripheral blood, plasma, tumor tissue, or puncture sample.

[0008] In a specific embodiment of the present invention, the sample is peripheral blood.

[0009] In this invention, the term "sample" refers to a composition obtained from or derived from a patient / subject that contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, a sample refers to any sample derived from a patient / subject that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell cultures, cell supernatants, cell lysates, platelets, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, pancreatic juice, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tissue culture fluid, tissue extracts, homogenized tissue, cell extracts, and combinations thereof.

[0010] Furthermore, the reagents include reagents for detecting gene-level mutations in PIK3CA and ESR1 in the sample and / or reagents for detecting protein-level mutations in PIK3CA and ESR1 in the sample.

[0011] In this invention, the term "gene-level mutation" refers to changes in genetic material at the DNA level, including but not limited to point mutations (missense mutations, nonsense mutations, synonymous mutations), insertion mutations, deletion mutations, frameshift mutations, splice site mutations, repetitive sequence abnormalities, copy number variations (CNVs), and structural variations (such as translocations, inversions, large fragment deletions or insertions) that result in changes to gene sequences or gene structures.

[0012] In a specific embodiment of the present invention, the co-mutation of the PIK3CA and ESR1 genes refers to the simultaneous occurrence of any one of the following gene mutations in both the PIK3CA and ESR1 genes, including but not limited to: frameshift deletion, in-frame insertion / deletion, non-termination nonsense mutation, frameshift insertion, missense mutation, nonsense mutation, in-frame insertion, splice site mutation, in-frame deletion, splice region mutation, amplification, deletion, multiple hit, and complex events.

[0013] The term "protein-level mutation" refers to changes in protein sequence, three-dimensional structure, stability, expression level, subcellular localization, or function caused by gene sequence variations, splicing abnormalities, translation errors, or post-translational modifications. These changes include, but are not limited to, amino acid substitutions, truncation, deletion, conformational changes, overexpression, underexpression, or gain / loss of function. Furthermore, the reagent is selected from the group consisting of: (1) Primers for specific amplification of PIK3CA and ESR1; (2) Probes that specifically recognize PIK3CA and ESR1; (3) A binding agent that specifically binds to mutant proteins encoded by PIK3CA and ESR1.

[0014] Furthermore, the binding agent comprises an antibody, an antibody functional fragment, or a conjugated antibody that specifically binds to mutant proteins encoded by PIK3CA and ESR1.

[0015] In this invention, the term "primer" refers to a short oligonucleotide sequence used to specifically bind to a target nucleic acid template and initiate DNA polymerization in nucleic acid amplification reactions (such as PCR, qPCR, digital PCR, etc.). The primers typically include forward and reverse primers for specific amplification of the target nucleic acid region. The term "probe" refers to an oligonucleotide molecule that is specifically complementary to the target nucleic acid sequence and used for detecting, quantifying, or recognizing the target sequence. The probe may carry fluorescent groups, quenching groups, chemically modified groups, or other detectable tags and can be used in techniques such as qPCR, digital PCR, FISH, hybridization capture, and microarray detection. Furthermore, the product includes reagents for detecting the co-mutation of PIK3CA and ESR1 using nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, sequencing technology, chromatography technology, and mass spectrometry technology.

[0016] In this invention, the nucleic acid hybridization technology refers to a technology that achieves detection based on the principle of complementary pairing of nucleic acids, through the specific binding of probes to target nucleic acid sequences, including but not limited to: in situ hybridization, fluorescence in situ hybridization, nucleic acid capture hybridization, Southern blot, Northern blot, hybridization chips, etc.; the nucleic acid amplification technology refers to a technology that uses enzymatic reactions to exponentially amplify target nucleic acid sequences for detection, including but not limited to: polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), reverse transcription PCR (RT-PCR), nested PCR, LAMP isothermal amplification technology, rolling circle amplification (RCA), and the amplification step in high-throughput sequencing (NGS); the protein immunoassay technology refers to a technology that achieves qualitative or quantitative detection of proteins based on antigen-antibody specific recognition, including but not limited to: enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunochromatographic assay (LFIA), Western blot, immunohistochemistry (IHC), flow cytometry, etc. Chromatography techniques include, but are not limited to, high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gel filtration chromatography, ion exchange chromatography, and affinity chromatography.

[0017] Furthermore, the products also include chips, reagent kits, and nucleic acid modules.

[0018] Furthermore, the chip includes gene chips and protein chips.

[0019] Furthermore, the gene chip includes oligonucleotide probes for detecting PIK3CA and ESR1.

[0020] Furthermore, the protein chip includes a binder for the mutant protein encoded by PIK3CA and ESR1.

[0021] Furthermore, the kit includes a gene detection kit and a protein detection kit.

[0022] Furthermore, the gene detection kit includes reagents or chips for detecting PIK3CA and ESR1; the protein detection kit includes reagents or chips for detecting mutant proteins encoded by PIK3CA and ESR1.

[0023] Furthermore, the kit includes reagents for detecting PIK3CA and ESR1 mutations or corresponding encoded protein mutations using RT-PCR, qRT-PCR, microarray detection, DNA blotting, in situ hybridization, immunoblotting, and enzyme-linked immunosorbent assay.

[0024] Furthermore, the kit also includes excipients and / or carriers.

[0025] In this invention, the excipients and / or carriers refer to pharmaceutically or biologically acceptable components used to stabilize, dilute, protect, or assist the reagents of this invention in functioning, including but not limited to: buffer solutions, stabilizers, protein protectants, surfactants, salts, solvents, excipients, biocompatible polymers, microspheres, liposomes, or other carrier materials suitable for the preparation of nucleic acid or protein reagents.

[0026] Furthermore, the kit also includes instructions.

[0027] Furthermore, the product also includes reagents for processing samples.

[0028] Furthermore, the breast cancer is HR+ / HER2- advanced breast cancer.

[0029] In this invention, the term "breast cancer" refers to a malignant tumor originating from mammary epithelial cells, including but not limited to molecular subtypes classified according to hormone receptor status (ER, PR) and HER2 expression status, namely Luminal breast cancer, HER2-positive breast cancer, and triple-negative breast cancer; in a specific embodiment of this invention, the breast cancer is hormone receptor-positive (HR+) and HER2-negative (HER2-) breast cancer, more preferably, HR+ / HER2- advanced endocrine-resistant breast cancer.

[0030] A second aspect of the present invention provides a system for predicting the sensitivity of breast cancer patients to combined treatment with piroxicillin and fulvestrant, the system comprising an input unit, an analysis unit, and an output unit.

[0031] Furthermore, the input unit is used to acquire mutation status data of PIK3CA and ESR1 in the breast cancer patient to be tested.

[0032] Furthermore, the analysis unit is used to input the mutation status data obtained by the input unit into the prediction model. The prediction model is a pre-trained model. The prediction model determines the sensitivity of the breast cancer patient to the combined treatment of piroxicam and fulvestrant based on the mutation status data of PIK3CA and ESR1. When the breast cancer patient to be tested has co-mutations of PIK3CA and ESR1, the prediction result of the breast cancer patient's sensitivity to the combined treatment of piroxicam and fulvestrant is obtained.

[0033] Furthermore, the output unit is used to output the prediction result.

[0034] Furthermore, the steps for constructing the prediction model in the analysis unit include: collecting breast cancer patient samples with known response status to pilociclib and fulvestrant treatment, detecting their co-mutation status of PIK3CA and ESR1 as input feature data, providing the input feature data to a machine learning algorithm, and obtaining the prediction model.

[0035] Furthermore, the machine learning algorithm includes algorithmic models developed using various development tools; these development tools include, but are not limited to, TensorFlow, Scikit-Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, and Spell; the algorithmic models include, but are not limited to, linear regression models, logistic regression models, Lasso regression models, Ridge regression models, linear discriminant analysis models, nearest neighbor models, decision tree models, perceptron models, neural network models, support vector machine models, Naive Bayes models, AdaBoost models, GBDT models, XGBoost models, LightGBM models, CatBoost models, and random forest models.

[0036] Furthermore, the output unit also includes a receiving unit, which can be used to receive prediction results; the receiving unit includes a display screen, a computer client, a mobile client, or a tablet.

[0037] In this invention, the input unit is used to acquire PIK3CA and ESR1 mutation data of breast cancer patients, and can support multiple detection sources and data types. The PIK3CA and ESR1 mutation data includes, but is not limited to, data obtained through Sanger sequencing, polymerase chain reaction (PCR) and its derivatives, high-resolution melting curve analysis (HRM), probe-based nucleic acid hybridization, high-throughput sequencing (NGS), third-generation sequencing, liquid biopsy, etc. The input unit may include a data interface module for receiving data or images output by the detection instrument; a sample information management module for entering the basic information of the subject (number, gender, age, pathological origin, etc.); and a data preprocessing module for standardizing, background subtracting, or numerically transforming the raw test results.

[0038] The analysis unit can use statistical algorithms (such as Z-score judgment, t test) or machine learning algorithms (such as logistic regression, support vector machine SVM) to perform pattern recognition on the mutation status of PIK3CA and ESR1; when the input is multidimensional data, the analysis unit can realize multimodal fusion judgment to improve diagnostic accuracy.

[0039] The output unit is used to output diagnostic results, including but not limited to quantitative results, qualitative judgments, graphical displays (ROC curves, AUC values), and suggestive prompts (such as "further pathological review is recommended"). The output unit can transmit the results to the clinical information system via a display screen, printed report, or electronic interface, and can generate a PDF format diagnostic report containing the patient number, testing method, result interpretation, and data traceability information. The output unit supports cloud storage and multi-center data sharing to enable AI training and model updates.

[0040] The advantages and beneficial effects of this invention are as follows: (1) Improving the precision and individualization of drug therapy: This invention, by detecting the co-mutation characteristics of PIK3CA and ESR1, can accurately identify patient subgroups that are more sensitive to piroxicillin and fulvestrant, enabling precise drug use and individualized treatment for breast cancer patients, significantly improving the treatment response rate, and reducing the adverse reactions caused by the use of commonly used first-line clinical combination regimens of phosphatidylinositol 3-kinase (PI3K) inhibitors, CDK4 / 6 inhibitors, and endocrine three-drug regimens. Due to the high heterogeneity of the genetic background of breast cancer patients, traditional clinical indicators are difficult to accurately predict the efficacy of piroxicillin and fulvestrant combination therapy. The mutation characteristics provided by this invention, as predictive biomarkers, can be used to screen patients who are truly likely to benefit from piroxicillin and fulvestrant combination therapy, helping to reduce ineffective treatment and improve treatment efficiency.

[0041] (2) Achieving early prediction of drug response and providing a basis for clinical decision-making: This invention can obtain the patient's co-mutation status before treatment through gene testing, thereby enabling prediction of drug sensitivity before treatment. Compared with efficacy judgment methods based on follow-up or imaging changes, this invention can guide the adjustment of treatment strategies earlier and improve the timeliness of clinical decision-making.

[0042] (3) The detection method is flexible and widely applicable, and can be adapted to a variety of clinical samples and technical platforms: The data used by this invention to detect gene mutations can be obtained through a variety of nucleic acid detection methods, including but not limited to PCR, qPCR, digital PCR, NGS, sequencing chips, etc., and can be adapted to a variety of clinical samples such as tissue biopsy samples, puncture samples, plasma cfDNA, ctDNA, etc., which facilitates its widespread application in clinical and laboratory settings. Attached Figure Description

[0043] Figure 1 Flowchart for clinical research and translational research; Figure 2 Gene mutation heatmaps for the baseline experimental group and the control group; Figure 3 PFS and OS-KM curves for different baseline PIK3CA / ESR1 paired mutation states in the experimental and control groups; Figure 4 To test the interaction between PIK3CA and ESR1 mutation status and intervention in the experimental group (with PFS as the endpoint); Figure 5 To test the interaction between ESR1 mutation status and intervention measures in PIK3CA-mutated patients in the experimental group (with overall survival as the endpoint); Figure 6 The effects of dynamic changes in PIK3CA and ESR1 on PFS and OS in the experimental and control groups; Figure 7 ROC curves predicting the sensitivity of patients with PIK3CA and ESR1 co-mutations to combination therapy with piroxicillin and fulvestrant; where the blue line represents the training set and the red line represents the validation set. Figure 8 This study investigated the mutation status of the ESR1 gene in PIK3CA-mutated patients in the training and validation sets. Detailed Implementation

[0044] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0045] Example 1: Analysis and screening of co-mutations in PIK3CA and ESR1 genes BRIGHT-2 is a randomized, double-blind, phase III clinical trial designed to evaluate the efficacy of piroxicillin plus fulvestrant versus placebo plus fulvestrant in women with HR-positive, HER2-negative advanced breast cancer. Eligible patients were randomized 2:1 to receive either piroxicillin or placebo plus fulvestrant. The primary endpoint was progression-free survival (PFS) as assessed by investigators according to RECIST version 1.1 criteria. Inclusion criteria for this trial were: women aged ≥18 years and ≤75 years with a confirmed diagnosis of locally advanced, recurrent, or metastatic HR+ / HER2- breast cancer who experienced disease progression following or during prior endocrine therapy, regardless of their menstrual status. Premenopausal or perimenopausal women were receiving gonadotropin-releasing hormone analogues (goserelin recommended) during the trial. Eligible patients had at least one measurable lesion as defined by RECIST version 1.1, or only bone metastases, and an Eastern Cooperative Oncology Group (ECCG) performance status score of 0 or 1. Patients in advanced stages should not receive more than one chemotherapy regimen. Patients must meet one of the following criteria: relapse or disease progression during (neo)adjuvant endocrine therapy, relapse within 12 months of the end of adjuvant endocrine therapy, or disease progression after first-line endocrine therapy in advanced stages.

[0046] Key exclusion criteria included: visceral crisis, inflammatory breast cancer, symptomatic brain metastases not treated with radiation or surgery, leptomeningeal metastases, and prior treatment with mTOR inhibitors, CDK4 / 6 inhibitors, fulvestrant, or similar drugs. This study was approved by the ethics committees and local institutional review committees of the participating centers and was conducted in accordance with the Declaration of Helsinki. All patients signed written informed consent forms prior to enrollment.

[0047] This study enrolled 305 patients with HR+, HER2- advanced breast cancer who had progressed after prior endocrine therapy. Patients were randomly assigned in a 2:1 ratio to either the experimental group (n=204) or the control group (n=101). The experimental group received piroxicillin 360 mg orally every 12 hours and fulvestrant 500 mg intramuscularly on days 1 and 15 of the first cycle, followed by day 1 in subsequent cycles, with each cycle lasting 28 days. The control group received placebo 360 mg orally every 12 hours and fulvestrant 500 mg intramuscularly on days 1 and 15 of the first cycle, followed by day 1 in subsequent cycles, with each cycle lasting 28 days. Patients were randomly assigned in a 2:1 ratio to the experimental and control groups, using the following stratification factors: 1. Endocrine therapy resistance (primary resistance, secondary resistance); 2. Visceral metastasis (yes, no).

[0048] At two predetermined time points (baseline and during treatment), 10 ml of peripheral blood samples were collected from the patient into StreckCell-Free DNA BCT® blood collection tubes. The tubes were gently inverted 8-10 times to mix thoroughly. The tubes were stored at 6-37°C and transported in batches, arriving at the Beijing Geneplus Medical Testing Laboratory within 2-3 days. Plasma separation was performed in the laboratory. The plasma was centrifuged at 1600 g for 10 minutes at room temperature, and the supernatant was transferred to a new centrifuge tube. The tubes were then centrifuged again at 1600 g for 10 minutes at 4°C. The plasma was then aliquoted and stored in an ultra-low temperature freezer at -80°C.

[0049] After all samples were collected and isolated, the sequencing process was initiated. Circulating cell-free DNA was isolated using the QIAamp Cyclic Nucleic Acid Kit (MagMAX Cell-Free DNA Isolation Kit, Qiagen) according to the manufacturer's instructions, and its concentration, purity, and integrity were assessed. The samples underwent end repair, A-tailing was added, sequencing adapters were ligated, and the DNA was purified. PCR amplification was then performed (TruSeq Nano Library Preparation Kit, Illumina, San Diego, CA). Subsequently, a targeted panel containing 1021 genes (including all exon regions, specific introns for translocation detection, and coverage regions for improved copy number calling) was used to enrich tumor-related genes through hybridization capture, thus constructing a library for sequencing. The sequencing platform used was a DNESEQ T7, and the sequencing mode was PE100 sequencing.

[0050] FastP was used to remove end adapter sequences and low-quality reads from the raw data of paired samples. Clean reads were aligned to the reference human genome (GRCh37) using Burrows-Wheeler Aligner (BWA, version 0.7.15-r1140), duplicate reads were labeled using realSeq, and normal samples were labeled using Picard (version 2.6.0). Somatic SNVs and Indels were detected using MuTect2 to obtain single nucleotide variants (SNVs) and short insertions / deletions (Indels). Germline mutations and false positives were filtered out using publicly available dbSNP and ExAC databases. Somatic copy number variations (Somatic CNVs) in paired samples were detected using FACETS software, taking into account ploidy and ctDNA content. Amplification was defined as focal events ≥6 copies and nonfocal events ≥7 copies. Deletion was defined as 0 copies. SV information was detected in Tumor and Normal paired samples using Lumpy software. Pathway enrichment analysis was performed using software such as clusterProfiler, with databases including KEGG, GO, DO, and Reactome.

[0051] Statistical analysis was performed using R software (version 4.3.3, as specified) and Excel software. The Kaplan-Meier method was used to estimate PFS and OS for each group, and survival curves were plotted. The Cox regression method was used to calculate HR and 95% CI. The log-rank test was used to estimate p-values, and p < 0.05 was considered statistically significant. Interaction tests were performed using the "TableSubgroupMultiCox" function in the "jstable" package, and the resulting interaction p-values ​​were corrected for multiple tests using the false discovery rate (FDR) method. The bootstrap resampling test was performed by repeatedly sampling with replacement from the original sample to create multiple "pseudo-samples" to estimate the distribution of the statistic. The Cox regression method was used to calculate HR, and the log-rank method was used to calculate p-values. 1000 samplings were performed, with each sampling number equal to the original total sample size of the comparison group. This was used to assess the stability of the co-mutation analysis results.

[0052] As of February 22, 2024, the number of PFS events in the BRIGHT-2 study reached the pre-specified final analysis threshold. This study uses efficacy and survival data from this cutoff date. See [link to ctDNA sample collection procedure] for details. Figure 1 In the experimental group of 204 patients, samples were collected at baseline and during treatment from 197 and 139 patients, respectively. In the control group of 101 patients, samples were collected at baseline and during treatment from 79 and 46 patients, respectively. All samples underwent ctDNA analysis, detecting 1021 tumor-related genes. Specifically, ctDNA was detected in 152 and 73 samples at baseline and during treatment in the experimental group, respectively, while in the control group, ctDNA was detected in 67 and 35 samples, respectively.

[0053] Sequencing results as follows Figure 2 As shown, the three genes with the highest mutation frequency in the experimental group were PIK3CA (45%), TP53 (41%), and ESR1 (24%), while the three genes with the highest mutation frequency in the control group were PIK3CA (54%), TP53 (27%), and ESR1 (19%). No significant correlation was observed between individual mutations of PIK3CA and ESR1 and survival outcomes. These three high-frequency mutant genes often exhibited co-mutation, and based on the analysis results of the INAVO-120 study, there were significant interactions between the PI3K pathway, cell cycle pathway, and ER pathway. Since ESR1 mutation is one of the most common causes of ER pathway drug resistance, PIK3CA and ESR1 genes were selected for co-mutation analysis.

[0054] Example 2: Intergroup analysis of different PIK3CA / ESR1 paired mutation states Baseline co-mutation analysis results indicated that patients with co-mutations of PIK3CA and ESR1 receiving piroxicillin combined with fulvestrant had significantly prolonged PFS and OS, followed by patients with wild-type PIK3CA and ESR1. Patients with PIK3CA+ / ESR1- and PIK3CA- / ESR1+ had the worst PFS and OS. Figure 3 A, 3B, p=0.003 and p=0.024. Pairwise comparisons showed that patients with co-mutations of PIK3CA and ESR1 had significantly longer progression-free survival (PFS) than PIK3CA+ / ESR1- and PIK3CA- / ESR1+ patients (Table 1, p<0.001 and p=0.031), and patients with co-mutations of PIK3CA and ESR1 had significantly longer overall survival (OS) than PIK3CA+ / ESR1- patients (Table 2, NA vs 19.2 months, p=0.03). No similar results were found in the control group. Wild-type or mutant, single mutation or co-mutation of both genes had no significant effect on prognosis. Figure 3 (C, 3D). The above results suggest that in the HR+ / HER2- patient population with PIK3CA mutations, patients with ESR1 mutations have a more significant survival benefit than ESR1 wild-type patients when treated with CDK4 / 6 inhibitors in combination with SERD drugs.

[0055] Table 1. Comparative analysis of PFS among different PIK3CA / ESR1 paired mutation states in the experimental groups.

[0056] Table 2. Comparison of OS among different PIK3CA / ESR1 paired mutation states in the experimental groups.

[0057] To further confirm the above results, we performed multivariate Cox regression analysis on different gene mutation subgroups, and adjusted for clinical factors such as Eastern Cooperative Oncology Group (ECOG) score, hormone receptor status, Ki-67 expression, measurable disease, visceral metastasis, number of metastatic sites, previous endocrine therapy, and type of endocrine therapy resistance. The interaction test between gene mutation subgroups and interventions revealed that the improvement in PFS by piroxicillin compared to placebo varied with mutation combination: patients with PIK3CA+ / ESR1+ double mutations showed the most significant benefit. Figure 4The median PFS was not reached in the PIK3CA+ / ESR1+ subgroup (HR=0.02, 95% CI 0–0.34, p=0.007). The next most effective subgroup was the PIK3CA+ / ESR1- subgroup (HR=0.56, 95% CI 0.32–0.99, p=0.047). While the other two subgroups showed that the pelosicillin group was superior to the placebo group, the difference was not statistically significant. The p-value for the interaction test was 0.201, suggesting that the therapeutic effect of pelosicillin combined with fulvestrant showed a consistent trend across different gene subgroups, but the benefit was more significant in the PIK3CA-mutant population compared to placebo combined with fulvestrant. Subgroup analysis in PIK3CA-mutant patients showed that in the PIK3CA+ / ESR1+ subgroup, the median PFS was not reached in the pelosicillin group, while it was reached in the placebo group at 6 months (HR=0.02, 95% CI 0–0.34, p=0.007). Figure 5 (HR = 0.02, 95% CI 0–0.34, P = 0.007). In PIK3CA+ / ESR1 In the subgroups, the median PFS was 8.8 months in the piroxicamib group and 2.6 months in the placebo group (HR = 0.56, 95% CI 0.32–0.99, P = 0.047). Further analysis showed a significant interaction between ESR1 mutation status and piroxicamib efficacy (P = 0.03), suggesting that ESR1 mutation status may serve as a predictive biomarker for CDK4 / 6 inhibitor efficacy in the PIK3CA-mutated population.

[0058] Example 3: Predictive power of co-mutations of PIK3CA and ESR1 genes in the experimental group and Bootstrap resampling test Dynamic analysis of PIK3CA and ESR1 mutation status at baseline and during treatment (day 1 of cycle 5) showed that patients in the experimental group with C1D1+ / C5D1- PIK3CA and ESR1 gene status had a significantly better prognosis than the C1D1- / C5D1- group and the C1D1+ / C5D1+ group. Figure 6 A, 6C, PFS: PIK3CA p=0.025, ESR1 p=0.003; Figure 6 B, 6D, OS: PIK3CA p=0.005, ESR1 p=0.012). In the control group, there were no significant differences in PFS and OS among subgroups with different mutation statuses of PIK3CA and ESR1. Figure 6 These results support the existence of a subgroup of patients with baseline ESR1 and PIK3CA co-mutations who were sensitive to CDK4 / 6 inhibitors in combination with fulvestrant, experienced clonal clearance during treatment, and showed improved PFS and OS benefits.

[0059] ROC analysis was used to predict the efficacy of PIK3CA and ESR1 gene co-mutations in the combination therapy of piroxicillin and fulvestrant for breast cancer. PIK3CA and ESR1 gene mutation information for all patients is as follows: Figure 8 As shown in the figure. In the experimental group, patients' PFS was divided into ≥12 months and <12 months. In the PIK3CA mutant population, stratified sampling for co-mutations was used. After the training set and validation set were split at an 8:2 ratio, the training set sample size was 56 people and the validation set sample size was 13 people. The results are as follows. Figure 7 As shown, the AUC value of the training set was 0.63, the specificity was 0.844, and the sensitivity was 0.417. The AUC value of the validation set was 0.702, the specificity was 0.833, and the sensitivity was 0.571. These results indicate that co-mutations of the PIK3CA and ESR1 genes can predict the efficacy of piroxicillin and fulvestrant combination therapy for breast cancer.

[0060] In addition, we performed a bootstrap resampling test on all patients in the experimental group, using PIK3CA+ / ESR1+ as the reference group, and conducted three group comparisons: PIK3CA- / ESR1- vs PIK3CA+ / ESR1+, PIK3CA- / ESR1+ vs PIK3CA+ / ESR1+, and PIK3CA+ / ESR1- vs PIK3CA+ / ESR1+. The bootstrap resampling test results for the experimental group, with PFS as the endpoint, indicated that the PIK3CA+ / ESR1+ group receiving piroxicillin treatment had a better PFS benefit than the other three groups, especially the PIK3CA+ / ESR1- subgroup (Table 3, P = 0.001), while no significant differences were observed among the groups in the control group (Table 4).

[0061] Table 3. Bootstrap resampling test of PFS in different PIK3CA / ESR1 mutation status subgroups of the experimental group.

[0062] Table 4. OS Bootstrap resampling test comparing different PIK3CA / ESR1 mutation status subgroups in the experimental group

[0063] In conclusion, patients with co-mutations of PIK3CA and ESR1 showed the best efficacy with piroxicillin combined with fulvestrant. This not only outperformed other gene mutation subgroups but also provided a more significant benefit compared to the control group. In particular, among patients with PIK3CA mutations, those with co-mutations of ESR1 showed significantly better efficacy with CDK4 / 6 inhibitors combined with fulvestrant than those with wild-type ESR1.

[0064] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting co-mutations of PIK3CA and ESR1 in samples in the preparation of products for predicting the sensitivity of breast cancer patients to combination therapy with piroxicillin and fulvestrant.

2. The application according to claim 1, characterized in that, The sample includes any one of peripheral blood, plasma, tumor tissue, or puncture sample; Preferably, the sample is peripheral blood.

3. The application according to claim 1, characterized in that, The reagents include reagents for detecting gene-level mutations in PIK3CA and ESR1 in samples and / or reagents for detecting protein-level mutations in PIK3CA and ESR1 in samples.

4. The application according to claim 1, characterized in that, The reagents are selected from the following group: (1) Primers for specific amplification of PIK3CA and ESR1; (2) Probes that specifically recognize PIK3CA and ESR1; (3) A binding agent that specifically binds to mutant proteins encoded by PIK3CA and ESR1. Preferably, the binding agent comprises an antibody, an antibody functional fragment, or an antibody-drug conjugate that specifically binds to mutant proteins encoded by PIK3CA and ESR1.

5. The application according to claim 1, characterized in that, The product includes reagents for detecting the co-mutation of PIK3CA and ESR1 using nucleic acid hybridization, nucleic acid amplification, protein immunoassay, sequencing, chromatography, and mass spectrometry.

6. The application according to claim 1, characterized in that, The products include chips, reagent kits, and nucleic acid modules; Preferably, the chip includes a gene chip and a protein chip; Preferably, the gene chip includes oligonucleotide probes for detecting PIK3CA and ESR1; Preferably, the protein chip includes a binder for the mutant protein encoded by PIK3CA and ESR1; Preferably, the kit includes reagents for detecting PIK3CA and ESR1 mutations or corresponding encoded protein mutations by RT-PCR, qRT-PCR, microarray detection, DNA blotting, in situ hybridization, immunoblotting, or enzyme-linked immunosorbent assay.

7. The application according to claim 1, characterized in that, The product also includes reagents for processing samples.

8. The application according to claim 1, characterized in that, The breast cancer in question is HR+ / HER2- advanced breast cancer.

9. A system for predicting the sensitivity of breast cancer patients to combined treatment with piroxicillin and fulvestrant, characterized in that, The system includes an input unit, an analysis unit, and an output unit: The input unit is used to acquire mutation status data of PIK3CA and ESR1 in the breast cancer patient to be tested; The analysis unit is used to input the mutation status data obtained by the input unit into the prediction model. The prediction model is a pre-trained model. The prediction model determines the sensitivity of the breast cancer patient to piroxicillin and fulvestrant treatment based on the mutation status data of PIK3CA and ESR1. When the breast cancer patient to be tested has co-mutations of PIK3CA and ESR1, the prediction result of the breast cancer patient's sensitivity to combined piroxicillin and fulvestrant treatment is obtained. The output unit is used to output the prediction results.

10. The application according to claim 9, characterized in that, The steps for constructing the prediction model in the analysis unit include: collecting breast cancer patient samples with known response status to combined treatment with pilocciribé and fulvestrant, detecting their co-mutation status of PIK3CA and ESR1 as input feature data, providing the input feature data to a machine learning algorithm, and obtaining the prediction model.

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