Biomarkers for guiding treatment medication of colorectal cancer cetuximab and application of biomarkers

Abstract

The invention belongs to the technical field of biomarkers, and particularly relates to detection for predicting responsiveness of colorectal cancer patients to cetuximab targeted therapy. The invention provides a novel lipid biomarker Cer d (18: 1 / 26: 0) and a corresponding detection method, aiming at solving the technical problems that the EGFR activation state cannot be directly reflected only by detecting gene mutation of downstream pathways such as KRAS / BRAF and the like, the detection method is large in invasiveness, high in cost and the like in the prior art, and the invention provides the novel lipid biomarker Cer d (18: 1 / 26: 0) and the corresponding detection method. The content of Cer d (18: 1 / 26: 0) in peripheral plasma of a colorectal cancer patient is detected, and the treatment responsiveness of the patient is judged according to a preset threshold value. Experimental results show that the content of Cer d (18: 1 / 26: 0) in tumors and plasma of CRC patients is remarkably increased, and the Cer d is closely related to the treatment effect of cetuximab. According to the scheme provided by the invention, the operation is simple and convenient, the wound is small, dynamic monitoring can be realized, and cetuximab treatment can be guided more accurately.

Description

Technical Field

[0001] The present invention relates to the field of biomarkers, and in particular, to a group of biomarkers for predicting the prognosis of cetuximab targeted therapy for colon cancer and applications thereof. Background Art

[0002] Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide, imposing a significant morbid, economic, and societal burden. Mortality rates have steadily declined over the past few decades, driven by the implementation of cancer screening programs, standardization of preoperative and postoperative care, advances in surgical techniques, and increasingly effective systemic therapies for both early-stage and advanced-stage CRC. However, approximately half of CRC patients will develop metastatic disease. At initial diagnosis, 25–30% of patients already have stage IV synchronous metastases, and an additional 15–20% develop metastases during disease progression. For these patients, comprehensive treatment with chemotherapy and targeted therapies, in addition to possible surgery, is indicated. 5-Fluorouracil, oxaliplatin, and irinotecan are the mainstays of chemotherapy for advanced CRC, and the median overall survival for patients with metastatic CRC receiving sequential treatment with these drugs ranges from 18 to 20 months. Targeted therapy combined with mainstream chemotherapy has gradually become the first-line treatment for advanced CRC. Targeted drugs such as epidermal growth factor receptor (EGFR) inhibitors and vascular endothelial growth factor (VEGF) inhibitors have been shown to enhance treatment and increase the median survival time of patients with metastatic CRC to 30 months.

[0003] Cetuximab is the most commonly used anti-EGFR monoclonal antibody for cancer treatment in clinical practice in my country. Currently, the indication for cetuximab use in patients with advanced CRC depends on the presence of MAPK pathway-related gene mutations, as these mutations can lead to intrinsic drug resistance. Detection of MAPK pathway gene mutations is primarily performed through targeted panel sequencing or whole-exome sequencing of patient tumor tissue. However, these assays are cumbersome and costly, and a high proportion of patients with advanced CRC who lack relevant mutations still do not respond to treatment. The CRC patient population that will benefit from cetuximab remains unclear. Currently, there are no predictive biomarkers for prioritizing targeted therapies, including cetuximab.

[0004] Currently, the indication for the use of cetuximab for patients with advanced CRC depends on the presence of MAPK pathway-related gene mutations, such as BRAF, KRAS, NRAS, and PIK3. The main disadvantages are as follows:

[0005] 1. It can only indicate the situation where mutations downstream of EGFR, the target of cetuximab, render anti-EGFR treatment ineffective, leading to drug resistance. It cannot accurately indicate the potential groups that will benefit from cetuximab treatment, and its sensitivity is low.

[0006] 2. Currently, there are no biomarkers in clinical practice that can directly indicate the degree of EGFR activation. This is the most direct target of cetuximab, but it has poor specificity.

[0007] 3. Genetic testing for BRAF, KRAS, NRAS, PIK3, etc. requires the use of tumor biopsy tissue for targeted panel sequencing or whole exome sequencing, which has a long cycle and high cost. In addition, biopsy often requires colonoscopy, tumor puncture, etc., which is complicated and has many side effects, making it difficult to achieve regular monitoring during treatment.

[0008] Ceramide is a regulator of mitochondrial function and plays a central role in sphingolipid metabolism. Ceramide acts as a signaling lipid in various cellular processes, including cell survival, metabolic homeostasis, and inflammation regulation. Ceramides of different chain lengths can exhibit distinct signaling functions, primarily mediating apoptosis, and an imbalance between ceramides of different chain lengths plays a crucial role in cancer progression. However, the value of ceramide as a diagnostic and therapeutic biomarker for CRC remains unclear.

[0009] Currently, there is a lack of reliable predictive markers for the targeted treatment of advanced colorectal cancer with cetuximab. Existing technologies primarily assess treatment suitability by detecting mutations in genes downstream of the EGFR pathway, such as KRAS, BRAF, and NRAS. However, these tests cannot directly reflect the activation status of the EGFR pathway. Furthermore, these tests rely on invasive tumor tissue sampling, which is time-consuming and expensive, making them unsuitable for dynamic monitoring. Summary of the Invention

[0010] Based on this, the purpose of the present invention is to provide a new prediction method to more accurately and conveniently predict the responsiveness of colorectal cancer patients to cetuximab treatment.

[0011] To achieve the above object, the present invention proposes the following technical solutions:

[0012] In a first aspect, the present invention provides a use of Cer d (18:1 / 26:0) in the preparation of an in vitro detection kit for predicting the responsiveness of colorectal cancer patients to cetuximab treatment.

[0013] In one embodiment, the kit is used to detect the content of Cer d (18:1 / 26:0) in peripheral plasma samples from colorectal cancer patients.

[0014] In one embodiment, the prediction is an in vitro evaluation of the patient's EGFR pathway activation status.

[0015] In one embodiment, the detection method of Cer d (18:1 / 26:0) is liquid chromatography-mass spectrometry (LC-MS / MS).

[0016] In one embodiment, the threshold Cer d (18:1 / 26:0) level for determining treatment responsiveness is about 16410 nM.

[0017] A second aspect of the present invention provides an in vitro detection kit comprising a Cer d (18:1 / 26:0) detection reagent, wherein the kit is used to evaluate the potential responsiveness of colorectal cancer patients to cetuximab.

[0018] In one embodiment, the detection reagent includes components for extracting plasma samples, a solvent containing a quality control internal standard, and reagent components suitable for LC-MS / MS analysis.

[0019] The present invention has the following significant improvements over the prior art:

[0020] 1. Currently, biomarkers for cetuximab therapy in clinical applications primarily detect mutations in EGFR downstream pathway indicators. However, since they do not directly indicate the degree of EGFR activation, patients whose tumors primarily proliferate through non-EGFR pathways cannot be excluded, making it difficult to accurately define the appropriate population for cetuximab. The present invention uses Cerd (18:1 / 26:0) to directly detect the degree of EGFR activation in patients, providing more accurate guidance for cetuximab use.

[0021] 2. Currently, the main indicators tested are BRAF, KRAS, NRAS, PIK3, etc., which are detected through targeted panel sequencing or whole-exome sequencing. These tests require tumor tissue obtained through surgery, colonoscopy, tumor puncture, etc., which is complex, risky, and invasive. Targeted panel sequencing or whole-exome sequencing also has long processing times and high costs. The present invention detects Cer d (18:1 / 26:0) levels in peripheral plasma, which is more convenient, less invasive, and easier to implement regular monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1The Cer d (18:1 / 26:0) content in tumor tissue and non-tumor colon mucosal tissue of CRC patients was statistically analyzed using the Wilcoxon signed-rank test;

[0024] Figure 2 Serum Cer d (18:1 / 26:0) levels in CRC patients and healthy controls were statistically analyzed using the independent sample T test;

[0025] Figure 3 For the ATP cell viability assay of the 22 patient-derived organoids in the discovery set, the statistical method was the independent sample T test;

[0026] Figure 4 The plasma Cer d (18:1 / 26:0) levels of high-responsive and low-responsive CRC patients were found, and the statistical method was independent sample T test;

[0027] Figure 5 The receiver operating characteristic (ROC) curve of plasma Cer d (18:1 / 26:0) level in predicting the response to cetuximab treatment was found.

[0028] Figure 6 ROC curve of the validation set plasma Cer d (18:1 / 26:0) content in predicting cetuximab treatment response.

[0029] Figure 7 The TR-FRET signal levels of phosphorylated EGFR (Y1068) protein in the control group and the Cer d (18:1 / 26:0) stimulation group were analyzed using the independent sample T test. DETAILED DESCRIPTION

[0030] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0031] Example 1. Detection of Cer d (18:1 / 26:0) content in samples from different populations

[0032] 1. Sample: Forty-three patients with CRC diagnosed by the Department of General Surgery at Peking University Third Hospital based on colonoscopic biopsy pathology and healthy controls were enrolled. Inclusion criteria for colorectal cancer patients included: 1) age ≥18 years and ≤75 years; 2) histopathologically confirmed colorectal adenocarcinoma; 3) surgical treatment required; and 4) normal or acceptable baseline laboratory tests, including blood count, liver and kidney function. Exclusion criteria included: 1) history of other malignancies; 2) patients receiving neoadjuvant therapy before surgery, including chemotherapy, targeted therapy, immunotherapy, and radiotherapy; 3) patients with severe underlying medical conditions (e.g., severe heart failure, active pulmonary tuberculosis, cirrhosis); and 4) previous gastrointestinal surgery. Inclusion criteria for healthy controls included: 1) age ≥18 years and ≤75 years. Exclusion criteria included: 1) history of malignancy; 2) patients with severe underlying medical conditions (e.g., severe heart failure, active pulmonary tuberculosis, cirrhosis); and 3) previous gastrointestinal surgery. Tumor samples, non-tumor colon tissue samples, and plasma samples were collected from patients and matched with plasma samples from 41 healthy individuals with no significant differences in age, gender, or underlying diseases. Cer d (18:1 / 26:0) levels were measured.

[0033] 2. Methods: Tumor and non-tumor colon tissues of CRC patients were ground into homogenates, and plasma samples from CRC patients and healthy controls were centrifuged at 3000 rpm at 4 degrees Celsius to extract serum. 50 μl of methanol was added to each sample and mixed thoroughly, centrifuged at 12,000 rpm, and the supernatant was passed through a membrane. The Cer d (18:1 / 26:0) content was detected by mass spectrometry (SCIEX, 200 mg of tissue was shaken into a homogenate, or 50 μl of serum was taken and added to 200 μl of ice-cold chloroform / methanol (2:1) solution containing 1 μM internal standard LM6002 (purchased from Avanti Polar Lipids). After vortexing for 30 seconds, the mixture was centrifuged at 15,000 g for 5 minutes at 4°C. The lower organic phase was collected and evaporated to dryness. The dried residue was dissolved in 100 μl of isopropanol / acetonitrile (IPA / ACN) (1:1) mixed solution. The samples were analyzed using an Eksigent LC100 ultra-performance liquid chromatography (UPLC) system equipped with a Waters XBridge Peptide BEH C18 column (3.5 μm, 2.1×100 mm) and an AB SCIEX TripleTOF Analyze with 5600 mass spectrometer system. Specific chromatographic conditions are as follows: mobile phase:

[0034] 3. Phase A: Aqueous solution containing 10 mM ammonium formate and 0.1% formic acid; Phase B: A mixed solution of acetonitrile (ACN) / isopropyl alcohol (IPA) containing 10 mM ammonium formate and 0.1% formic acid (ACN:IPA = 49.9%:49.9%). Gradient elution program: 0 min: 35% B; 2 min: 80% B; 9 min: 100% B; 15 min: 100% B; 16 min: 35% B; 20 min: 35% B; Flow rate: 0.4 mL / min, injection volume: 2 μL. Statistical method: Wilcoxon signed-rank test. 4. Experimental results: Compared with non-tumor colon tissue, the Cer d (18:1 / 26:0) content in tumor tissue of CRC patients was significantly increased ( Figure 1 ); Compared with the serum samples of healthy controls, the Cer d (18:1 / 26:0) content in the serum samples of CRC patients was significantly increased ( Figure 2 ).

[0035] Example 2. Correlation between the difference in responsiveness of CRC patient tumor tissue to cetuximab treatment and the Cer d (18:1 / 26:0) content

[0036] 1. Samples: Patients with CRC diagnosed by the Department of General Surgery at Peking University Third Hospital based on colonoscopic biopsy pathology were enrolled. 22 CRC patients served as the discovery set, and 21 CRC patients served as the validation set. Tumor tissues from patients undergoing radical colorectal cancer surgery were collected and cultured in vitro in 96-well plates in a 37°C incubator for organoids. On the third day of culture, each sample was stimulated with cetuximab (added to the culture medium at a concentration of 10 nM) for 72 hours. Organoids from the same patients who did not receive cetuximab stimulation served as a blank control group.

[0037] 2. Methods: Surgically resected colorectal cancer tumor tissue was separated into small pieces and digested with digestion buffer containing 200U / ml type IV collagenase (STEMCELL technologies, #07247) and 125μg / ml type II dispase (Millipore Sigma, #D4693) at 37 degrees Celsius for 5-8 minutes. After confirming that most cells had been reduced to single cells under a microscope, they were collected by centrifugation at 800g for 5 minutes. After filtering through a 70μm filter, the cells were centrifuged again and resuspended in matrigel. The single-cell suspension was then distributed into a 96-well plate at approximately 3000 cells per well. After solidification in a 37°C incubator for 10 minutes, 50μL Advanced DMEM / F12 medium was added to each well. The experimental group was given cetuximab (added to the culture medium at a concentration of 10 nM) on the third day of culture (the kit was purchased from Promega), and the blank control group was given 0.1% DMSO (cetuximab solvent). After 72 hours, the ATP level of each group was measured, and the difference in ATP between each patient's experimental group and its own blank control group was statistically analyzed. Statistical differences were considered to be responses to cetuximab treatment. Cer d (18:1 / 26:0) content detection: Plasma samples from CRC patients were centrifuged at 3000 rpm at 4 degrees Celsius to extract serum. 50 ul of methanol was added and shaken to mix, centrifuged at 12000 rpm, the supernatant was passed through a membrane, and the Cer d (18:1 / 26:0) content was detected by mass spectrometry. ROC analysis of the data was performed using R software (version 4.2.1) and the pROC package, and the results were visualized using ggplot2.

[0038] 3. Experimental results: Using an ATP-based cell viability assay, we identified 14 patients who were highly responsive to cetuximab treatment and 8 patients who were poorly responsive to cetuximab in the discovery set ( Figure 3 ). The detection of plasma Cer d (18:1 / 26:0) levels in the two groups of tumors showed that the serum Cer d (18:1 / 26:0) levels in the high-responsiveness group were significantly higher than those in the low-responsiveness group ( Figure 4). The receiver operating characteristic curve (ROC) analysis was used to evaluate the ability of serum Cer d (18:1 / 26:0) content as a diagnostic model to discriminate the responsiveness to cetuximab treatment. Based on the experimental results of the organoid samples mentioned above, the patients were divided into high and low responsiveness groups (14 patients with high response to cetuximab treatment and 8 patients with low response). The plasma Cer d (18:1 / 26:0) content was used as the discriminant index, the ROC curve was drawn, and the area under the curve (AUC) was calculated to reflect its diagnostic performance. ROC analysis showed that the sensitivity of the plasma Cer d (18:1 / 26:0) content in the concentrated patients for predicting the responsiveness to cetuximab treatment was 0.75, the specificity was 0.93, the AUC value was 0.866, the confidence interval (CI) was 0.692–1.000, and the cut-off value was 16410 (nM) ( Figure 5 ). The same method was used to detect the validation set samples. ROC analysis of 21 CRC patients as the validation set (14 patients with high response to cetuximab treatment and 7 patients with low response) showed that when the same cut-off value as the discovery set was used, the sensitivity of the plasma Cer d (18:1 / 26:0) content of patients in the validation set for predicting the response to cetuximab treatment was 0.71, the specificity was 1, the AUC value was 0.847, and the confidence interval (CI) was 0.639–1.000 ( Figure 6 ).

[0039] Example 3: Relationship between Cer d (18:1 / 26:0) and EGFR pathway activation

[0040] 1. Materials: Three patients with CRC diagnosed by colonoscopic biopsy pathology at the Department of General Surgery at Peking University Third Hospital, included in the discovery set, served as independent samples. Tumor tissue was collected after radical surgery and cultured into organoids. Organoids were then stimulated with 50 μM Cer d (18:1 / 26:0) for 72 hours. The fluorescence resonance energy transfer (FRET) signal of phosphorylated EGFR (Y1068) was measured using the fluorescence resonance energy transfer (FRET) method.

[0041] 2. Methods: Tumor organoids were digested with collagenase at 37 degrees Celsius for 5-8 minutes. After confirming under a microscope that most cells had been reduced to single cells, they were collected by centrifugation at 800g for 5 minutes. After filtering through a 70μm filter, the cells were centrifuged again and resuspended in matrigel. The single-cell suspension was then distributed into a 96-well plate at approximately 3000 cells per well. After solidification in a 37°C incubator for 10 minutes, 50μL of culture medium was added to each well, and 0.1% DMSO was used as a negative control. The experimental group was intervened with cetuximab (added to the culture medium at a concentration of 10nM), and the control group was intervened with the corresponding solvent. After 72 hours of Cer d (18:1 / 26:0) stimulation, 1x lysis buffer containing 1x phosphatase inhibitor cocktail was added to each well. After lysis at room temperature with shaking for 30 minutes, the lysate was transferred to a 384-well assay plate and labeled antibodies Eu-Ab1 and FR-Ab2 (Thunderbolt kit, Cat. No. KIT-EGFRP-100) were added to detect phosphorylated EGFR (Y1068). After incubation at room temperature for 1 hour, TR-FRET signals were detected at wavelengths of 665 nm and 615 nm.

[0042] 3. Experimental results: After stimulation with Cer d (18:1 / 26:0), the TR-FRET signal of phosphorylated EGFR (Y1068) protein increased significantly, indicating that the EGFR pathway was activated ( Figure 7 ).

[0043] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of Cer d (18:1 / 26:0) in the preparation of an in vitro detection kit for predicting the responsiveness of colorectal cancer patients to cetuximab treatment.

2. The use according to claim 1, wherein the kit is used to detect the Cer d (18:1 / 26:0) content in peripheral plasma samples from patients with colorectal cancer.

3. The use according to claim 1, wherein the prediction is an in vitro evaluation of the patient's EGFR pathway activation status.

4. The use according to claim 1, wherein the detection method of Cer d (18:1 / 26:0) is liquid chromatography-mass spectrometry (LC-MS / MS). The use according to claim 1 , wherein the threshold value of Cer d (18:1 / 26:0) content for determining treatment responsiveness is about 16410 nM.

6. An in vitro detection kit comprising a Cer d (18:1 / 26:0) detection reagent, said kit being used for evaluating the potential responsiveness of colorectal cancer patients to cetuximab.

7. The kit according to claim 6, wherein: The detection reagent includes components for extracting plasma samples, a solvent containing a quality control internal standard, and reagent components suitable for LC-MS / MS analysis.

Images