Use of protein combination in preparation of precise prognosis stratification kit for patients with hepatocellular carcinoma

Abstract

The application discloses application of a protein combination in preparation of a precise prognosis stratification kit for a hepatocellular carcinoma patient. It is found by the application that the expression amount of myeloperoxidase (MPO) and pyruvate kinase (PKM) in a liver cancer tissue of the hepatocellular carcinoma patient can be used for prognosis or precise prognosis stratification of the hepatocellular carcinoma patient. In the MPO high-expression patient, only the HCC patient with simultaneous high expression of PKM has a poor prognosis, and when the PKM is low expression, the prognosis of the patient is still good. The application can utilize MPO and PKM to precisely stratify the hepatocellular carcinoma patient.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically the application of protein assemblies in the preparation of a precise prognostic stratification kit for hepatocellular carcinoma patients. Background Technology

[0002] The dual role of neutrophils in tumor progression has increasingly attracted the attention of researchers. As a result, two polarization states of neutrophils have been proposed to distinguish the different functions of tumor-associated neutrophils (TANs)—anti-tumor neutrophils (N1) and pro-tumor neutrophils (N2). 1 (Similar to macrophages in different polarization states). Recent studies have shown that TANs promote tumor progression by directly promoting tumor cell proliferation and invasion, enhancing extracellular matrix remodeling and angiogenesis, and maintaining an immunosuppressive microenvironment. 2-4 However, the existence of truly meaningful N1 and N2 neutrophil subsets and their significance in tumor development and prognosis remain controversial. Furthermore, the dynamic heterogeneity of neutrophils is greatly influenced by the tissue microenvironment in which they colonize, which further limits their application as biomarkers in tumor diagnosis and treatment.

[0003] Recent studies have found that patients with malignant hepatocellular carcinoma (HCC) have an increased neutrophil-to-lymphocyte ratio (NLR) in peripheral blood. 5 Furthermore, the patient's liver cancer tissue will show extensive diffuse neutrophil infiltration. 6 Therefore, the latest 2019 WHO Classification of Tumors (Digestive System) guidelines defined a new pathological subtype of HCC—neutrophilic infiltrative type—and used neutrophils as a marker of poor prognosis. However, according to the guideline criteria (neutrophil infiltration rate >50%), this type of HCC accounts for <1% of the total patient population. This significantly limits the application of neutrophils as a marker for HCC diagnosis and treatment in clinical practice, and it does not take into account the different significance of neutrophil heterogeneity for patient prognosis. 7 . Summary of the Invention

[0004] The technical problem to be solved by this invention is how to accurately stratify the prognosis of HCC patients with high neutrophil infiltration.

[0005] To address the aforementioned technical problems, this invention first provides the application of a substance for detecting the expression levels of a protein combination in the preparation of a prognostic or precise prognostic stratification kit for hepatocellular carcinoma patients, wherein the protein combination is myeloperoxidase (MPO, Uniprot number: P05164) and / or pyruvate kinase (PKM, Uniprot number: P14618).

[0006] Among them, precise prognostic stratification refers to the prognostic analysis of hepatocellular carcinoma patients' survival period or survival rate at different time points (such as 12 months, 24 months, 60 months, etc.) using the expression abundance of MPO and PKM.

[0007] In the above applications, the substance used to detect the combined expression levels of proteins may include substances that detect the expression levels of myeloperoxidase and pyruvate kinase by mass spectrometry and / or immunohistochemical staining.

[0008] The mass spectrometer may be a data-dependent tandem mass spectrometer.

[0009] In the above applications, the substances used for immunohistochemical staining can be myeloperoxidase-specific antibodies and / or pyruvate kinase-specific antibodies.

[0010] In the above applications, the expression level of myeloperoxidase can be its expression level in liver cancer tissue.

[0011] In the above applications, the expression level of pyruvate kinase can be compared to its expression level in liver cancer tissue.

[0012] In the above applications, the substance for detecting the combined expression levels of proteins may also include a data analysis model, which is used to input the combined expression level data of proteins into a Cox regression model and classify patients into high-risk or low-risk groups based on the model results.

[0013] In this invention, the prognostic stratification method for hepatocellular carcinoma can be obtained by inputting the expression levels of MPO and PKM detected by label-free quantitative mass spectrometry (or immunohistochemistry) into a Cox regression model.

[0014] In this invention, the expression level of protein by label-free quantitative mass spectrometry can be obtained by the following method:

[0015] 1. Data generation methods

[0016] First, we obtained sample cohorts and prognostic follow-up data of hepatocellular carcinoma patients, and simultaneously obtained surgically resected hepatocellular carcinoma tissue samples. After protein extraction and enzymatic digestion, the samples were prepared using the iBAQ label-free quantification method for high-throughput sample preparation. Subsequently, mass spectrometry data were acquired using data-dependent mass spectrometry acquisition technology, and the raw spectrum files were quantitatively analyzed using MaxQuant software to obtain quantitative information on the sample proteome.

[0017] 2. Data Preprocessing Methods

[0018] The obtained protein expression matrix was standardized using the quantile algorithm from the R software package limma, and missing values ​​were imputed using the global minimum.

[0019] This invention reveals that among patients with high MPO expression, HCC patients with high PKM expression alone have a poorer prognosis, while those with low PKM expression still have a better prognosis. MPO and PKM can be used for precise prognostic stratification of hepatocellular carcinoma patients. By combining the biomarkers MPO and PKM, the prognosis of HCC patients with high neutrophil infiltration can be precisely stratified, thus supplementing current clinical guidelines for neutrophil-based diagnosis of HCC prognosis.

[0020] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0021] Figure 1 This is a flowchart for process analysis.

[0022] Figure 2 This study included Kaplan-Meier survival analysis of HCC patients in an independent HCC population, specifically those with low MPO expression, high MPO expression, and those with high or low PKM expression within the high MPO expression HCC group. The gray boxes in "All" represent patients with low MPO expression, the black boxes in "All" represent patients with high MPO expression, the gray dashed boxes in "High MPO" represent patients with low PKM expression, and the black dashed boxes in "High MPO" represent patients with high PKM expression.

[0023] Figure 3 This study included Kaplan-Meier survival analysis of HCC patients with low MPO expression, high MPO expression, and high and low PKM expression in HCC with high MPO expression, as determined by IHC. The gray boxes in "All" represent patients with low MPO expression, the black boxes in "All" represent patients with high MPO expression, the gray dashed boxes in "High MPO" represent patients with low PKM expression, and the black dashed boxes in "High MPO" represent patients with high PKM expression. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0025] Example 1: Precise screening of HCC patients with poor prognosis and high neutrophil infiltration using the MPO+PKM combined biomarker.

[0026] Analysis process as follows Figure 1 As shown.

[0027] 1. Sample inclusion

[0028] With informed consent, patients with early-stage hepatocellular carcinoma who underwent surgical resection were recruited from Zhongshan Hospital of Fudan University and Peking University Cancer Hospital. Exclusion criteria were as follows: (1) preoperative chemotherapy or radiotherapy; (2) not the first surgery; (3) not Barcelona stage 0 or A; (4) HCV infection; (5) lost to follow-up or incomplete clinical data. A total of 101 patients with hepatocellular carcinoma were ultimately included, with the following inclusion criteria: (1) Barcelona stage 0 or A hepatocellular carcinoma; (2) sufficient resection tissue for proteomics data production; (3) complete follow-up information and clinical data. This study was approved by the ethics committees of Zhongshan Hospital of Fudan University and Peking University Cancer Hospital.

[0029] Postoperative follow-up was conducted on patients. If the patient died, the prognostic time was recorded as the time interval from postoperative surgery to death. If the patient did not die, the prognostic time was recorded as the time interval from postoperative surgery to the last follow-up visit, and this was recorded as right-censored data.

[0030] 2. Proteomics Data Acquisition and Preprocessing

[0031] Protein extraction from hepatocellular carcinoma tissue. Frozen tumor tissue from hepatocellular carcinoma patients was collected and lysed with T-PER tissue protein extraction reagent (Thermo, catalog number 78510) containing protease inhibitor (Roche, catalog number 04693116001) and phosphatase inhibitor (Roche, catalog number 04906837001). The tissue was then disrupted using an ultrasonic homogenizer (Scientz) (25% amplitude sonication for 1 min, including 3 seconds of sonication followed by 3 seconds of intermittent sonication). The resulting liquid was then centrifuged at 16000 g for 10 min at 4 degrees Celsius. The supernatant was collected as the tissue protein extract, and the protein concentration was determined using Bradford protein quantification reagent (Thermo, catalog number 23246).

[0032] Ultrafiltration-assisted cathepsin digestion and subsequent lyophilization of peptides. The specific steps are as follows: 500 μg of the above-mentioned liver cancer tissue protein extract was diluted to 500 μL with 8M urea solution (prepared with 0.1M Tris-HCl, pH 8.5). The diluted sample solution was placed in a 30 kDa ultrafiltration tube (Millipore, catalog number UFC503096) and centrifuged for 20 minutes. Then, 200 μL of urea solution containing 10 mM dithiothreitol was added to the ultrafiltration tube, and the mixture was incubated at 37°C for 4 hours for reductive alkylation. After incubation, the reaction solvent was discarded by centrifugation. Then, urea solution containing 50 mM iodoacetamide was added to the ultrafiltration tube, and the mixture was incubated at room temperature in the dark for 30 minutes. The ultrafiltration tube was then washed three times sequentially with 200 μL of urea solution and 200 μL of 50 mM ammonium bicarbonate solution, centrifuged at 14000 g for 15 minutes at room temperature after each wash. Next, 100 μL of a 50 mM ammonium bicarbonate solution containing 0.1 μg / μL trypsin (Promega, catalog number V528A) was added to the ultrafiltration tube and incubated at 37°C for 12 hours. After enzyme digestion, the digested peptides were collected by centrifugation at 14000g for 15 minutes and washed twice with 100 μL of 50 mM ammonium bicarbonate solution. The washings were collected by centrifugation and mixed with the peptides collected in the previous step. The peptide concentration was determined using a NanoDrop 2000C (Thermo). The digested peptides were lyophilized using a vacuum centrifuge (Eppendorf) and stored at -80°C. Before mass spectrometry detection, the obtained peptide samples were redissolved in ammonia (pH 10) and detected using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. iBAQ standard-free quantitative mass spectrometry data were obtained using data-dependent mass spectrometry acquisition technology. The raw spectral files were quantitatively analyzed using MaxQuant software to obtain quantitative information of the sample proteome. The obtained protein expression matrix was standardized using the quantile algorithm from the R software package limma, and missing values ​​were imputed using the global minimum.

[0033] 3. Utilizing the MPO+PKM combined biomarker for precise screening of HCC patients with poor prognosis and high neutrophil infiltration.

[0034] Quantitative data of myeloperoxidase (MPO, Uniprot ID: P05164) and pyruvate kinase (PKM, Uniprot ID: P14618) were extracted from the protein expression matrix. First, the MPO expression level was input into a Cox regression model. Based on the MPO expression level and the Cox regression results, 24 patients (23.8%) with HCC were defined as the MPO high-expression group (high neutrophil infiltration group), and the remaining 77 patients (76.2%) were defined as the MPO low-expression group (low neutrophil infiltration group). The definition method was as follows: the protein abundance was taken as the logarithm to base 2; MPO abundance values ​​greater than or equal to 22 were classified as high-expression, and values ​​less than 22 were classified as low-expression.

[0035] In 24 HCC patients with high MPO expression, based on PKM expression levels detected by label-free quantitative mass spectrometry and Cox regression model results, 16 HCC patients (66.7%) were defined as high-risk, and 8 patients (33.3%) were defined as low-risk. The definition method was as follows: the protein abundance was taken as the logarithm to base 2, and PKM abundance values ​​greater than or equal to 28.5 were classified as high-expression group, and values ​​less than 28.5 were classified as low-expression group.

[0036] Table 1 shows the MPO and PKM expression and prognostic information of each patient.

[0037] Table 1. MPO and PKM expression levels and prognostic analysis in patients.

[0038]

[0039] Kaplan-Meier survival analysis showed that for all enrolled patients, the 12-month survival rate for the 24 patients with high MPO expression was 87.5%, the 24-month survival rate was 75.0%, and the 60-month survival rate was 75%. For the 77 patients with low MPO expression, the 12-month survival rate was 98.7%, the 24-month survival rate was 94.8%, and the 60-month survival rate was 89.6%. The prognostic hazard ratio for high MPO expression was 2.63 (95% CI: 0.911–7.57), with a p-value of 6.47e-02.

[0040] Of the 24 patients with high MPO expression, the 12-month survival rate was 81.3% for the 16 patients with high PKM expression, the 24-month survival rate was 62.5%, and the 60-month survival rate was 62.5%. All 8 patients with low PKM expression survived during the follow-up period. The prognostic hazard ratio for high PKM expression was 1.06e+09 (95% CI: 0-1nf), with a p-value of 5.86e-02.

[0041] The above results indicate that the combined use of MPO and PKM can differentiate HCC patients with high neutrophil infiltration into two groups with drastically different prognoses. HCC patients with high expression of both MPO and PKM have the worst prognosis, while HCC patients with high MPO expression but low PKM expression still have a relatively good prognosis. Figure 2 ).

[0042] 2. Verification by immunohistochemical staining

[0043] HCC tissue microarrays (HLivH180Su11, HLivH180Su18) commercially available from Shanghai Xinchao Biotechnology Co., Ltd. were used to verify HCC tissues through immunohistochemical staining. The specific detection steps are as follows: (1) Dewaxing: The slides were placed in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then washed with distilled water. (2) Antigen retrieval: The tissue slides were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 9.0) and subjected to antigen retrieval in a microwave oven: medium heat for 9 min until boiling, turn off the heat for 7 min, keep warm and then turn to medium-low heat for 6 min. After natural cooling, the slides were placed in PBS and washed 3 times, 5 min each time. (3) Blocking: Block endogenous peroxidase activity with 3% H2O2 (Zhongshan Jinqiao, catalog number PV-6001) and block with normal sheep serum (Zhongshan Jinqiao, catalog number ZLI-9056) at room temperature for 20 min. (4) Primary antibody: Incubate overnight at 4 degrees Celsius with ready-to-use rabbit anti-MPO antibody (Zhongshan Jinqiao, catalog number ZA-0197) or mouse anti-PKM2 antibody (Proteintech, catalog number 60268-1-Ig, 1:40000). (5) Secondary antibody: Place the slide in PBS and wash three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add HRP-labeled secondary antibody (Zhongshan Jinqiao, catalog number PV-6001, 6002) of the corresponding species as the primary antibody to cover the tissue and incubate at room temperature for 50 min. (6) DAB staining and counterstaining: Place the slide in PBS and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution (Servicebio, catalog number G1211) to the circle. Control the staining time under the microscope. The positive color is brownish-yellow. Rinse the slide with tap water to stop the staining. Then counterstain with hematoxylin (Servicebio, catalog number G1004) for about 3 minutes, rinse with tap water, differentiate with hematoxylin differentiation solution (Servicebio, catalog number G1039) for a few seconds, rinse with tap water, and then re-blue with hematoxylin blue solution (Servicebio, catalog number G1040) and rinse with running water. (7) Mounting: Mount the slide with neutral resin (Servicebio, catalog number G1403). (8) Image acquisition and result interpretation: Images were acquired using a NanoZoomer 2.0-RS scanner (HAMAMATSU). The staining score of PKM was calculated by multiplying the staining ratio integral (0 points for less than 5%; 1 point for 5%-25%; 3 points for 26%-50%; 3 points for 51%-75%; and 4 points for greater than 75%) by the staining intensity integral (0 points for negative; 1 point for weak positive; 2 points for moderate positive; and 3 points for strong positive).MPO staining intensity was obtained by directly counting the number of MPO-positive cells in the HCC interstitium.

[0044] Table 2 shows the MPO and PKM immunohistochemical staining results and prognostic information for each patient.

[0045] Table 2. Immunohistochemical staining of MPO and PKM in patients and prognostic analysis

[0046]

[0047] Kaplan-Meier survival analysis of immunohistochemical staining results of MPO and PKM in HCC tissues showed that, for all patients, the 12-month survival rate was 61.5% for the 52 patients with high MPO expression, the 24-month survival rate was 51.9%, and the 60-month survival rate was 28.8%. For the 121 patients with low MPO expression, the 12-month survival rate was 78.5%, the 24-month survival rate was 62.0%, and the 60-month survival rate was 47.9%. The prognostic hazard ratio for high MPO expression was 1.68 (95% CI: 1.12–2.53), with a p-value of 1.13e-02.

[0048] Of the 52 patients with high MPO expression, the 12-month survival rate was 59.4% for the 37 patients with high PKM expression, the 24-month survival rate was 45.9%, and the 60-month survival rate was 18.9%. Of the 15 patients with low PKM expression, the 12-month survival rate was 66.7%, the 24-month survival rate was 66.7%, and the 60-month survival rate was 53.3%. The prognostic hazard ratio for high PKM expression was 2.57 (95% CI: 1.11–5.93), with a p-value of 2.36e-02.

[0049] The above immunohistochemical staining results validated the accurate prognostic value of the MPO+PKM combined marker for HCC with high neutrophil infiltration, further confirming a significant prognostic difference in HCC patients with high MPO expression. Although both patients exhibited high MPO expression, those with HCC exhibiting high PKM expression alone had a poorer prognosis, while those with low PKM expression still had a better prognosis. Figure 3 ).

[0050] These results not only further demonstrate the different prognostic significance of neutrophil infiltration for HCC patients, but also provide a clinically applicable strategy for accurately screening HCC patients with high neutrophil infiltration and poor prognosis.

[0051] References:

[0052] 1.Coffelt, SB, Wellenstein, MD&de Visser, KE Neutrophils incancer: neutral no more. Nat. Rev. Cancer 16, 431-446 (2016).

[0053] 2.Geh, D.et al. Neutrophils as potential therapeutic targets inhepatocellular carcinoma. Nat. Rev. Gastroenterol. Hepatol. 19, 257-273 (2022).

[0054] 3.Zhou, SLet al. A Positive Feedback Loop Between Cancer Stem-LikeCells and Tumor-Associated Neutrophils Controls Hepatocellular CarcinomaProgression. Hepatology 70, 1214-1230 (2019).

[0055] 4.Donne, R.&Lujambio, A. The liver cancer immune microenvironment: Therapeutic implications for hepatocellular carcinoma. Hepatology https: / / doi.org / 10.1002 / hep.32740 (2022).

[0056] 5.Chu, HHet al. Neutrophil-to-Lymphocyte Ratio as a BiomarkerPredicting Overall Survival after Chemoembolization for Intermediate-StageHepatocellular Carcinoma. Cancers (Basel) 13, 2830 (2021).

[0057] 6.Torbenson, MS Morphologic Subtypes of Hepatocellular Carcinoma. Gastroenterol. Clin. North. Am. 46, 365-391 (2017).

[0058] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of a substance for detecting the expression levels of a protein combination in the preparation of a precise prognostic stratification kit for hepatocellular carcinoma patients, wherein the protein combination is myeloperoxidase and pyruvate kinase; Precise prognostic stratification refers to the prognostic analysis of hepatocellular carcinoma patients' survival time or survival rate at different time points by using the expression abundance of MPO and PKM proteins. The substances used to detect the combined expression levels of proteins include those that can detect the expression levels of myeloperoxidase and pyruvate kinase by mass spectrometry and / or immunohistochemical staining. The expression level of myeloperoxidase is its expression level in liver cancer tissue; The expression level of pyruvate kinase is its expression level in liver cancer tissue.

2. Use according to claim 1, characterized in that: The mass spectrometer is a data-dependent tandem mass spectrometer.

3. Use according to claim 1, characterized in that: The substances used for immunohistochemical staining are myeloperoxidase-specific antibodies and / or pyruvate kinase-specific antibodies.

4. Use according to any one of claims 1 to 3, characterized in that: The substance used to detect the combined expression levels of proteins also includes a data analysis model, which is used to input the combined expression level data of proteins into a Cox regression model and classify patients into high-risk or low-risk groups based on the model results.

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