Use of uchl1 in the preparation of a detection product for predicting the curative effect and prognosis of hcc treated by atezolizumab combined with bevacizumab

Abstract

The application relates to the field of biological medicine, discloses an application of UCHL1 in preparation of a detection product for predicting the curative effect and prognosis of hepatocellular carcinoma atezolizumab combined with bevacizumab, and further discloses a kit for predicting the curative effect and prognosis of hepatocellular carcinoma atezolizumab combined with bevacizumab. The kit based on UCHL1 can be used as a curative effect prediction tool of Atezo / Bev immune combined treatment, provides precise treatment guidance for HCC patients, and improves the individualized treatment level.

Description

Technical Field

[0001] This application relates to the biomedical field, and more specifically, to the use of UCHL1 in the preparation of a assay product for predicting the efficacy and prognosis of hepatocellular carcinoma treated with atezolizumab in combination with bevacizumab. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common and life-threatening malignant tumors worldwide, with more than 782,000 new cases diagnosed annually. In recent years, the incidence of liver cancer has been on the rise in the United States and Europe, while China still accounts for nearly half of all liver cancer-related deaths globally. The main curative treatments for HCC include surgical resection, local ablation therapy, and liver transplantation; however, because most patients are diagnosed at an advanced stage, only a small percentage are able to receive curative treatment.

[0003] Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, significantly improving the prognosis of some patients. However, a large number of patients remain insensitive to treatment or develop resistance, leading to disease progression. Therefore, exploring combination therapies that can enhance anti-tumor immune responses, such as strategies combining immunotherapy with targeted therapy, radiotherapy, or metabolic interventions, is crucial to overcome the limitations of current immunotherapy and improve clinical benefits for patients, potentially further increasing remission rates and survival. For example, anti-angiogenic drugs can enhance the efficacy of immunotherapy by improving the tumor microenvironment and reducing the infiltration of immunosuppressive cells. Furthermore, combination strategies that target and regulate tumor metabolism, microbiota, or inflammatory signaling pathways may also play a key role in improving immunotherapy responses. Current immunotherapy combined with targeted therapy strategies, exemplified by immunotherapy combined with anti-angiogenic therapy—atezolizumab + bevacizumab (Atezo / Bev)—has been recommended as a first-line preferred regimen for patients with advanced HCC by the IMBrave150 Phase III clinical trial, significantly improving patient survival and quality of life. However, the Atezo / Bev regimen still faces bottlenecks in achieving durable clinical benefits, with a median overall survival (OS) of approximately 20 months and an objective response rate (ORR) that fluctuates between 20-25%, consistently failing to break through 30%.

[0004] Therefore, identifying biomarkers that can predict the efficacy of combined immunotherapy, i.e., Atezo / Bev treatment, is crucial for optimizing patient selection. Due to the high heterogeneity in individual responses to immunotherapy, relying solely on a single treatment regimen is insufficient to achieve ideal efficacy. Therefore, integrating multi-omics data analysis to identify biomarkers indicating immunotherapy sensitivity or tolerance from the perspectives of gene expression, proteomics, and metabolomics will help in the precise selection of suitable patient populations for immunotherapy. For example, the proportion of specific immune cells within certain tumors, the expression levels of inflammatory factors, or changes in tumor metabolic status may all be potential predictors.

[0005] In conclusion, by developing more effective combination immunotherapy strategies and accurately identifying treatment response-related biomarkers, we can not only improve the effectiveness of immunotherapy but also avoid unnecessary treatment side effects, ultimately achieving precision treatment for HCC patients, improving long-term survival rates, and enhancing their quality of life. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to explore a molecular biomarker based on peripheral plasma protein levels, detectable in peripheral venous blood, that can effectively predict the response and overall prognosis of HCC patients receiving Atezo / Bev treatment, accurately identify patient groups who respond well to combined immunotherapy, avoid unnecessary treatment side effects, improve the prognosis of HCC patients, and achieve patient survival benefits.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of UCHL1 in the preparation of a detection product for predicting the efficacy and prognosis of hepatocellular carcinoma treated with atezolizumab in combination with bevacizumab.

[0009] Furthermore, the testing product includes a reagent kit.

[0010] In a second aspect, the present invention provides a kit for predicting the efficacy and prognosis of atezolizumab combined with bevacizumab in hepatocellular carcinoma, including reagents for detecting UCHL1 levels.

[0011] Furthermore, the kit includes a 96-well ELISA plate coated with UCHL1 antibody; diluent for standards and UCHL1 detection samples; enzyme-labeled antibody working solution, washing buffer, substrate chromogenic solution and stop solution; and an instruction manual.

[0012] Furthermore, the test sample for the kit is a plasma sample.

[0013] The technical solution of this invention is proposed based on the following:

[0014] The role of UCHL1 in HCC:

[0015] UCHL1 is a ubiquitin hydrolase that plays an important role in protein degradation, cellular metabolism, and immune regulation. Studies have shown that UCHL1 is aberrantly expressed in various tumor types and is closely related to the immune microenvironment, tumor drug resistance, and patient prognosis.

[0016] The relationship between UCHL1 and combination immunotherapy:

[0017] The effectiveness of immune checkpoint inhibitor (ICI) therapy depends on the immune status of the tumor microenvironment, and UCHL1 can affect the immunotherapy response by regulating the metabolic patterns of tumor cells, the secretion of immunosuppressive factors, and T cell infiltration.

[0018] UCHL1 may be associated with HIF1α stability, glycolysis levels, and lactate production, thereby affecting the function of tolerogenic dendritic cells (DCs) and regulatory T cells (Tregs), ultimately determining the sensitivity to immunotherapy. Preliminary analysis in this study indicates that patients with high UCHL1 expression have a lower response rate and shorter survival to Atezo / Bev treatment, while patients with low expression have better immunotherapy benefits.

[0019] The rationale for UCHL1 as a biomarker:

[0020] Peripheral blood UCHL1 testing can assess a patient's immune status without invasive tumor biopsy, which is clinically feasible and practical.

[0021] Combining UCHL1 levels for risk stratification of HCC patients helps optimize Atezo / Bev treatment decisions, improve treatment efficiency, and avoid the side effects and economic burden caused by ineffective treatment. The detection method provided by this invention is simple to operate, stable and reliable, and has high specificity and sensitivity, and can be widely used in clinical practice.

[0022] In summary, compared with the prior art, this application has the following beneficial effects:

[0023] Peripheral circulation UCHL1 biomarkers provide an accurate, effective, simple, convenient, and easily applicable quantitative indicator and risk stratification model for assessing the efficacy and overall prognosis of HCC immunotherapy combination therapy, namely Atezo / Bev.

[0024] This invention relates to a reagent kit based on UCHL1, which can serve as a predictive tool for the efficacy of Atezo / Bev combined immunotherapy, providing precise treatment guidance for HCC patients and improving the level of individualized treatment. Attached Figure Description

[0025] Figure 1 Correlation analysis of UCHL1 levels with treatment efficacy and survival prognosis;

[0026] Figure 2 Multifactor regression model and Kaplan-Meier survival curve. Detailed Implementation

[0027] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0028] The specific embodiments of this invention provide a method and kit based on UCHL1 protein level detection for predicting the efficacy and overall prognosis of hepatocellular carcinoma (HCC) patients treated with atezolizumab in combination with bevacizumab (Atezo / Bev).

[0029] 1. Detection indicators: This invention is based on the expression level of UCHL1 (Ubiquitin C-terminal Hydrolase L1) protein in the peripheral blood circulation of HCC patients, and uses UCHL1 as a biomarker to predict the patient's response to Atezo / Bev immunotherapy.

[0030] 2. Sample collection and processing: Peripheral venous blood was collected from HCC patients using anticoagulant blood collection tubes (EDTA purple-tipped tubes); the plasma samples were separated by centrifugation at 4°C for 20 min and stored at -80°C for later use.

[0031] 3. Detection method: UCHL1 protein level was detected by enzyme-linked immunosorbent assay (ELISA) using a specific anti-UCHL1 monoclonal antibody; UCHL1 protein concentration was calculated using a standard curve, and the optimal cutoff value of UCHL1 was determined by combining biostatistical methods to distinguish between Atezo / Bev treatment responders and non-responders.

[0032] 4. Data Analysis and Risk Assessment: Logistic regression analysis was used to analyze the relationship between UCHL1 levels and the efficacy of Atezo / Bev treatment; Kaplan-Meier survival curves were used to analyze the differences in survival prognosis among patients with different UCHL1 levels; the sensitivity, specificity, and optimal cutoff value of UCHL1 in predicting the efficacy of Atezo / Bev were calculated by combining receiver operating characteristic (ROC) curves; and a risk stratification model for HCC patients based on UCHL1 levels was constructed to provide precise guidance for clinical decision-making.

[0033] 5. Kit components: 96-well ELISA plate coated with UCHL1 antibody; diluent for standards and UCHL1 detection samples; enzyme-labeled antibody working solution, washing buffer, substrate chromogenic solution and stop solution; detailed experimental instructions.

[0034] This invention can be used for preoperative UCHL1 level detection, screening of potential beneficiaries of Atezo / Bev treatment, and providing guidance on efficacy prediction and risk stratification to optimize HCC immunotherapy regimens.

[0035] Example 1

[0036] 1. Study Subjects: This study included 85 HCC patients who received Atezo / Bev treatment at Zhongshan Hospital affiliated with Fudan University between January 2020 and December 2023. All patients provided written informed consent. Inclusion and exclusion criteria were as follows:

[0037] (a) Underwent radical resection and pathological diagnosis of hepatocellular carcinoma;

[0038] (b) Has not received any other cancer-related treatments prior to surgery;

[0039] (c) No history of other malignant tumors;

[0040] (d) Possesses complete clinical pathology data and follow-up information;

[0041] (e) Patients who have been followed up for 1 year post-surgery.

[0042] 2. Research Methods:

[0043] 2.1 Surgically resected tumor tissue specimens were collected from 85 patients with hepatocellular carcinoma and subjected to frozen section analysis. Peripheral venous blood was also collected from the patients to separate and obtain plasma samples. Postoperative follow-up included complete blood count, liver function tests, serum tumor marker detection, abdominal ultrasound, and chest X-ray. Overall postoperative survival was defined as the time interval from the date of surgery to death or the last follow-up visit.

[0044] 2.2 Statistical Methods: Continuous variables were expressed as means (SDs) with standard deviation or medians (IQRs) with interquartile range. Student's t-test or Mann-Whitney U test was used for continuous variables, and Pearson's chi-square test or Fisher's exact test was used for categorical variables. The Kolmogorov-Smirnov test was used to verify normality. Odds ratios (ORs) were calculated using logistic regression for 95% confidence intervals (CIs). Receiver operating characteristic (ROC) curves were used to determine the optimal cutoff values ​​for variables predicting the efficacy and survival outcomes of Atezo / Bev treatment. Survival analysis was performed using Kaplan-Meier curves and the log-rank test. Univariate Cox regression analysis was used to initially analyze risk factors for AAEs; variables statistically significant (p<0.2) in the univariate Cox analysis were included in multivariate Cox regression to identify independent risk factors. Multiple imputation was used to handle missing data. P<0.05 was considered statistically significant.

[0045] 2.3 Peripheral circulation UCHL1 level determination: Peripheral venous blood was collected from enrolled patients preoperatively using an anticoagulant blood collection tube (purple tip). Approximately 2 ml of peripheral blood was collected, centrifuged at 4°C for 20 min, and the supernatant was collected and incubated at 37°C for 40 min. 350 μl of 1× washing buffer was added, and the mixture was allowed to stand for 15 seconds before being discarded and the plate was dried. The mixture was washed 5 times. 50 μl each of CitH3 primary antibody working solution and deionized water were added, and the mixture was reacted at 37°C for 20 min. The plate was washed as before.

[0046] Add 100 μl of enzyme-labeled antibody working solution and react at 37°C for 10 minutes. Wash the plate as before. Add 100 μl of substrate working solution and incubate at 37°C in the dark for 15 minutes. Then add 100 μl of stop solution and mix well. After 1 minute, place the plate in a microplate reader, read the OD value, and calculate the concentration.

[0047] 3. Research Results:

[0048] TALENTop (NCT04649489) was a neoadjuvant trial in patients with hepatocellular carcinoma (HCC) comparing the efficacy of perioperative Atezolizumab (Atezo) / Bevacizumab (Bev) combined with surgical resection versus standard systemic Atezolizumab / Bev therapy. Based on blood samples from this cohort of patients, we investigated whether UCHL1 could be used to predict prognostic outcomes and treatment response in HCC patients receiving Atezolizumab / Bev therapy.

[0049] In this prospective cohort, a total of 85 patients were closely monitored and underwent multiple blood tests and imaging evaluations during a follow-up period of mean (±SD) 13.03 (±0.85) months. Of all patients, 30 were sensitive to Atezo / Bev, while 55 were resistant to Atezo / Bev.

[0050] Most patients resistant to Atezo / Bev showed elevated UCHL1 levels after treatment, while plasma UCHL1 levels in treatment responders typically decreased after treatment. Figure 1 AB). Using the maximum selection log-rank statistical method, the optimal cutoff value for UCHL1 was determined to be 0.815 ng / mL, which can be used to predict the response of HCC patients to Atezo / Bev treatment. The sensitivity and specificity of this cutoff value were 0.85 and 0.80, respectively, and the area under the curve (AUC) was 0.85 ( ). Figure 1 C). In patients with high UCHL1 levels (≥0.815 ng / mL), 88.68% (47 / 53) presented with SD / PD, while in patients with low UCHL1 levels (<0.815 ng / mL), 75.00% (24 / 32) achieved CR / PR. Figure 1 C). Furthermore, the optimal cutoff value for UCHL1 was determined to be 1.188 ng / mL to predict overall survival (OS) in HCC patients. At this cutoff value, the sensitivity and specificity were 0.71 and 0.79, respectively. Figure 1 D). Among patients with high UCHL1 levels (≥1.188 ng / mL), 46.15% (12 / 26) died, while among patients with low UCHL1 levels (<1.188 ng / mL), 91.53% (65 / 59) remained alive. Figure 1 D).

[0051] LASSO (minimum absolute contraction and selection operator) regression analysis and multivariate Cox regression model showed that baseline plasma UCHL1 levels (HR 5.75, 95% CI 1.37–24.14, p = 0.017) were independent risk factors for overall survival (OS) and progression-free survival (PFS). Figure 2 A). Kaplan-Meier survival curves showed that patients with elevated plasma UCHL1 levels had significantly lower overall survival (OS) and progression-free survival (PFS) than patients with lower UCHL1 levels. Figure 2 B).

[0052] In summary, UCHL1 plays a crucial role in the onset, progression, and outcome of HCC. Baseline peripheral circulation UCHL1 markers can effectively predict the efficacy of immunotherapy before treatment and accurately identify high-risk patients with poor prognosis. Therefore, enhanced perioperative management and more rigorous follow-up strategies for these patients should be considered, which will help improve the efficacy of combined immunotherapy and overall prognosis in HCC patients, providing a more scientific, objective, and detailed basis for personalized precision treatment of HCC.

[0053] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Use of UCHL1 in the preparation of a assay product for predicting the efficacy and prognosis of atezolizumab combined with bevacizumab in hepatocellular carcinoma.

2. The use according to claim 1, characterized in that, The testing products include reagent kits.

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