Application of serum TERT protein as marker for early diagnosis and prognosis evaluation of resectable non-small cell lung cancer

Abstract

The invention relates to application of serum TERT as a marker for early diagnosis and prognosis evaluation of resectable non-small cell lung cancer, and belongs to the field of medical health. The invention discloses application of serum TERT as a marker for early diagnosis of resectable non-small cell lung cancer. The invention discloses application of serum TERT as a marker for prognosis evaluation of resectable non-small cell lung cancer. The invention aims to evaluate the preoperative or postoperative circulating TERT level of healthy donors and resectable NSCLC patients and evaluate the diagnosis and prognosis values of the healthy donors and the resectable NSCLC patients, and researches prove that the circulating TERT is a promising biomarker. Based on the research, the invention provides a kit which can be used for early diagnosis of the resectable NSCLC and accurate evaluation of patient prognosis in a rapid, non-invasive and quantitative manner.

Description

Technical Field

[0001] The present invention relates to the application of serum TERT protein as a marker for early diagnosis and prognosis evaluation of resectable non-small cell lung cancer, and belongs to the field of medical care and health. Background Art

[0002] Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide, with the majority of patients presenting at metastatic or locally advanced stages at the time of diagnosis, but 25% to 30% (including early-stage and oligometastatic NSCLC) have resectable disease. For these patients, complete surgical resection offers the best chance of cure. However, 30-75% of these patients eventually relapse, often systemic, equivalent to stage IV disease, with a short post-relapse survival (PRS) of 8-14 months. Therefore, there is an urgent need to develop clinically available biomarkers to diagnose relapse and progressive disease (PD) as easily as possible.

[0003] Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and contributes to the maintenance of telomere length and cellular self-renewal. This function is of great significance for cancer cells, which have acquired the ability to bypass senescence by maintaining telomere length, primarily through the activation of telomerase. Telomerase dysregulation is a ubiquitous feature of human cancer, with more than 90% of malignancies showing upregulated expression of this enzyme. As a determinant of telomerase activity, increased TERT expression has been consistently shown to be an essential requirement for cellular transformation and is considered a key event in cellular immortalization and a hallmark of tumorigenesis. Therefore, TERT upregulation and telomerase activation have attracted much attention due to their potential as diagnostic and therapeutic targets.

[0004] A cancer biomarker is any measurable molecular indicator of cancer initiation, progression, transformation, and outcome. Emerging evidence supports the potential of TERT as a cancer biomarker due to its critical role in telomere maintenance. 1 For example, TERT C228T and C250T mutations found in a range of cancers are associated with reduced overall survival (OS), suggesting that they could serve as genomic cancer biomarkers. 2,3 Nevertheless, most biomarker-related studies have focused on TERT promoter mutations rather than its circulating protein, which is more feasible and convenient for biosensor development. Summary of the invention

[0006] In view of the detection-related problems existing in the early diagnosis and prognosis evaluation of resectable non-small cell lung cancer at this stage, this application aims to evaluate the circulating TERT level in healthy donors and resectable NSCLC patients before or after surgery, and evaluate its diagnostic and prognostic value for resectable NSCLC. Studies have confirmed that circulating TERT is a promising biomarker. Based on this study, the present invention provides a kit that can be used for early diagnosis of resectable NSCLC and accurate assessment of patient prognosis in a rapid, non-invasive and quantitative manner.

[0007] The technical solution of this application is as follows:

[0008] The first purpose of the present application is to protect the use of serum TERT as a marker for early diagnosis of resectable non-small cell lung cancer.

[0009] The second purpose of this application is to protect the use of serum TERT as a marker for prognosis assessment of resectable non-small cell lung cancer.

[0010] The third purpose of the present application is to protect a kit for early diagnosis of resectable non-small cell lung cancer, which comprises the serum TERT described in claim 1.

[0011] The fourth object of the present application is to protect a kit for evaluating the prognosis of resectable non-small cell lung cancer, wherein the kit comprises the serum TERT as described in claim 2.

[0012] The present application further provides a detection method of the above kit, which is characterized by comprising the following steps:

[0013] (1) Separating serum;

[0014] (2) Determination of protein TERT concentration;

[0015] (3) enzyme-linked immunosorbent assay for circulating TERT expression levels;

[0016] (4) Data analysis

[0017] Beneficial effects of the present invention

[0018] 1. Serum circulating TERT expression levels are significantly increased in patients with resectable NSCLC

[0019] The present invention proposes for the first time that compared with healthy donors, the serum circulating TERT expression level in patients with resectable NSCLC is significantly increased, and ROC curve analysis shows that the AUC is 0.9.

[0020] 2. Serum circulating TERT expression levels are significantly increased in patients with early resectable NSCLC

[0021] Compared with healthy donors, TERT expression level was significantly increased in patients with early-stage resectable NSCLC, and ROC curve analysis showed an AUC of 0.89.

[0022] 3. TERT changes before and after resection were significantly correlated with overall survival (OS) and disease-free survival (DFS).

[0023] Compared with preoperatively, circulating TERT levels increased significantly after surgery. All patients were divided into high and low groups according to the median of preoperative and postoperative TERT changes (TERT changes). Compared with the low group, the high group had longer DFS and OS (p = 0.022, p = 0.046, respectively). In addition, TERT changes and lymph node metastasis were independent prognostic factors for DFS and OS. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of enrolled patients;

[0025] Figure 2 Graph of circulating TERT expression levels in NSCLC patients;

[0026] Figure 3 Graph showing the expression level of circulating TERT in early-stage NSCLC;

[0027] Figure 4 ROC curve of circulating TERT in the diagnosis of NSCLC patients;

[0028] Figure 5 ROC curve of circulating TERT in diagnosing early-stage NSCLC patients;

[0029] Figure 6 Graph of circulating TERT expression levels before and after resectable NSCLC;

[0030] Figure 7 The relationship between preoperative TERT level and OS and DFS in NSCLC;

[0031] Figure 8 The relationship between TERT level and OS and DFS after NSCLC surgery;

[0032] Fig. 9 The relationship between TERT changes before and after NSCLC surgery and OS and DFS. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available products.

[0034] Case Study: Application of Circulating TERT as a Novel Diagnostic and Prognostic Biomarker for Resectable NSCLC

[0035] 1. Experimental design

[0036] The experimental design was to collect serum samples from patients with NSCLC who were diagnosed with NSCLC at the Affiliated Tumor Hospital of Shandong First Medical University and healthy volunteers, and separate the serum. The expression of circulating TERT was detected by ELISA, and the difference in its expression between healthy volunteers and lung cancer was statistically analyzed, and its diagnostic efficacy was analyzed. TERT was quantitatively evaluated in patients who were followed up for 3 months after surgery. The prognostic value was evaluated by disease-free survival (DFS) and overall survival (OS).

[0037] 2. Patients and samples involved in the experiment

[0038] A total of 85 NSCLC patients were recruited from January 2017 to 2019 by the Affiliated Tumor Hospital of Shandong First Medical University. After screening, 69 patients were eligible for study participation. Finally, 69 NSCLC patients and 58 healthy donors were included. The patient enrollment flow chart is shown in the figure below. Figure 1 As shown. All patients were paired preoperatively and postoperatively (follow-up evaluation 3 months after surgery) and did not receive any anti-tumor treatment or suffer from any other endocrine, immune or metabolic diseases before peripheral blood collection. Healthy volunteers did not show any disease. Patients were followed up by telephone or clinical visits. The eighth edition of the UICC / AJCC TNM classification was used to stage the tumor, and the review of electronic medical records provided us with clinical and pathological data. Serum was separated by centrifuging the collected blood at 3000g for 10 minutes. The samples were then collected in centrifuge tubes and stored at -80°C for future use.

[0039] 3. Protein concentration detection

[0040] Serum protein concentration was determined according to the instructions of the BCA protein assay kit (Thermo Fisher, MA, USA). The absorbance of the protein sample was measured using a fluorescence microplate reader (Molecular Devices, CA, USA), corrected by blank absorbance, and then converted into protein concentration.

[0041] 4. Determination of circulating TERT levels in serum

[0042] The expression level of circulating TERT was measured using a human TERT ELISA kit (E-EL-H0706c, Elabscience, China). The experimental operation was performed according to the kit instructions and the sample was diluted according to the actual situation.

[0043] 4. Mathematical statistical analysis

[0044] Statistical analysis was performed using SPSS 26.0 (IBM Corp., NY, USA) and GraphPad Prism 9.0 (GraphPad, CA, USA). Disease-free survival was measured from the time of surgery to clinical or radiological progression or death from any cause. Overall survival (OS) was the time from the start of treatment to death from any cause. The normality of the distribution was checked using the Kolmogorov-Smirnov test. The comparison of continuous data was performed using parametric tests if they followed a normal distribution and expressed as mean ± standard deviation; if they did not follow a normal distribution, the nonparametric test was performed using the Mann-Whitney test and expressed as median and interquartile range. The differentially expressed TERT was determined by the receiver operating characteristic curve (ROC curve) and the area under the curve (AUC). The Wilcoxon signed-rank test was used for paired determinations. The log-rank test and univariate and multivariate Cox proportional hazards regression were used to analyze DFS and OS. P < 0.05 was considered statistically significant.

[0045] 5. Summary of case characteristics of NSCLC patients and their relationship with TERT expression levels

[0046] The clinical characteristics of NSCLC patients, including age, sex, smoking status, pathological type, stage, lymph node and distant metastasis, were analyzed in Table 1. Circulating TERT expression levels were not associated with these clinicopathological parameters.

[0047] Table 1 Summary of NSCLC patient characteristics and their relationship with TERT expression levels

[0048]

[0049]

[0050] 6. Differences in serum TERT levels between healthy volunteers and NSCLC patients

[0051] The serum TERT levels of NSCLC patients and healthy donors were measured by ELISA to determine whether circulating TERT can be used as a biomarker for diagnosing lung cancer. The results showed that the expression level of TERT was significantly decreased in NSCLC patients, and the difference was statistically significant (p<0.0001). Figure 2 Similarly, the serum TERT levels of early NSCLC patients and healthy donors were quantified to determine whether circulating TERT can be used as a biomarker for diagnosing early NSCLC. The results showed that the expression level of TERT was significantly decreased in early NSCLC patients, and the difference was statistically significant (p<0.0001). Figure 3 .

[0052] 7. ROC curve analysis of the diagnostic efficacy of serum TERT for NSCLC

[0053] The present invention further uses ROC curve analysis to detect the diagnostic efficacy of TERT for NSCLC. The ROC curve analysis shows that the AUC is 0.9. Figure 4 .

[0054] 8. Diagnostic efficacy of serum TERT for early NSCLC

[0055] The present invention further uses ROC curve analysis to detect the diagnostic efficacy of TERT for early NSCLC. The ROC curve analysis shows that the AUC is 0.89. Figure 5 .

[0056] 9. Differences in circulating TERT before and after NSCLC surgery

[0057] The results of ELISA showed that the circulating TERT level was significantly increased after surgery compared with that before surgery, and the difference was statistically significant (P<0.0001). Figure 6 .

[0058] 10. Relationship between TERT changes before and after NSCLC surgery and OS and DFS

[0059] The survival rate of preoperative TERT and postoperative TERT was analyzed, but no correlation was observed in either DFS or OS ( Figure 7 and 8 In addition, the preoperative and postoperative TERT changes of all patients (TERT changes) were further divided into high group and low group according to the median. Compared with the low group, the high group had longer DFS and OS, and the difference was statistically significant (p = 0.022, p = 0.046, respectively). Fig. 9 .

[0060] In addition, Table 2 performed univariate and multivariate regression analysis of each clinicopathological factor (including TERT changes) and DFS of NSCLC. In the multivariate analysis, TERT changes (p = 0.03) and lymph node metastasis (p = 0.01) were both independent prognostic factors for DFS. In the univariate analysis, lymph node metastasis (p = 0.02) was significantly associated with DFS, but not with TERT changes (p = 0.05). Similarly, Table 3 performed univariate and multivariate regression analysis of each clinicopathological factor (including TERT changes) and OS of NSCLC. TERT changes were also significant factors in the univariate (p = 0.01) and multivariate (p = 0.004) analyses, but in the multivariate analysis, lymph node metastasis was only associated with OS (p = 0.03).

[0061] Table 2 Univariate and multivariate analyses (Cox proportional hazards) of TERT and clinicopathological factors associated with DFS in NSCLC patients.

[0062]

[0063] Table 3 Univariate and multivariate analyses (Cox proportional hazards) of TERT and clinicopathological factors associated with OS in NSCLC patients.

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[0065]

Claims

1. Application of serum TERT as a marker for early diagnosis of resectable non-small cell lung cancer.

2. Application of serum TERT as a marker for prognostic assessment of resectable non-small cell lung cancer.

3. A kit for early diagnosis of resectable non-small cell lung cancer, characterized in that: The kit comprises the serum TERT according to claim 1.

4. A kit for evaluating the prognosis of resectable non-small cell lung cancer, characterized in that: The kit comprises the serum TERT according to claim 2.

5. The detection method of the kit according to claim 3 or 4, characterized in that: The following steps are involved: (1) Separating serum; (2) Determination of protein TERT concentration; (3) enzyme-linked immunosorbent assay for circulating TERT expression levels; (4) Data analysis

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