Application of FBH1 protein in judging the efficacy of somatostatin analogs adjuvant therapy after surgery for pancreatic neuroendocrine tumors

Abstract

The application of FBH1 protein in judging the efficacy of postoperative somatostatin analogue adjuvant therapy for pancreatic neuroendocrine tumors relates to the fields of proteomics and clinical detection technology. The present invention finds that the disease-free survival and total survival time of the postoperative SSA adjuvant therapy group of PNETs patients with high FBH1 expression are significantly better than those of the non-adjuvant therapy group. By detecting the relative content of FBH1 protein in the tumor tissue of PNETs patients, it is effective to evaluate whether the patients use SSA drug adjuvant therapy after surgery, which is of great significance for the guidance of postoperative medication for patients.

Description

Technical Field

[0001] The present invention belongs to the field of proteomics and clinical detection technology, and specifically relates to the application of FBH1 protein in judging the efficacy of somatostatin analogues adjuvant therapy after surgery for pancreatic neuroendocrine tumors. Background Art

[0002] Pancreatic neuroendocrine tumors (PNETs) are the second most common type of pancreatic tumors, accounting for about 2-5% of all pancreatic tumors. Their detection rate has gradually increased in recent years. Radical surgical resection is currently the main treatment for PNETs, ​​but the prognosis of patients after surgery varies greatly and shows a high degree of heterogeneity.

[0003] Somatostatin analogues (SSA) are a class of synthetic peptides that function similarly to natural somatostatin, delaying tumor progression and downregulating overproduced hormones. Using somatostatin analogues for postoperative adjuvant therapy of PNETs is a first-line treatment for clinically delaying the recurrence of PNETs. However, there is currently no predictive scheme for the efficacy of postoperative adjuvant therapy with SSA.

[0004] F-box DNA helicase 1 (FBH1) is a DNA helicase that plays a key role in responding to damaged replication forks. Currently, some related studies focus on the exploration of biomarkers for PNETs, ​​including proteomic analysis of specific protein levels in the blood to distinguish PNETs from other types of tumors, as well as to evaluate treatment response and prognosis; however, there are no direct reports on the role of FBH1 protein in predicting the efficacy of postoperative SSA (somatostatin analogue) adjuvant therapy in patients with pancreatic neuroendocrine tumors (PNETs). Summary of the invention

[0005] In order to accurately analyze the prognostic effect of SSA adjuvant therapy on PNETs, ​​the present invention has discovered new markers and can accurately predict the effect of SSA adjuvant therapy on the survival of PNETs after surgery. Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention is to provide the use of FBH1 protein in the preparation of preparations related to predicting the prognosis of postoperative SSA adjuvant therapy in pancreatic neuroendocrine tumors, characterized in that, for PNETs patients with high expression of FBH1 protein, the disease-free survival time and overall survival time of the SSA adjuvant therapy group are significantly better than those of the non-adjuvant therapy group.

[0007] Furthermore, the method for determining the expression level of the FBH1 protein includes, but is not limited to, a mass spectrometer, a spectrophotometer, a chemiluminescence meter signal reading device, or an antibody-based immunohistochemistry or ELISA quantitative device.

[0008] Furthermore, high expression of FBH1 protein refers to a relative quantitative value of FBH1 protein detected by mass spectrometry using a data independent acquisition (DIA) mode of ≥60.

[0009] The second aspect of the present invention is to provide a system for predicting the efficacy of SSA adjuvant therapy after surgery for PNETs patients, characterized in that the system includes a device for detecting FBH1 protein and a data analysis device; wherein the device for detecting FBH1 protein includes but is not limited to a mass spectrometer, a spectrophotometer, a chemiluminescence signal reading device, or an antibody-based immunohistochemistry, ELISA quantification device; the device can quantify the abundance of the target protein.

[0010] Furthermore, the device for detecting FBH1 protein is a mass spectrometer; the mass spectrometer is configured to include but not limited to the following modes: data independent acquisition (DIA), data dependent acquisition (DDA), mass spectrometer targeted quantification; the above modes can quantify the abundance of target protein.

[0011] Furthermore, the mass spectrometer is configured as a data independent acquisition (DIA) mode, wherein when the relative quantitative value of the detected FBH1 protein is ≥60, it is defined as high expression, and PNETs patients can significantly benefit from postoperative SSA adjuvant therapy; otherwise, PNETs patients have no significant therapeutic effect from postoperative SSA adjuvant therapy.

[0012] The third aspect of the present invention is to provide a use of a substance for specifically detecting FBH1 protein in the preparation of a preparation for evaluating the prognosis of postoperative SSA adjuvant therapy for PNETs patients, characterized in that the substance for specific detection is suitable for mass spectrometry, wherein when the relative quantitative value of FBH1 protein detected by the data-independent acquisition mode in the mass spectrometry detection is ≥60, it is defined as high expression, and PNETs patients can significantly benefit from postoperative SSA adjuvant therapy; otherwise, PNETs patients have no significant therapeutic effect from postoperative SSA adjuvant therapy;

[0013] The use comprises the following steps:

[0014] S1. Obtain tumor tissue samples from patients with PNETs to be tested;

[0015] S2, protein extraction and enzymatic hydrolysis;

[0016] S3, based on mass spectrometry, use liquid chromatography-mass spectrometry system to collect DIA mode for all samples;

[0017] S4. Spectronaut uses the spectral library to search the DIA data of all samples and gives protein identification and quantification results.

[0018] Furthermore, the sample is a FFPE tumor tissue sample, and the protein extraction step is:

[0019] 1) Dewaxing in xylene for 10-20 min;

[0020] 2) Gradient ethanol hydration: hydration in 100%, 85%, 70%, and 50% ethanol for 3-8 minutes;

[0021] 3) Lysis: Heat the cells in a lysis buffer consisting of 0.1 M Tris-HCl, pH 8.0, 100 mM DTT, and 1% SDS for 20-40 min.

[0022] 4) Collection: The lysate formed in step 3) is sonicated for 3-5 minutes and the supernatant is collected by centrifugation.

[0023] Furthermore, it is characterized in that the proteolysis step is:

[0024] 1) Dilute the sample with 8 M urea, 100 mM Tris-Cl, pH 8.5 UA buffer and add it to the FASP ultrafiltration tube. Centrifuge at 12,000-16,000 g for 15-25 min and then add UA buffer again for washing.

[0025] 2) After centrifugation, add UA buffer containing 8-12 mM DTT, incubate at 37°C for 4 h, and then replace with UA solution containing 50 mM iodoacetamide;

[0026] 3) Keep in dark place for 30 min and centrifuge at 14,000 g for 15 min;

[0027] 4) After three washes with 50 mM ammonium bicarbonate, trypsin was added and reacted at 37 degrees for 16 hours. Peptides were collected by centrifugation at 14000 g, vacuum dried and stored in a -80°C refrigerator.

[0028] Furthermore, it is characterized in that the operation of using the liquid chromatography-mass spectrometry system to collect all samples in DIA mode is:

[0029] The LC-MS system is a Dionex UltiMate 3000 rapid separation liquid chromatography system connected to a timsTOF Pro mass spectrometer; the liquid phase gradient is 70-80 min, and the flow rate is 250-350 nl / min; the diaPASEF scanning mode is used, the isolation window is 25 Th, and there is a 1 Da overlap between adjacent windows.

[0030] The beneficial effects of the present invention include:

[0031] The present invention discloses a marker for judging the efficacy of postoperative SSA adjuvant therapy for PNETs patients, and specifically relates to a system and method for predicting the efficacy of postoperative SSA adjuvant therapy for PNETs patients based on the relative abundance of FBH1 protein. Experiments have shown that the disease-free survival time and total survival time of the postoperative SSA adjuvant therapy group of PNETs patients with high FBH1 expression are significantly better than those of the non-adjuvant therapy group. By detecting the relative content of FBH1 protein in the tumor tissue of PNETs patients, it is possible to effectively evaluate whether the patients use SSA drug adjuvant therapy after surgery, which is of great significance for the guidance of postoperative medication for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A is the Kaplan–Meier curve of disease-free survival between the SSA adjuvant treatment group and the no-treatment group in all PNETs patients; Figure 1 B is the Kaplan–Meier curve of overall survival between the SSA adjuvant treatment group and the no treatment group in all PNETs patients.

[0033] Figure 2 A is the Kaplan–Meier curve of disease-free survival between the SSA adjuvant treatment group and the no-treatment group in PNETs patients with high FBH1 expression; Figure 2 B is the Kaplan–Meier curve of overall survival between the SSA adjuvant treatment group and the no-treatment group in patients with PNETs with high FBH1 expression.

[0034] Figure 3 A is the Kaplan–Meier curve of disease-free survival between the SSA adjuvant treatment group and the no-treatment group in PNETs patients with low FBH1 expression; Figure 3 B is the Kaplan–Meier curve of overall survival between the SSA adjuvant treatment group and the no-treatment group in patients with PNETs with low FBH1 expression. DETAILED DESCRIPTION

[0035] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example: Determination of FBH1 protein content in tumor tissues of PNETs patients using mass spectrometry based on data-independent mode

[0039] 1. Experimental Materials

[0040] FFPE tumor tissue samples were obtained from 139 patients with G1 or G2 PNETs and provided by the General Hospital of the Chinese People's Liberation Army.

[0041] 2. Experimental Methods

[0042] 1. Protein extraction: After FFPE tissue was dewaxed in xylene for 15 minutes, it was hydrated in 100%, 85%, 70%, and 50% ethanol for 5 minutes. The sample was then heated at 99 degrees in a lysis buffer (0.1 M Tris-HCl, pH 8.0, 100 mM DTT, 1% SDS 1% SDS) for 30 minutes. After 5 minutes of ultrasonication, the sample was further centrifuged at 16000g for 10 minutes, and the supernatant was collected.

[0043] 2. Protein enzymatic hydrolysis: Dilute the sample with UA buffer (8 M urea, 100 mM Tris-Cl, pH 8.5) and add it to the FASP ultrafiltration tube. Centrifuge at 14000 g for 20 min and then add UA to wash again. After centrifugation, add 200 μl of UA solution containing 10 mM DTT, incubate at 37°C for 4 h, and then replace with UA solution containing 50 mM iodoacetamide. Centrifuge at 14000 g for 15 min after 30 min in the dark. After three washes with 50 mM ammonium bicarbonate, add trypsin (Promega, 1 / 50 of the protein amount) and react at 37 degrees for 16 hours. Collect the peptides by centrifugation at 14000 g, vacuum dry and store in a -80°C refrigerator.

[0044] 3. Mass spectrometry data acquisition: All samples were collected in DIA mode using a liquid chromatography-mass spectrometry system, which was a Dionex UltiMate 3000 rapid separation liquid chromatography system connected to a timsTOF Pro mass spectrometer (Thermo Fisher Scientific). The liquid phase gradient was 80 min and the flow rate was 300 nl / min. The diaPASEF scan mode (m / z 300–1500) was used, with an isolation window of 25Th and 1 Da overlap between adjacent windows. Eight samples with the largest number of identified proteins were selected for data-dependent acquisition of a 140-min gradient to establish a spectral library.

[0045] 4. Mass spectrometry data analysis: The spectra of the eight data-dependent samples were compiled into a spectral library using Spectronaut (version 16.1, Biognosys). Spectronaut used the spectral library to search the DIA data of all samples to give protein identification and quantification results, with protein and peptide FDR less than 0.01.

[0046] The relative quantitative results of the identified FBH1 were extracted, and the missing values ​​were filled with 1 for the samples where FBH1 was not identified. The results are shown in Table 1.

[0047] Table 1. Relative abundance and clinical information of FBH1 protein in tumor tissues of PNETs patients

[0048]

[0049] The prognosis of PNETs patients was analyzed by Kaplan–Meier curve analysis, and the patients were divided into SSA adjuvant therapy group and no adjuvant therapy group according to the postoperative adjuvant therapy method (such as Figure 1 The results showed that the disease-free survival time ( Figure 1 A) and overall survival ( Figure 1 B) There was no significant difference (median disease-free survival time was 40 months vs. 50 months, log-rank P = 0.9; median overall survival time was 44.5 months vs. 59 months, log-rank P = 0.198). However, the disease-free survival time of PNETs patients with high FBH1 expression in the SSA adjuvant treatment group was Figure 2 A) and overall survival ( Figure 2 B) were significantly better than those in the group without adjuvant therapy (median disease-free survival time was 44.5 months vs. 25 months, log-rank P = 0.001; median overall survival time was 44.5 months vs. 31.5 months, log-rank P = 0.001). The disease-free survival time of PNETs patients with low FBH1 expression was different between the SSA adjuvant therapy group and the non-adjuvant therapy group ( Figure 3 A) and overall survival ( Figure 3 B) There was no significant difference (median disease-free survival time was 38 months vs. 60 months, log-rank P = 0.368; median overall survival time was 44.5 months vs. 61 months, log-rank P = 0.891). When FBH1 was highly expressed, the recurrence rate in the non-adjuvant therapy group was 40%, and the mortality rate was 30%; while the recurrence rate in the SSA adjuvant therapy group was only 10%, and the mortality rate was 0%.

[0050] The above results indicate that the relative content of FBH1 protein in tumor tissue of PNETs patients can be used to determine the efficacy of SSA adjuvant therapy.

[0051] The FBH1 high expression group consisted of PNETs patients whose relative quantitative value of FBH1 protein detected by mass spectrometry DIA mode was greater than or equal to 60;

[0052] The FBH1 low expression group consisted of PNETs patients whose relative quantitative value of FBH1 protein detected by mass spectrometry DIA mode was less than 60;

[0053] The above embodiments specifically describe the present invention and are not intended to limit the present invention. Without departing from the principle of the present invention, modifications and alterations to the present invention should all be included in the protection scope of the present invention.

Claims

1. The use of FBH1 protein in the preparation of a preparation related to predicting the prognosis of postoperative SSA adjuvant therapy in pancreatic neuroendocrine tumors, characterized in that: In patients with PNETs with high FBH1 protein expression, the disease-free survival and overall survival time in the SSA adjuvant treatment group were significantly better than those in the non-adjuvant treatment group.

2. The use according to claim 1, wherein the method for determining the expression level of the FBH1 protein includes but is not limited to a mass spectrometer, a spectrophotometer, a chemiluminescence instrument signal reading device, or an antibody-based immunohistochemistry or ELISA quantitative device.

3. The use according to claim 1 or 2, characterized in that: High expression of FBH1 protein refers to a relative quantitative value of FBH1 protein detected by mass spectrometry using data independent acquisition (DIA) mode ≥ 60.

4. A system for predicting the efficacy of SSA adjuvant therapy after surgery for PNETs patients, characterized in that: The system includes a device for detecting FBH1 protein and a data analysis device; The device for detecting FBH1 protein is a mass spectrometer; The mass spectrometer is configured as a data independent acquisition (DIA) mode, wherein when the relative quantitative value of the detected FBH1 protein is ≥60, it is defined as high expression, and PNETs patients can significantly benefit from postoperative SSA adjuvant therapy; conversely, PNETs patients postoperative SSA adjuvant therapy has no significant therapeutic effect.

5. Use of a substance for specifically detecting FBH1 protein in the preparation of a preparation for evaluating the prognosis of postoperative SSA adjuvant therapy in PNETs patients, characterized in that: The specifically detected substance is suitable for mass spectrometry, wherein when the relative quantitative value of FBH1 protein detected by mass spectrometry using a data-independent acquisition mode is ≥60, it is defined as high expression, and PNETs patients can benefit significantly from postoperative SSA adjuvant therapy; conversely, PNETs patients have no significant therapeutic effect from postoperative SSA adjuvant therapy; The use comprises the following steps: S1. Obtain tumor tissue samples from patients with PNETs to be tested; S2, protein extraction and proteolysis; S3, based on mass spectrometry, use liquid chromatography-mass spectrometry system to collect DIA mode for all samples; S4. Spectronaut uses the spectral library to search the DIA data of all samples and gives protein identification and quantification results.

6. The use according to claim 5, characterized in that The sample is a FFPE tumor tissue sample, and the protein extraction steps are: 1) Dewaxing in xylene for 10-20 min; 2) Gradient ethanol hydration: hydration in 100%, 85%, 70%, and 50% ethanol for 3-8 minutes; 3) Lysis: Heat and lyse for 20-40 min in a lysis buffer consisting of 0.1 M Tris-HCl, pH 8.0, 100 mM DTT, and 1% SDS. 4) Collection: The lysate formed in step 3) is sonicated for 3-5 minutes and the supernatant is collected by centrifugation.

7. The use according to claim 5, characterized in that The proteolysis step is: 1) Dilute the sample with 8 M urea, 100 mM Tris-Cl, pH 8.5 UA buffer and add it to the FASP ultrafiltration tube. Centrifuge at 12,000-16,000 g for 15-25 min and then add UA buffer again for washing. 2) After centrifugation, add UA buffer containing 8-12 mM DTT, incubate at 37°C for 4 h, and then replace with UA solution containing 50 mM iodoacetamide; 3) Keep in dark place for 30 min and centrifuge at 14,000 g for 15 min; 4) After three washes with 50 mM ammonium bicarbonate, trypsin was added and reacted at 37 degrees for 16 hours. The peptides were collected by centrifugation at 14,000 g, vacuum dried and stored in a -80°C refrigerator.

8. The use according to claim 7, characterized in that The operation of using LC-MS system to collect DIA mode for all samples is as follows: The LC-MS system is a Dionex UltiMate 3000 rapid separation liquid chromatography system connected to a timsTOFPro mass spectrometer; the liquid phase gradient is 70-80 min, and the flow rate is 250-350 nl / min; the diaPASEF scanning mode is used, the isolation window is 25 Th, and there is a 1 Da overlap between adjacent windows.

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