Pancreatic cancer chemotherapeutic drug administration guidance marker and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a pancreatic cancer chemotherapeutic drug administration guidance marker and application thereof. The molecular marker for evaluating the sensitivity of the pancreatic cancer chemotherapeutic drug is as follows: a marker group 1, which is composed of hsa-miR-203a-3p, hsa-miR-1246 and hsa-miR-205-5p, and a marker group 2, which is composed of hsa-miR-1246 and hsa-miR-205-5p; and the marker group 2 is composed of hsa-miR-150-5p and hsa-miR-143-3p, and the marker group 2 is composed of hsa-miR-150-5p and The molecular marker for evaluating the drug resistance of the pancreatic cancer chemotherapeutic drug is as follows: a marker group A, a marker group B and a marker group C, wherein the marker group A is composed of hsa-miR-376c-3p and hsa-miR-1262; the marker group B is composed of hsa-miR-122-3p, hsa-miR-618 and hsa-miR-455-3p, and the marker group B is composed of hsa-miR-122-3p, hsa-miR-618 and The pancreatic cancer chemotherapeutic drug is a combined drug of gemcitabine and albumin-bound paclitaxel.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a guideline biomarker for the use of chemotherapy drugs for pancreatic cancer and its application. Background Technology

[0002] The poor prognosis of pancreatic cancer patients is due not only to the difficulty in detecting the disease and the fact that patients are often diagnosed at an advanced stage, but also to the high resistance of pancreatic cancer to various treatments. The efficacy of postoperative chemotherapy is a crucial factor affecting the prognosis of pancreatic cancer patients. Discovering biomarkers for precise postoperative chemotherapy in pancreatic cancer and accurately screening patients who will benefit from chemotherapy is an important direction for improving the survival rate of pancreatic cancer patients. Due to the complex mechanisms of pancreatic cancer, there is currently a lack of biomarkers in clinical practice to guide precise chemotherapy for pancreatic cancer, especially biomarkers suitable for the Chinese population. Therefore, discovering biomarkers for precise treatment of pancreatic cancer remains a challenging and cutting-edge issue.

[0003] Several reports have been published regarding the role of miRNAs in guiding cancer drug use. For example, miR-203a-3p expression is reduced in gastric cancer patients after drug treatment; miR-150-5p and miR-1246 are highly expressed in breast cancer patients and can regulate drug sensitivity and resistance; miR-205-5p expression is reduced in gallbladder cancer patients, while high miR-205-5p expression reduces drug sensitivity; miR-455-3p regulates drug resistance in prostate cancer; miR-376c-3p increases drug resistance in ovarian cancer cells; and miR-1262 participates in the regulation of drug response in non-small cell lung cancer cells. Based on these literature reviews, it is clear that miRNAs have significant potential for application in guiding cancer drug use.

[0004] Gemcitabine plus albumin-bound paclitaxel is a commonly used chemotherapy regimen for treating malignant tumors such as pancreatic cancer. Through the synergistic effect of these two drugs, in cases of drug sensitivity, they can effectively inhibit tumor cell proliferation and disrupt their structure. Clinical studies have shown that they effectively improve patient survival and quality of life. However, primary drug resistance significantly impacts the efficacy of this regimen. Screening and validating miRNA biomarkers associated with gemcitabine plus albumin-bound paclitaxel sensitivity using appropriate methods is of positive significance for guiding precision medicine in pancreatic cancer and improving patient survival. miRNAs can participate in the pancreatic cancer drug resistance process through related gene targets and signaling pathways, and can serve as prognostic biomarkers for cancer chemotherapy. However, current research in this area of ​​pancreatic cancer is not fully clear, and many biomarkers have low sensitivity and specificity.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a biomarker for guiding the use of chemotherapy drugs for pancreatic cancer and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a molecular biomarker for assessing the sensitivity of pancreatic cancer to chemotherapy drugs, wherein the pancreatic cancer chemotherapy drugs are a combination of gemcitabine and albumin-bound paclitaxel, and the molecular biomarker comprises biomarker group 1 and biomarker group 2: Marker group 1: consists of hsa-miR-203a-3p, hsa-miR-1246 and hsa-miR-205-5p; Marker group 2: consists of hsa-miR-150-5p and hsa-miR-143-3p; Preferably, the molecular marker is miRNA in serum or plasma.

[0008] A second aspect of the present invention provides the use of reagents for detecting the above-mentioned molecular markers in the preparation of products for assessing or assisting in the assessment of the sensitivity of pancreatic cancer patients to chemotherapy drugs, wherein the pancreatic cancer chemotherapy drugs are a combination of gemcitabine and albumin-bound paclitaxel. Preferably, the product includes reagents, chips, test strips, or kits; Patients with pancreatic cancer who showed high miRNA expression in marker group 1 and low miRNA expression in marker group 2 were assessed to determine their sensitivity to the gemcitabine + albumin-bound paclitaxel regimen.

[0009] A third aspect of the present invention provides a kit for assessing the sensitivity of pancreatic cancer to chemotherapy drugs, wherein the pancreatic cancer chemotherapy drugs are gemcitabine and albumin-bound paclitaxel in combination, and the kit includes reagents for detecting the expression levels of the aforementioned molecular markers; preferably, the reagents include miRNA probes.

[0010] According to a preferred embodiment, the probe sequences corresponding to the above-mentioned molecular markers are as follows: The probe sequence corresponding to hsa-miR-203a-3p is shown in SEQ ID NO.1. The probe sequence corresponding to hsa-miR-1246 is shown in SEQ ID NO.5. The probe sequence corresponding to hsa-miR-205-5p is shown in SEQ ID NO.9. The probe sequence corresponding to hsa-miR-150-5p is shown in SEQ ID NO.13. The probe sequence corresponding to hsa-miR-143-3p is shown in SEQ ID NO.17.

[0011] According to a preferred embodiment, the reagents in the kit further include miRNA primers, which include reverse transcription primers, quantitative PCR forward primers, and reverse primers; The preferred miRNA primer sequences corresponding to the above molecular markers are as follows: The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-203a-3p are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-1246 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-205-5p are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-150-5p are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively. The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-143-3p are shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively.

[0012] A fourth aspect of the present invention provides a molecular marker for assessing drug resistance to chemotherapy in pancreatic cancer, wherein the chemotherapy drug is a combination of gemcitabine and albumin-bound paclitaxel, and the molecular marker comprises marker group A and marker group B: Marker group A: consists of hsa-miR-376c-3p and hsa-miR-1262; Marker group B: consists of hsa-miR-122-3p, hsa-miR-618 and hsa-miR-455-3p; Preferably, the molecular marker is miRNA in serum or plasma.

[0013] The fifth aspect of the present invention provides the use of reagents for detecting the above-mentioned molecular markers for assessing resistance to chemotherapy drugs in pancreatic cancer in the preparation of products for assessing or assisting in the assessment of resistance to chemotherapy drugs in pancreatic cancer patients, wherein the chemotherapy drugs for pancreatic cancer are a combination of gemcitabine and albumin-bound paclitaxel. Preferably, the product includes reagents, chips, test strips, or kits; Patients with pancreatic cancer who showed low miRNA expression in marker group A and high miRNA expression in marker group B were assessed to determine resistance to the gemcitabine + albumin-bound paclitaxel regimen.

[0014] A sixth aspect of the present invention provides a kit for assessing drug resistance to chemotherapy drugs in pancreatic cancer, wherein the chemotherapy drugs for pancreatic cancer are gemcitabine and albumin-bound paclitaxel in combination, and the kit includes reagents for detecting the expression levels of the aforementioned molecular markers for assessing drug resistance to chemotherapy drugs in pancreatic cancer; preferably, the reagents include miRNA probes.

[0015] According to a preferred embodiment, the probe sequences corresponding to the molecular markers for assessing resistance to chemotherapy drugs in pancreatic cancer described above are as follows: The probe sequence corresponding to hsa-miR-376c-3p is shown in SEQ ID NO.21. The probe sequence corresponding to hsa-miR-1262 is shown in SEQ ID NO.25. The probe sequence corresponding to hsa-miR-122-3p is shown in SEQ ID NO.29. The probe sequence corresponding to hsa-miR-618 is shown in SEQ ID NO.33. The probe sequence corresponding to hsa-miR-455-3p is shown in SEQ ID NO.37.

[0016] According to a preferred embodiment, the reagents in the kit for assessing resistance to chemotherapy drugs in pancreatic cancer further include miRNA primers, which include reverse transcription primers, quantitative PCR forward primers, and reverse primers; The preferred miRNA primer sequences corresponding to the above-mentioned molecular markers for assessing resistance to chemotherapy drugs in pancreatic cancer are as follows: The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-376c-3p are shown in SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-1262 are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively. The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-122-3p are shown in SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.32, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-618 are shown in SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, respectively. The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-455-3p are shown in SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, respectively.

[0017] The beneficial effects of this invention are: This invention provides biomarkers for sensitivity or resistance to the "gemcitabine + albumin-bound paclitaxel" regimen in pancreatic cancer. These biomarkers are characteristic markers for determining whether pancreatic cancer patients benefit from chemotherapy with this drug. Experiments using this invention have confirmed that differentially expressed miRNAs before and after chemotherapy in these biomarker groups are significantly correlated with sensitivity / resistance to gemcitabine + albumin-bound paclitaxel chemotherapy in pancreatic cancer, serving as characteristic biomarkers for determining whether pancreatic cancer patients benefit from chemotherapy with this drug. By detecting these miRNA molecular biomarkers, patients sensitive to "gemcitabine + albumin-bound paclitaxel" treatment can be accurately screened, thereby improving chemotherapy efficacy and providing positive guidance for clinicians in the precise administration of medication to pancreatic cancer patients. Attached Figure Description

[0018] Figure 1 This is the result of differential expression analysis of gemcitabine + albumin-bound paclitaxel in Example 1.

[0019] Figure 2 This describes the differential expression of miRNA markers before and after treatment in 10 sensitive patients in Example 1.

[0020] Figure 3 This is the ROC curve of marker group 1 in Example 1.

[0021] Figure 4 This is the ROC curve of marker group 2 in Example 1.

[0022] Figure 5 The results of ΔCT values ​​for biomarker group 1 in each group of samples in Example 1 are as follows: A. Comparison of expression levels of biomarker group 1 in patients and healthy individuals; B. Distribution of ΔCT values ​​for all miRNAs in biomarker group 1 in samples from sensitive patients before treatment.

[0023] Figure 6 The results of ΔCT values ​​for biomarker group 2 in each group of samples in Example 1 are as follows: A. Comparison of expression levels of biomarker group 2 in patients and healthy individuals; B. Distribution of ΔCT values ​​for all miRNAs in biomarker group 2 in samples from sensitive patients before treatment.

[0024] Figure 7 This is a volcano diagram of the differential miRNAs between groups R0 and R1 in Example 2.

[0025] Figure 8 This shows the differential expression of miRNA markers before and after treatment in drug-resistant patients in Example 2.

[0026] Figure 9 This refers to the changes in ΔCT before and after treatment with the miRNA biomarker combination in Example 2.

[0027] Figure 10 This is the ROC curve of marker group A in Example 2.

[0028] Figure 11 This is the ROC curve of marker group B in Example 2.

[0029] Figure 12 The results of ΔCT values ​​for biomarker group A in each group of samples in Example 2 are as follows: A. Comparison of expression levels of biomarker group A in patients and healthy individuals; B. Distribution of ΔCT values ​​for all miRNAs in biomarker group A in samples from sensitive patients before treatment.

[0030] Figure 13 The results of ΔCT values ​​for biomarker group B in each group of samples in Example 2 are as follows: A. Comparison of expression levels of biomarker group B in patients and healthy individuals; B. Distribution of ΔCT values ​​for all miRNAs in biomarker group B in samples from sensitive patients before treatment. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Unless otherwise specified, the methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials used are conventional reagents and materials in the art and can be obtained commercially.

[0033] Example 1: Gemcitabine + Albumin-Bound Paclitaxel-Sensitive miRNA Markers I. Screening of gemcitabine + albumin-bound paclitaxel-sensitive miRNA biomarkers S1. Sample selection and grouping The case group consisted of newly diagnosed pancreatic cancer patients meeting the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Pancreatic Cancer" (2018 edition). Serum samples were collected from pancreatic cancer patients before and after receiving gemcitabine + albumin-bound paclitaxel chemotherapy. Treatment efficacy was evaluated after one cycle of chemotherapy. Twelve patients sensitive to gemcitabine + albumin-bound paclitaxel chemotherapy were selected, and serum samples were obtained from collaborating hospitals. These samples were divided into two groups: before gemcitabine + albumin-bound paclitaxel-sensitive chemotherapy (S0) and after gemcitabine + albumin-bound paclitaxel-sensitive chemotherapy (S1).

[0034] The sensitivity of pancreatic cancer patients to gemcitabine plus albumin-bound paclitaxel was determined using the following methods: 1. Imaging assessment Tumor shrinkage: Changes in tumor size, shape, and metastases are observed through examinations such as CT and MRI. If the tumor volume shrinks by ≥30% after treatment (according to RECIST criteria), the treatment is considered effective.

[0035] Decreased metabolic activity: In PET-CT scans, the level of glucose metabolism in tumors is significantly reduced, reflecting suppressed cell activity.

[0036] 2. Changes in tumor markers Serum tumor markers such as CA19-9 are important references. If CA19-9 levels continue to decrease to the normal range or are significantly lower than baseline after treatment, it indicates that the treatment regimen is sensitive. However, it is necessary to consider imaging results, as some patients may experience a decrease in markers but no significant tumor shrinkage.

[0037] 3. Improvement in clinical symptoms Pain relief: Pancreatic cancer is often accompanied by abdominal pain and back pain. If the pain intensity is reduced and the frequency of attacks decreases, it indicates that the treatment is effective.

[0038] Jaundice subsides: If jaundice is caused by bile duct obstruction, a decrease in the jaundice index and a reduction in yellowing of the skin and sclera after treatment indicate that the tumor's compression of the bile duct has been relieved.

[0039] S2 and Small RNA sequencing: The above samples were subjected to small RNA sequencing. Detailed methods are as follows: 1) Total RNA was extracted from the samples; 2) RNA concentration and purity were detected using an Agilent 2100 Bioanalyzer; 3) A small RNA library was constructed using Illumina's TruSeq Small RNA Sample prep Kit; 4) Expand and enrich the library using PCR, and add sequencing adapters and index portions; 5) Select and purify libraries by gel electrophoresis; 6) Use the Agilent 2100 Bioanalyzer to perform quality checks on the library. A qualified library should have a single peak. 7) The library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit; 8) Using the Illumina platform, bridge PCR amplification, sequencing primer annealing, and sequencing while synthesizing were performed using a single-stranded library as a template.

[0040] S3. Data analysis to screen chemotherapy-related miRNAs as candidate biomarkers. First, the sequencing data underwent adapter removal and quality filtering. The filtered sequences were then deduplicated (i.e., identical sequences were merged, and their abundance was recorded). The analysis focused on miRNAs, including miRNA characterization and miRNA expression level statistical analysis, to screen for specifically expressed miRNAs associated with chemotherapy efficacy. (a) Analysis of known miRNA expression levels: Based on the number of sequences aligned to mature human miRNAs, the Reads Count of the detected miRNAs was counted. Because a miRNA precursor may be located at multiple locations in the genome, the mature miRNAs produced by the same precursor at different locations may be the same or slightly different. These precursors are named the same miRNA. Therefore, the same miRNA may appear repeatedly in the abundance list and there may be very small differences. However, randomly selecting one of them has little impact on the expression level analysis. Therefore, the abundance of the first occurrence of the same miRNA was selected as the abundance of that miRNA for subsequent analysis. The expression level was divided into different intervals, and the number of genes in different expression level intervals for each sample was counted.

[0041] (b) Expression density analysis: The miRNA density distribution of all samples was statistically analyzed to examine the expression patterns of all miRNAs in the samples. If the miRNA density distribution pattern of the samples shows that moderately expressed miRNAs account for the vast majority, while low- and high-expressed miRNAs account for a small portion, then it conforms to the general pattern of miRNA expression. If the results do not conform to this pattern, the samples need to be resubmitted for sequencing to confirm the results.

[0042] (c) Differential expression analysis: Based on the miRNA expression data in each sample, DESeq (version 1.18.0, Anders S and Huber W, 2010) was used to perform differential expression analysis on miRNAs, and conserved miRNAs with differential expression were screened according to the fold change in expression level (|log2FoldChange|>1) and the significance of expression difference (P-value <0.05).

[0043] Figure 1 The results showed that in patients sensitive to gemcitabine plus albumin-bound paclitaxel treatment, 11 miRNAs were expressed at low levels and 4 miRNAs were expressed at high levels before and after chemotherapy. Specifically, in sensitive patients, the expression levels of hsa-miR-203a-3p, hsa-miR-1246, and hsa-miR-205-5p decreased significantly after treatment, while the expression levels of hsa-miR-150-5p and hsa-miR-143-3p increased significantly. The specific differentially expressed miRNAs are shown in Table 1.

[0044] Table 1. Differentially expressed miRNAs among groups

[0045] II. Validation of gemcitabine + albumin-bound paclitaxel-sensitive miRNA biomarkers Another 20 patients who received gemcitabine plus albumin-bound paclitaxel were selected; 10 were sensitive and 10 were not. Serum samples were collected before and after chemotherapy. Primers and probes (sequences shown in Table 2) were designed for the five miRNAs with significantly different expression levels obtained in Table 1 of Example 1. Total RNA was extracted from the serum samples and reverse transcribed. The expression levels of each miRNA in the serum samples before and after chemotherapy were detected by qPCR as validation.

[0046] The screened miRNA biomarkers were further validated by qPCR, and miRNAs with significant differential expression (P-value <0.05) were used as a biomarker combination sensitive to pancreatic cancer. Results ( Figure 2The results showed that in patients with pancreatic cancer sensitive to gemcitabine plus albumin-bound paclitaxel treatment, the expression levels of hsa-miR-150-5p and hsa-miR-143-3p increased significantly, while the expression levels of hsa-miR-203a-3p, hsa-miR-1246 and hsa-miR-205-5p decreased significantly.

[0047] Table 2 Primer and probe sequences

[0048] S1. Preparation before the experiment Environment: The entire experiment must be conducted in a clean room at room temperature (20-25℃). Instruments: High-speed centrifuge, Nanodrop, PCR amplification instrument, quantitative PCR instrument; Consumables: 1.5 mL EP tubes of RNAase-free PCR, 0.1 mL 8-strip PCR tubes, 1 mL / 200 μL / 10 μL tips, 96-well plates; Reagents: TRIzol™ LS Reagent (Invitrogen 10296028), RNase-free ddH2O TaqMan™ MicroRNA Reverse Transcription Kit (ABI 4366596), Premix ExTaq™ (Probe qPCR) TAKARARR390; isopropanol, chloroform, anhydrous ethanol, RT primers (U6, RT primers for miRNAs in Table 1), qPCR primers (forward and reverse qPCR primers for miRNAs in Table 1), probes (Fam: U6, VIC: probes for miRNAs in Table 1).

[0049] S2, qPCR detection 1. Serum RNA extraction - Serum RNA was extracted using the RNA extraction reagent TRIzol™ LS Reagent. 2. Reverse transcription Combine any miRNA from Table 1 with U6, prepare primer working solution at a concentration of 5 μM per primer, and perform reverse transcription using a TaqMan™ MicroRNA Reverse Transcription Kit (ABI, 4366596). The reverse transcription system is 15 μL, containing: 50 ng RNA, 1.5 μL 10×Buffer, 0.15 μL dNTP mix, 1 μL RT enzyme, 0.19 μL RNase inhibitor, 1 μL U6 RT primer (5 μM), 1 μL miRNA RT primer (5 μM), and ddH2O to bring the total to 15 μL. Perform the following PCR program: 16℃ for 30 min, 42℃ for 30 min, 85℃ for 5 min, and store at 4℃. After completion, briefly centrifuge to the bottom of the tube to obtain cDNA.

[0050] 3. qPCR The assay was performed using the Premix Ex Taq™ (Probe qPCR) kit (TAKARA, RR390).

[0051] The reaction mixture was prepared as follows: 3 μL of cDNA obtained from reverse transcription, 5 μL of Premix Ex Taq (Probe qPCR) (2×), 0.2 μL of U6 forward primer (10 μM), 0.2 μL of U6 reverse primer (10 μM), 0.2 μL of miRNA forward primer (10 μM), 0.2 μL of miRNA reverse primer (10 μM), 0.4 μL of U6 probe (10 μM), 0.4 μL of miRNA probe (10 μM), and ddH2O to a final volume of 10 μL. PCR amplification was performed on a quantitative PCR instrument under the following conditions: pre-denaturation, 1 cycle, 95℃ for 30 seconds; PCR reaction, 40 cycles, 95℃ for 5 seconds and 60℃ for 30 seconds; cooling, 1 cycle, 50℃ for 30 seconds.

[0052] Results of 10 patients with pancreatic cancer who were sensitive to gemcitabine plus albumin-bound paclitaxel treatment showed that the expression levels of hsa-miR-203a-3p, hsa-miR-1246, and hsa-miR-205-5p decreased significantly after treatment, while the expression levels of hsa-miR-150-5p and hsa-miR-143-3p increased significantly.

[0053] For hsa-miR-203a-3p, hsa-miR-1246, and hsa-miR-205-5p (group 1), whose expression levels significantly decreased after treatment in patients with sensitive drug use, and hsa-miR-150-5p and hsa-miR-143-3p (group 2), whose expression levels significantly increased after treatment, ROC curves were plotted using the pre-treatment qPCR results of all 20 patients to evaluate the accuracy of these five miRNA biomarkers in predicting the sensitivity of patients to gemcitabine + albumin-bound paclitaxel treatment. The ROC curve method is simple and intuitive; the clinical accuracy of the analysis method can be observed graphically and judged visually. The ROC curve combines sensitivity and specificity graphically, accurately reflecting the relationship between the specificity and sensitivity of an analytical method, and is a comprehensive representation of the test's accuracy. The more convex the ROC curve and the closer it is to the upper left corner, the greater its diagnostic value, facilitating comparisons between different indicators. The area under the curve (AUC) evaluates diagnostic accuracy; the larger the AUC, the greater the diagnostic efficacy of the test. The area under the ROC curve is between 1.0 and 0.5. When AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC between 0.5 and 0.7 indicates low accuracy, AUC between 0.7 and 0.9 indicates some accuracy, and AUC above 0.9 indicates high accuracy. An AUC of 0.5 indicates that the diagnostic method is completely ineffective and has no diagnostic value. An AUC < 0.5 is unrealistic and rarely occurs in practice.

[0054] ROC curve results showed that the AUC of the marker group 1, in pancreatic cancer patients sensitive to gemcitabine + albumin-bound paclitaxel, which showed a significant decrease in expression after treatment, was 1 (P<0.05), while the AUC of the miRNA marker alone ranged from 0.973 to 0.993 (P<0.05) (Table 3). Figure 3 In pancreatic cancer patients sensitive to the gemcitabine + albumin-bound paclitaxel regimen, the AUC of the marker group 2 that showed a significant increase in expression after treatment was 1 (P<0.05), while the AUC of the miRNA marker alone ranged from 0.973 to 1 (P<0.05) (Table 4). Figure 4 In summary, the biomarkers in groups 1 and 2 of pancreatic cancer patients sensitive to the gemcitabine + albumin-bound paclitaxel regimen showed good accuracy as prognostic markers for pancreatic cancer chemotherapy, and can predict the efficacy of chemotherapy in pancreatic cancer patients and guide precision medication.

[0055] Table 3. AUC values ​​of different miRNAs and biomarkers in group 1

[0056] Table 4. AUC values ​​of different miRNAs and biomarkers in group 2

[0057] III. Verification by Healthy Individuals To identify gemcitabine + albumin-bound paclitaxel-sensitive miRNA markers, we collected data from 10 patients who had undergone one cycle of gemcitabine + albumin-bound paclitaxel treatment and were confirmed to be sensitive to this regimen. A total of 20 clinical data and serum samples were collected before and after treatment (from collaborating hospitals). Simultaneously, we collected serum from 10 healthy individuals, both before and after chemotherapy in pancreatic cancer patients, for a total of 20 samples.

[0058] All 40 collected samples were tested using the aforementioned qPCR detection method.

[0059] Analysis of miRNA biomarker combination detection results: The ΔCT values ​​of biomarker group 1 (hsa-miR-203a-3p, hsa-miR-1246, and hsa-miR-205-5p) and biomarker group 2 (hsa-miR-150-5p and hsa-miR-143-3p) in all samples were statistically analyzed and categorized into four groups based on sample source: pancreatic cancer patients before treatment, pancreatic cancer patients after treatment, healthy individuals (serum collected concurrently with patients before treatment), and healthy individuals (serum collected concurrently with patients after treatment). A comparative analysis of the mean ΔCT values ​​of the two biomarkers was performed according to sample category. The results showed that the expression levels of the two biomarkers in healthy individuals were basically constant. The expression levels of the two biomarkers in patients after treatment were close to those in healthy individuals. However, in patients sensitive to the gemcitabine + albumin-bound taxane regimen, the expression levels of the two biomarkers before treatment were significantly different from those in healthy individuals, showing high expression of biomarker group 1 and low expression of biomarker group 2. Figure 5 , Figure 6 As shown.

[0060] Furthermore, a meta-analysis of the two biomarker results in healthy individuals revealed that biomarker group 1 showed low expression, with all miRNAs in group 1 having a ΔCT value greater than -6, while biomarker group 2 showed high expression, with all miRNAs in group 2 having a ΔCT value less than -3. Conversely, the data from the two biomarker groups before treatment in pancreatic cancer patients sensitive to the gemcitabine + albumin-bound taxane regimen showed the opposite: biomarker group 1 showed significantly high expression, with all miRNAs having a ΔCT value less than -6; while biomarker group 2 showed significantly low expression, with all miRNAs having a ΔCT value greater than -3. Therefore, ΔCT mean analysis can be performed on the two groups of biomarkers before treatment. If the ΔCT value of biomarker group 1 is < -6, it is recorded as high expression of biomarker group 1, while the ΔCT value of biomarker group 2 is > -3, it is recorded as low expression of biomarker group 2. This means that they are sensitive to the gemcitabine + albumin-bound taxane regimen. This screening method is called "pretreatment sorting". For this type of population, the gemcitabine + albumin-bound taxane regimen can be used for treatment.

[0061] IV. Clinical Sample Validation The five miRNA biomarkers obtained above, hsa-miR-203a-3p, hsa-miR-1246, hsa-miR-205-5p, hsa-miR-150-5p, and hsa-miR-143-3p, were used to verify their accuracy in predicting sensitivity to gemcitabine plus albumin-bound paclitaxel treatment using clinical samples.

[0062] We collected clinical information from 60 patients who had undergone one course of treatment with gemcitabine and albumin-bound paclitaxel, along with serum samples collected before treatment. Patients were categorized according to their response to one course of treatment, with 30 cases showing sensitivity and 30 cases showing insensitivity. Five miRNA markers in the serum were detected using the aforementioned qPCR method.

[0063] The miRNA detection results were summarized and analyzed. High expression of the markers hsa-miR-203a-3p, hsa-miR-1246, and hsa-miR-205-5p in serum before treatment, coupled with low expression of hsa-miR-150-5p and hsa-miR-143-3p, was considered a predictor of sensitivity to gemcitabine + albumin-bound paclitaxel treatment. Other results were considered insensitive to gemcitabine + albumin-bound paclitaxel treatment. In other words, if any of the aforementioned five miRNA markers was not present, the sample was not included in the sensitive sample. The predicted results were compared with the actual sensitivity results of clinical treatment, resulting in Table 5.

[0064] Table 5

[0065] Table 5 shows that the sensitivity of using two biomarkers, groups 1 and 2, to predict the sensitivity of patients treated with the gemcitabine + albumin-bound paclitaxel regimen was 90.00%, with a specificity of 93.33% and an overall concordance rate of 91.67%. These biomarkers can serve as characteristic markers for assessing the sensitivity of pancreatic cancer patients to the gemcitabine + albumin-bound paclitaxel regimen, and can provide positive guidance for clinicians in precision medicine for pancreatic cancer patients.

[0066] Example 2: Markers of gemcitabine + albumin-bound paclitaxel resistance miRNAs I. Screening of gemcitabine + albumin-bound paclitaxel resistance miRNAs The case group consisted of newly diagnosed pancreatic cancer patients meeting the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Pancreatic Cancer" (2018 edition). Serum samples were collected from pancreatic cancer patients before and after receiving gemcitabine + albumin-bound paclitaxel chemotherapy. Treatment efficacy was evaluated after one cycle of chemotherapy. Twelve patients resistant to gemcitabine + albumin-bound paclitaxel chemotherapy were selected; serum samples were obtained from collaborating hospitals. These samples were divided into two groups: before gemcitabine + albumin-bound paclitaxel resistance chemotherapy (R0) and after gemcitabine + albumin-bound paclitaxel resistance chemotherapy (R1).

[0067] The specific analytical method is the same as that in Example 1, “I. Screening of gemcitabine + albumin-bound paclitaxel-sensitive miRNA biomarkers”.

[0068] Figure 7 The results showed that 12 miRNAs were lowly expressed and 4 miRNAs were highly expressed in patients resistant to gemcitabine plus albumin-bound paclitaxel before and after chemotherapy. After treatment, the expression levels of hsa-miR-376c-3p and hsa-miR-1262 increased significantly, while the expression levels of hsa-miR-122-3p, hsa-miR-618, and hsa-miR-455-3p decreased significantly. The changes in these 5 miRNAs were significantly different, and the specific differentially expressed miRNAs are shown in Table 6.

[0069] Table 6. Differentially expressed miRNAs

[0070] Primers and probes (sequences shown in Table 7) were designed for the five miRNAs with significantly different expression levels obtained in Table 6 of Example 2.

[0071] Table 7 Primer and probe sequences

[0072] II. Validation of gemcitabine + albumin-bound paclitaxel resistance miRNA markers Serum samples (from a collaborating hospital) were collected from 27 patients with pancreatic cancer resistant to gemcitabine and albumin-bound paclitaxel. Serum samples were collected before and after chemotherapy. Detection was performed using the qPCR method described in Example 1.

[0073] result( Figure 8 , Figure 9 The study showed that in 27 patients with pancreatic cancer resistant to gemcitabine plus albumin-bound paclitaxel, the expression levels of hsa-miR-376c-3p and hsa-miR-1262 significantly increased after treatment, while the expression levels of hsa-miR-122-3p, hsa-miR-618, and hsa-miR-455-3p significantly decreased. Five screened miRNA biomarkers were further validated by qPCR, and miRNAs with significant differential expression (P-value < 0.05) were used to form a biomarker group for pancreatic cancer drug resistance. Biomarker group A consisted of hsa-miR-376c-3p and hsa-miR-1262; biomarker group B consisted of hsa-miR-122-3p, hsa-miR-618, and hsa-miR-455-3p.

[0074] Receiver operating characteristic (ROC) curves were plotted for the results obtained from five miRNA biomarkers and combinations of two miRNA biomarkers, respectively, to validate the sensitivity and specificity of miRNA combinations as prognostic biomarkers for pancreatic cancer chemotherapy.

[0075] ROC curve results ( Figure 10 , Figure 11 Tables 8 and 9 show that the AUC of marker group A, which showed a significant increase in expression after treatment with gemcitabine plus albumin-bound paclitaxel in pancreatic cancer patients, was 1 (P<0.05), with a sensitivity and specificity of 100% and 100%, respectively; the AUC of marker hsa-miR-376c-3p was 1 (P<0.05), with a sensitivity and specificity of 100% and 100%, respectively; and the AUC of hsa-miR-1262 was 1 (P<0.05), with a sensitivity and specificity of 100% and 100%, respectively. Figure 10Table 8). The AUC of markers showing a significant decrease in expression after treatment with gemcitabine plus albumin-bound paclitaxel in pancreatic cancer patients was 1 (P<0.05) in group B, with sensitivity and specificity of 100% and 100%, respectively; the AUC of hsa-miR-122-3p was 1 (P<0.05), with sensitivity and specificity of 100% and 100%, respectively; the AUC of hsa-miR-618 was 0.987 (P<0.05), with sensitivity and specificity of 89% and 100%, respectively; and the AUC of hsa-miR-455-3p was 1 (P<0.05), with sensitivity and specificity of 100% and 100%, respectively. Figure 11 (See Table 9). In summary, biomarkers for pancreatic cancer patients resistant to gemcitabine and albumin-bound paclitaxel show good sensitivity and specificity as prognostic markers for pancreatic cancer chemotherapy. They can predict the efficacy of chemotherapy in pancreatic cancer patients and guide precision medication.

[0076] Table 8. AUC values ​​of different miRNAs and biomarker groups A

[0077] Table 9. AUC values ​​of different miRNAs and biomarker groups B

[0078] III. Verification by Healthy Individuals By comparing the expression levels of the two biomarkers of this invention in pancreatic cancer patients and healthy individuals, their specificity can be further determined.

[0079] To investigate miRNA markers of gemcitabine + albumin-bound paclitaxel resistance, we collected data from 10 patients who had undergone one cycle of gemcitabine + albumin-bound paclitaxel treatment and were confirmed to be resistant to this regimen. A total of 20 clinical data and serum samples were collected before and after treatment (from collaborating hospitals). Simultaneously, we collected serum from 10 healthy individuals, both before and after chemotherapy in pancreatic cancer patients, for a total of 20 samples.

[0080] All 40 collected samples were tested using the aforementioned qPCR detection method.

[0081] Analysis of miRNA biomarker combination detection results: The ΔCT values ​​of biomarker group A (hsa-miR-376c-3p and hsa-miR-1262) and biomarker group B (hsa-miR-122-3p, hsa-miR-618 and hsa-miR-455-3p) in all samples were statistically analyzed and categorized into four groups based on sample source: pancreatic cancer patients before treatment, pancreatic cancer patients after treatment, healthy individuals (serum collected concurrently with patients before treatment), and healthy individuals (serum collected concurrently with patients after treatment). A comparative analysis of the mean ΔCT values ​​of the two biomarkers was performed according to sample category. The results showed that the expression levels of the two biomarkers in healthy individuals were basically constant. The expression levels of the two biomarkers in patients after treatment were close to those in healthy individuals. However, the expression levels of the two biomarkers in patients resistant to gemcitabine + albumin-bound taxane regimens before treatment were significantly different from those in healthy individuals, showing low expression of biomarker group A and high expression of biomarker group B. Figure 12 , Figure 13 As shown.

[0082] Furthermore, a meta-analysis of the two biomarker results in healthy individuals revealed that biomarker group A showed high expression, with all miRNAs in group A having a ΔCT value less than -5, while biomarker group B showed low expression, with all miRNAs in group B having a ΔCT value greater than -7. Conversely, the data from the two biomarker groups before treatment in pancreatic cancer patients resistant to gemcitabine plus albumin-bound taxane regimens showed the opposite: biomarker group A showed significantly low expression, with all miRNAs having a ΔCT value greater than -5; while biomarker group B showed significantly high expression, with all miRNAs having a ΔCT value less than -7. Therefore, ΔCT mean analysis can be performed on the two groups of biomarkers before treatment. If the ΔCT value of biomarker group A is greater than -5, it is recorded as low expression of biomarker group A, while the ΔCT value of biomarker group B is less than -7, it is recorded as high expression of biomarker group B. This means that they are resistant to the gemcitabine + albumin-bound taxane regimen. This screening method is called "pretreatment sorting". For this type of population, the gemcitabine + albumin-bound taxane regimen cannot be used for treatment.

[0083] IV. Clinical Sample Validation Clinical samples were used to validate the accuracy of biomarker groups A and B in predicting resistance to gemcitabine plus albumin-bound paclitaxel treatment. Information was collected from 100 patients (from collaborating hospitals) who had completed one cycle of gemcitabine plus albumin-bound paclitaxel treatment, along with serum samples collected before treatment. Patients were categorized according to their response to one cycle of treatment, with 50 cases showing resistance and 50 showing no resistance. Detection was performed using the qPCR method described in Example 1.

[0084] Analysis of miRNA biomarker combination detection results: Biomarker group A (hsa-miR-376c-3p, hsa-miR-1262) with low expression and biomarker group B (hsa-miR-122-3p, hsa-miR-618, hsa-miR-455-3p) with high expression were considered as samples predicted to be resistant to gemcitabine + albumin-bound paclitaxel treatment. Other cases were considered as samples not resistant to gemcitabine + albumin-bound paclitaxel treatment. That is, if any of the above 5 miRNA biomarkers did not meet the criteria, the sample was not included in the resistant sample. The predicted results were compared with the actual drug resistance results of clinical treatment, and the results are shown in Table 10.

[0085] Table 10

[0086] Table 10 shows that using two biomarker groups A and B to predict the sensitivity of patients treated with gemcitabine plus albumin-bound paclitaxel (ABTA) was 92.00% sensitive and 94.00% specific, with an overall concordance rate of 93.00%. These biomarkers can serve as characteristic markers for determining chemotherapy resistance in pancreatic cancer patients treated with gemcitabine plus ABTA, and can provide positive guidance for clinicians in precision medicine for pancreatic cancer patients.

Claims

1. A molecular biomarker for assessing the sensitivity of pancreatic cancer to chemotherapy drugs, characterized in that: The chemotherapy drugs for pancreatic cancer are a combination of gemcitabine and albumin-bound paclitaxel, and the molecular markers are marker group 1 and marker group 2: Marker group 1: consists of hsa-miR-203a-3p, hsa-miR-1246 and hsa-miR-205-5p; Marker group 2: consists of hsa-miR-150-5p and hsa-miR-143-3p; Preferably, the molecular marker is miRNA in serum or plasma.

2. The use of the reagent for detecting the molecular marker of claim 1 in the preparation of a product for assessing or assisting in the assessment of the sensitivity of pancreatic cancer patients to chemotherapy drugs, wherein the pancreatic cancer chemotherapy drug is a combination of gemcitabine and albumin-bound paclitaxel; Preferably, the product includes reagents, chips, test strips, or kits; Patients with pancreatic cancer who showed high miRNA expression in marker group 1 and low miRNA expression in marker group 2 were assessed to determine their sensitivity to the gemcitabine + albumin-bound paclitaxel regimen.

3. A kit for assessing the sensitivity of pancreatic cancer to chemotherapy drugs, characterized in that: The pancreatic cancer chemotherapy drug is a combination of gemcitabine and albumin-bound paclitaxel, and the kit includes reagents for detecting the expression level of the molecular markers of claim 1; preferably, the reagents include miRNA probes.

4. The reagent kit according to claim 3, characterized in that: The probe sequences corresponding to the molecular markers described in claim 1 are as follows: The probe sequence corresponding to hsa-miR-203a-3p is shown in SEQ ID NO.

1. The probe sequence corresponding to hsa-miR-1246 is shown in SEQ ID NO.

5. The probe sequence corresponding to hsa-miR-205-5p is shown in SEQ ID NO.

9. The probe sequence corresponding to hsa-miR-150-5p is shown in SEQ ID NO.

13. The probe sequence corresponding to hsa-miR-143-3p is shown in SEQ ID NO.

17.

5. The reagent kit according to claim 4, characterized in that: The reagent also includes miRNA primers, which include reverse transcription primers, quantitative PCR forward primers, and reverse primers; The preferred miRNA primer sequences corresponding to the molecular markers described in claim 1 are as follows: The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-203a-3p are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-1246 are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-205-5p are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-150-5p are shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively. The sequences of the forward and reverse primers and reverse transcription primers for quantitative PCR corresponding to hsa-miR-143-3p are shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively.

6. A molecular marker for assessing resistance to chemotherapy drugs in pancreatic cancer, characterized in that: The chemotherapy drugs for pancreatic cancer are a combination of gemcitabine and albumin-bound paclitaxel, and the molecular markers are marker group A and marker group B: Marker group A: consists of hsa-miR-376c-3p and hsa-miR-1262; Marker group B: consists of hsa-miR-122-3p, hsa-miR-618 and hsa-miR-455-3p; Preferably, the molecular marker is miRNA in serum or plasma.

7. The use of the reagent for detecting the molecular marker of claim 6 in the preparation of products for assessing or assisting in the assessment of pancreatic cancer patients' resistance to chemotherapy drugs, wherein the pancreatic cancer chemotherapy drug is a combination of gemcitabine and albumin-bound paclitaxel; Preferably, the product includes reagents, chips, test strips, or kits; Patients with pancreatic cancer who showed low miRNA expression in marker group A and high miRNA expression in marker group B were assessed to determine resistance to the gemcitabine + albumin-bound paclitaxel regimen.

8. A kit for assessing resistance to chemotherapy drugs in pancreatic cancer, characterized in that: The pancreatic cancer chemotherapy drug is a combination of gemcitabine and albumin-bound paclitaxel, and the kit includes reagents for detecting the expression level of the molecular markers of claim 6; preferably, the reagents include miRNA probes.

9. The reagent kit according to claim 8, characterized in that: The probe sequences corresponding to the molecular markers described in claim 6 are as follows: The probe sequence corresponding to hsa-miR-376c-3p is shown in SEQ ID NO.

21. The probe sequence corresponding to hsa-miR-1262 is shown in SEQ ID NO.

25. The probe sequence corresponding to hsa-miR-122-3p is shown in SEQ ID NO.

29. The probe sequence corresponding to hsa-miR-618 is shown in SEQ ID NO.

33. The probe sequence corresponding to hsa-miR-455-3p is shown in SEQ ID NO.

37.

10. The kit according to claim 8, characterized in that: The reagent also includes miRNA primers, which include reverse transcription primers, quantitative PCR forward primers, and reverse primers; The preferred miRNA primer sequences corresponding to the molecular markers described in claim 6 are as follows: The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-376c-3p are shown in SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-1262 are shown in SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively. The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-122-3p are shown in SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.32, respectively. The sequences of the forward and reverse primers for quantitative PCR and the reverse transcription primers corresponding to hsa-miR-618 are shown in SEQ ID NO. 34, SEQ ID NO. 35, and SEQ ID NO. 36, respectively. The sequences of the forward and reverse primers and the reverse transcription primers for quantitative PCR corresponding to hsa-miR-455-3p are shown in SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, respectively.

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