Application of TTK in the prognosis prediction of anaplastic thyroid cancer

Abstract

This invention discloses the application of TTK in predicting the prognosis of anaplastic thyroid cancer. It provides a method for accurately assessing the prognosis of anaplastic thyroid cancer patients by calculating a predicted value based on the expression of TTK in tumor tissue and the presence of distant metastasis. This method is simple to use and can provide a basis for clinically determining whether more proactive treatment measures should be taken for anaplastic thyroid cancer patients. This helps improve the prognosis of anaplastic thyroid cancer patients and avoid unnecessary medical expenses, thus addressing the shortcomings of existing technologies.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of TTK in the prognosis prediction of anaplastic thyroid cancer. Background Art

[0002] Anaplastic thyroid carcinoma (ATC), also known as anaplastic thyroid cancer, is an extremely aggressive and highly malignant tumor, with patients' median overall survival often measured in only a few months. Currently, histopathological morphological examination is considered the gold standard for the diagnosis of ATC, and surgical resection is the mainstream treatment for ATC. Although in recent years, small molecule targeted drugs such as dabrafenib and trametinib, as well as immunosuppressants such as PD-1 (programmed cell death-1) and PDL1 (programmed cell death-ligand 1) monoclonal antibodies have been tried in the treatment of ATC, unfortunately, these treatment options have only shown significant efficacy in some ATC patients, and the 2-year survival rate of ATC is still hovering at a low level below 50%.

[0003] The highly malignant nature of ATC, coupled with the lack of effective prognostic markers, leaves clinicians without reliable laboratory testing to inform their decisions about aggressive treatment. This situation not only leads to misdiagnosis and forgoing treatment for ATC patients with a better prognosis, but also results in the burden of overtreatment for some patients with a very poor prognosis, further exacerbating the financial burden on families and society.

[0004] In previous research explorations, the inventors have found that the increase in the proportion of double-positive exhausted T cells of TIM3 (T cell immunoglobulin domain and mucin domain 3) and CD8 (cluster of differentiation 8) in ATC tumor tissue is an important indicator for predicting poor prognosis of ATC (this discovery has been patented, patent number: CN202310797030.9, a marker combination for predicting the prognosis of anaplastic thyroid cancer and its application). In addition, there are currently no similar biomarkers that can be used for ATC prognosis prediction. The above-mentioned authorized invention is based on changes in the ATC immune microenvironment to predict the prognosis of ATC. To this end, the present invention will comprehensively consider the protein expression status of ATC tumor cells and the clinical characteristics of patients to jointly predict their prognosis. Further improve the accuracy of ATC prognosis prediction, thereby providing a laboratory basis for clinical improvement of ATC prognosis and avoiding unnecessary medical expenses. Summary of the Invention

[0005] To address these issues, the present invention provides a method for accurately assessing the prognosis of patients with anaplastic thyroid cancer by calculating a predicted value based on the expression of TTK in tumor tissue and the presence of distant metastasis. This method is simple to use and can provide a basis for clinically determining whether more proactive treatment measures should be taken for patients with anaplastic thyroid cancer. This method, therefore, helps improve the prognosis of patients with anaplastic thyroid cancer and avoid unnecessary medical expenses, thus addressing the shortcomings of existing technologies.

[0006] In a first aspect, the present invention provides the use of TTK in preparing a product for predicting the prognosis of anaplastic thyroid cancer.

[0007] Furthermore, the predicted value of the prognosis of the ATC patient is calculated by formula 1. When the predicted value is higher than the critical value, the ATC patient is assessed as having a highly poor prognosis.

[0008] Formula 1: Predicted value = TTK expression level + M × 0.1; wherein the TTK expression level is the expression level of TTK in the tumor tissue of the ATC patient; and M is the distant metastasis coefficient. If the ATC patient has distant metastasis, M is 1; if the ATC patient does not have distant metastasis, M is -1.

[0009] Furthermore, the critical value of the predicted value is 0.7.

[0010] Furthermore, when the prediction value is >0.7, it indicates that the predicted result is that the overall survival of the ATC patient is less than 10 months, and the prognosis is assessed to be highly poor.

[0011] Furthermore, obtaining the TTK expression level comprises the following steps:

[0012] Immunohistochemistry was used to detect TTK expression in tumor tissues of ATC patients;

[0013] Microscopic images of the tumor tissue area in each immunohistochemically stained section were collected, and at least 5 images were collected for each section;

[0014] Image analysis technology was used to analyze the average optical density value of the TTK positive signal in each image, and the average of the average optical density values of all images was taken as the expression level of TTK in the immunohistochemical staining section.

[0015] In a second aspect, the present invention provides a model for predicting the prognosis of ATC. The model uses the expression level of TTK in the tumor tissue of the ATC patient and whether the ATC patient has distant metastasis to calculate a prediction value using Formula 1. If the prediction value is higher than a critical value, the patient with anaplastic thyroid cancer is assessed to have a highly poor prognosis.

[0016] Formula 1: Predicted value = TTK expression level + M × 0.1; wherein the TTK expression level is the TTK expression level in the tumor tissue of the anaplastic thyroid cancer patient; M is the distant metastasis coefficient, when the anaplastic thyroid cancer patient has distant metastasis, M is 1, and when the anaplastic thyroid cancer patient does not have distant metastasis, M is -1;

[0017] The critical value of the predicted value is 0.7.

[0018] The above technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0019] The present invention detects the expression of TTK in tumor tissues of ATC cancer patients and, in combination with the presence of distant metastases, calculates the patient's prognostic value using a formula. When the predicted value is above a critical value, the patient is assessed as having a highly poor prognosis, thereby providing a more accurate ATC prognosis prediction method. The patented technology of the present invention is simple to operate and can provide a basis for whether more proactive measures should be taken in the treatment of ATC patients, thereby helping to improve the prognosis of ATC patients and avoid unnecessary medical expenses, thus addressing the shortcomings of existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0022] Figure 1 The results of TTK immunohistochemistry detection of ATC tumor tissue in the embodiment of the present invention; Figure 1 A shows the immunohistochemical staining results of 6 ATC tumor tissues, showing diffuse moderate to strong positive expression of TTK (scale bar = 100 μm); Figure 1 B: Compared with ATC patients in the OS≧10-month group, the TTK expression level in the OS<10-month group was significantly increased (P=0.000).

[0023] Figure 2 The comparison results of the overall survival (OS) of ATC patients with different TTK expression levels and the presence of distant metastasis in the examples of the present invention are as follows; Figure 2 A shows that compared with ATC patients with TTK expression >0.6, those with TTK expression ≤0.6 had significantly prolonged OS (P=0.009); Figure 2 B shows that compared with ATC patients with distant metastasis (M1), those without distant metastasis (M0) had a significantly prolonged OS (P=0.031).

[0024] Figure 3 This is the COX regression analysis result in the embodiment of the present invention.

[0025] Figure 4 Receiver operating characteristic curves (ROC) for predicting whether the OS of ATC patients is less than 10 months using different methods in the embodiments of the present invention; Figure 4 A is the ROC using the TTK expression level in tumor tissue of ATC patients as a predictive indicator, Figure 4 B is the ROC using the presence of distant metastasis in ATC patients as a predictive indicator, Figure 4 C is the ROC using the predicted value of the present invention as the prediction index.

[0026] Figure 5 This is a nomogram for predicting OS in ATC patients according to an embodiment of the present invention. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. If there are no corresponding national standards, the methods are carried out in accordance with general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0030] Explanation of terms in this invention:

[0031] TTK refers to threonine and tyrosine kinase (TTK).

[0032] The TNM staging system was proposed by French researcher Pierre Denoix between 1943 and 1952. It was subsequently standardized by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC), culminating in the publication of the first edition of the "TNM Classification of Malignant Tumors" manual in 1968. The TNM staging system is currently the standard method for malignant tumor staging by clinicians and medical researchers.

[0033] In the "TNM staging" system: T (the first letter of the word "tumor") refers to the primary tumor site. N (the first letter of the word "node") refers to the involvement of regional lymph nodes. M (the first letter of the word "metastasis") refers to distant metastasis. Specific stages are divided based on the combination (grouping) of the three TNM indicators. Due to the extremely high malignancy of ATC, all cases are classified as stage IV. Table 1 below shows the TNM staging criteria for anaplastic thyroid carcinoma proposed by the AJCC (8th edition).

[0034] Table 1 TNM staging criteria for anaplastic thyroid cancer

[0035]

[0036]

[0037] Example

[0038] In this study, immunohistochemistry and image analysis were used to detect TTK expression in tumor tissues of ATC patients. TTK expression and various clinical data of the patients were then used as indicators to predict overall survival (OS). The prediction effects of various methods were compared. The details are as follows:

[0039] 1. Clinically confirmed ATC cases were enrolled, and their gender, age, TNM stage, and paraffin-embedded tumor tissue blocks remaining after clinical pathological examination were collected.

[0040] 2. Paraffin blocks were routinely sectioned and then subjected to TTK immunohistochemical staining. The TTK primary antibody was purchased from Abcam (ab219068, USA, working concentration 10 μg / mL). pH 8.0 antigen retrieval solution (ZLI-9072), primary antibody diluent (ZLI-9029), secondary antibody and DAB color development kit (PV-8000), and phosphate buffer (ZLI-9061) were all purchased from Zhongshan Jinqiao (China). Staining procedures were performed according to the reagent instructions.

[0041] 3. Immunohistochemically stained sections were scanned using a pathology whole-slide scanning system (SQS12P, Shenzhen Shengqiang, China).

[0042] 4. Randomly select 5 images of ATC tumor tissue areas in each immunohistochemically stained slide scan.

[0043] 5. Image J software (National Institutes of Health, USA) was used to analyze the average optical density (AOD) of the TTK-positive signal in each image, and the average AOD of the five images was calculated as the TTK expression level of the case.

[0044] 6. The predicted value for ATC patients was further calculated using the formula: predicted value = TTK expression level + M × 0.1. M is assigned a value of 1 for patients with distant metastasis and -1 for those without distant metastasis. When the predicted value is > 0.7, the predicted OS for ATC patients is < 10 months; when the predicted value is ≤ 0.7, the predicted OS for ATC patients is ≥ 10 months.

[0045] 7. Data were analyzed and plotted using R language (University of Auckland, New Zealand), MedCalc software (MedCalc, Belgium), and GraphPadPrism software (GraphPad Software, USA). Differences between the two quantitative data groups were analyzed using the Mann-Whitney U test, and differences between the qualitative data were analyzed using the chi-square test. Correlations between the two data groups were analyzed using the Spearman correlation test. Cox regression analysis, 2×2 contingency table analysis, and receiver operator characteristic (ROC) curves were used to evaluate the effectiveness of various methods in predicting OS in ATC patients. A nomogram was further constructed to predict OS in ATC patients. A P value < 0.05 was considered statistically significant.

[0046] Here are the results:

[0047] A total of 19 ATC cases were enrolled, including 12 females and 7 males, with a median age of 65 years and a median overall survival (OS) of 11 months. The information of the enrolled patients is as follows (Table 2).

[0048] Table 2 Information of ATC patients enrolled

[0049]

[0050]

[0051] Note: Quantitative data in the table are expressed as medians (25% and 75% quantiles); qualitative data are expressed as n values (percentages).

[0052] 2. Image analysis and statistical analysis results showed that TTK was diffusely moderately to strongly positively expressed in ATC tumor tissues ( Figure 1 A). Compared with ATC patients with OS ≥ 10 months, the expression of TTK in tumor tissues of ATC patients with OS < 10 months was significantly increased (0.68 vs 0.53, P = 0.000) ( Figure 1 B).

[0053] 3. Further analysis results showed that compared with ATC patients with tumor tissue TTK expression > 0.6, ATC patients with TTK expression ≤ 0.6 had significantly longer OS (7 vs 15, P = 0.009) ( Figure 2 A). Compared with ATC patients with distant metastasis (M1), ATC patients without distant metastasis (M0) had significantly longer OS (9 vs 16, P = 0.031) ( Figure 2 B).

[0054] 4. COX regression analysis results showed that TTK expression >0.6 or ≤0.6 and the presence of distant metastasis were two independent risk factors affecting OS in ATC patients ( Figure 3 The hazard ratio (HR) for TTK expression was 4.09, with a 95% confidence interval (CI) of 1.17-14.28 (P = 0.027). The HR for the presence of distant metastasis was 5.69 (95% CI 1.48-6.11, P = 0.012).

[0055] The results of the 5.2×2 contingency table analysis showed that the sensitivity, specificity, total coincidence rate and kappa value of predicting whether the OS of ATC patients was less than 10 months based on whether the tumor had distant metastasis were 85.7%, 75.0%, 79.0% and 0.57, respectively (Table 3, Table 6). The sensitivity, specificity, total coincidence rate and kappa value predicted by TTK expression level were 71.4%, 83.3%, 79.0% and 0.55, respectively (Table 4, Table 6). The sensitivity, specificity, total coincidence rate and kappa value predicted by the method of the present invention were 71.4%, 100%, 89.5% and 0.76, respectively (Table 5, Table 6). The results show that the total coincidence rate and kappa value predicted by the present invention are the highest among the three methods, suggesting that the prediction effect of the present invention is the best among the three.

[0056] Table 3 2×2 contingency table of the presence or absence of distant metastasis to predict OS in ATC patients

[0057]

[0058] Table 4 2×2 contingency table of TTK expression level predicting OS of ATC patients

[0059]

[0060] Table 5 2×2 contingency table for predicting OS of ATC patients according to the present invention

[0061]

[0062] Table 6 Comparison of the three methods in predicting OS in ATC patients

[0063]

[0064]

[0065] 6. The ROC curves were drawn to predict whether the OS of ATC patients was less than 10 months based on the expression of TTK in tumor tissue, the presence of distant metastasis in patients, and the predicted value of the present invention. The areas under the ROC curves (AUCs) of the three methods were 0.881, 0.804, and 0.929, respectively (Table 6, Figure 4 Further Delong test results showed that compared with the presence of distant metastasis to predict ATC patient OS, the AUC for prediction using TTK expression increased but did not differ significantly (0.881 vs 0.804, P = 0.551). The AUC for prediction using the prediction value of the present invention increased further and showed a significant difference (0.929 vs 0.881, P = 0.015). This result suggests that the present invention has the best prediction effect among the three prediction methods.

[0066] 7. The age of ATC patients, primary tumor status (T), lymph node metastasis (N) and the predicted value of the present invention were included in the nomogram to predict OS ( Figure 5 ). The results showed that the predicted value of the present invention was an independent risk factor affecting the OS of ATC patients (P<0.001), but T (P=0.34), N (P=0.14) and patient age (P=0.17) were not independent risk factors affecting the OS of ATC patients. The following examples are given ( Figure 5 ): A certain ATC patient was clinically diagnosed with T2 (17.12 points), N1 (25.82 points), and was 65 years old (7.72 points). The predicted value evaluated by the method of the present invention was 0.7 (62.70 points). The total score = 17.12 + 25.82 + 7.72 + 62.70 = 113.36. The probability that the patient's OS exceeds 10 months is only about 0.05 (5%), that is, there is about a 95% probability that the patient's OS is less than 10 months.

[0067] The above results show that the method described in the present invention can be used to quickly and effectively assess the prognosis of ATC patients. Specifically, immunohistochemistry and image analysis techniques are used to obtain the expression of TTK protein in tumor tissue. Combined with the presence of distant metastasis in the patient, the predicted value of ATC patients is calculated using the formula: "Predicted value = TTK expression + M × 0.1" (M is calculated as 1 for distant metastasis and -1 for no distant metastasis). When the predicted value is > 0.7, the predicted result is that the OS of the ATC case is less than 10 months, which is a highly poor prognosis.

[0068] In summary, the present invention comprehensively considers the protein expression status of ATC tumor cells, particularly the introduction of TTK, a threonine and tyrosine kinase, for the first time, and simultaneously assesses the prognosis of anaplastic thyroid cancer in combination with the presence of distant metastasis in the patient, thereby constructing a more accurate prognostic prediction system. The patented technology of the present invention is simple to operate and can provide a basis for whether more proactive measures should be taken in the treatment of ATC patients, thereby helping to improve ATC prognosis and avoid unnecessary medical expenses, thus addressing the shortcomings of existing technologies.

[0069] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in a range format is only for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention; therefore, the range description should be considered to have specifically disclosed all possible subranges and single numerical values within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range.

[0070] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of reagents for detecting TTK protein expression in the preparation of prognosis prediction products for anaplastic thyroid cancer.

2. The use according to claim 1, characterized in that The predicted value of the prognosis of the patient with anaplastic thyroid cancer is calculated by formula 1. When the predicted value is higher than a critical value, the patient with anaplastic thyroid cancer is assessed as having a highly poor prognosis. Formula 1: predicted value = TTK expression level + M × 0.1; wherein the TTK expression level is the expression level of TTK in tumor tissue of patients with anaplastic thyroid cancer; and M is the distant metastasis coefficient. When the patient with anaplastic thyroid cancer has distant metastasis, M takes a value of 1; when the patient with anaplastic thyroid cancer does not have distant metastasis, M takes a value of -1.

3. The use according to claim 2, characterized in that The critical value of the predicted value is 0.

7.

4. The use according to claim 2, characterized in that When the predicted value is >0.7, the predicted result is that the overall survival of the patient with anaplastic thyroid cancer is less than 10 months, which is assessed as a highly poor prognosis.

5. The use according to claim 2, characterized in that The acquisition of the TTK expression level comprises the following steps: Immunohistochemistry was used to detect TTK expression in tumor tissues of patients with anaplastic thyroid cancer; Collect microscopic images of the tumor tissue area in immunohistochemically stained sections, and collect at least 5 images for each section; Image analysis technology was used to analyze the average optical density value of the TTK-positive signal in each image, and the average of the average optical density values of all images was taken as the expression level of TTK in the tumor tissue of the patient with anaplastic thyroid cancer.

Images