Application of TTK in prognosis prediction of thyroid undifferentiated cancer

Abstract

The invention discloses application of TTK in prognosis prediction of an undifferentiated thyroid cancer, and provides a method for accurately evaluating prognosis of a patient with the undifferentiated thyroid cancer by calculating a predicted value through a formula according to the expression quantity of TTK in tumor tissues and whether distant metastasis exists in tumors. The technical operation is simple and convenient, and a basis can be provided for whether more positive measures need to be taken clinically to treat the patient suffering from the thyroid undifferentiated cancer, so that the prognosis of the patient suffering from the thyroid undifferentiated cancer is helped to be improved, unnecessary medical expenditure is avoided, and the defects in the prior art are overcome.

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, and the median overall survival of patients is often only a few months. Currently, histopathological morphological examination is regarded as 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 and the lack of effective prognostic markers mean that clinicians lack reliable laboratory test basis when deciding whether to take more aggressive treatment measures for patients. This situation may not only lead to ATC patients who have the opportunity to have a better prognosis to give up treatment due to misdiagnosis, but also make some patients with extremely poor prognosis bear the burden of overtreatment, which in turn increases the economic pressure on families and society.

[0004] In previous research and exploration, 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 predicts the prognosis of ATC based on changes in the ATC immune microenvironment. 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 solve the above problems, the present invention provides a method for accurately evaluating the prognosis of patients with anaplastic thyroid cancer by calculating the predicted value through a formula based on the TTK expression level in tumor tissue and whether the tumor has distant metastasis. The technology of the present invention is simple to operate and can provide a basis for whether more active measures should be taken in the clinic to treat patients with anaplastic thyroid cancer, thereby helping to improve the prognosis of patients with anaplastic thyroid cancer and avoid unnecessary medical expenses, making up for the shortcomings of the existing technology.

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

[0007] Furthermore, the predicted value of the prognosis of the ATC patient is calculated by formula 1, and when the predicted value is higher than the critical value, the ATC patient is evaluated as having a highly poor prognosis;

[0008] The 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, and 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 predicted value is >0.7, it indicates that the predicted result is that the overall survival of the ATC patient is less than 10 months, which is assessed as a highly poor prognosis.

[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, wherein the model calculates a prediction value by formula 1 based on the expression level of TTK in the tumor tissue of the ATC patient and whether the ATC patient has distant metastasis. If the prediction value is higher than the critical value, the patient with anaplastic thyroid cancer is evaluated as having a highly poor prognosis.

[0016] The 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; the M is the distant metastasis coefficient, when the anaplastic thyroid cancer patient has distant metastasis, M takes a value of 1, and when the anaplastic thyroid cancer patient does not have distant metastasis, M takes a value of -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 the tumor tissue of ATC cancer patients, and combines whether the patient has distant metastasis, and calculates the patient's prognosis prediction value through a formula. When the prediction value is higher than the critical value, the patient is evaluated as having a highly poor prognosis, thereby providing a more accurate ATC prognosis prediction method. The patented technology of the present invention is easy to operate and can provide a basis for whether the clinic should take more active measures to treat ATC patients, thereby helping to improve the prognosis of ATC patients and avoid unnecessary medical expenses, making up for the shortcomings of the existing technology. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 TTK immunohistochemical detection results of ATC tumor tissue in the embodiment of the present invention; Figure 1 A is 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 expression of TTK 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 or absence of distant metastasis in the embodiments of the present invention; Figure 2 A shows that compared with ATC patients with TTK expression >0.6, those with TTK expression ≤0.6 had significantly longer OS (P=0.009); Figure 2 B shows that compared with ATC patients with distant metastasis (M1), those without distant metastasis (M0) had 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 The receiver operating characteristic curve (ROC) of different methods in the embodiments of the present invention for predicting whether the OS of ATC patients is less than 10 months; 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 or absence 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 of ATC patients in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the 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 creative work are 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 only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0030] Explanation of terms in this invention:

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

[0032] The "TNM staging" was proposed by French researcher Pierre Denoix between 1943 and 1952. Subsequently, it was gradually standardized by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC), and the first edition of the "TNM Classification of Malignant Tumors" manual was officially published in 1968. The TNM staging is currently the standard method for clinicians and medical researchers to stage malignant tumors.

[0033] In the "TNM staging" system: T (the first letter of the word "tumor") refers to the primary tumor. 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 drawn 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 AJCC (8th edition).

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

[0035]

[0036]

[0037] Example

[0038] In this case, immunohistochemistry and image analysis techniques were used to detect the expression of TTK in tumor tissues of ATC patients. The overall survival (OS) was predicted using the TTK expression and various clinical data of the patients, and 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 grade information and paraffin-embedded tumor tissue blocks remaining after clinical pathological examination were collected.

[0040] 2. Paraffin blocks were routinely sectioned and then TTK immunohistochemically stained. 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), phosphate buffer (ZLI-9061) were all purchased from Zhongshan Jinqiao Company (China). The staining steps were carried out according to the reagent instructions.

[0041] 3. Immunohistochemical staining sections were scanned using a pathology whole-slice scanning system (SQS12P, Shenzhen Shengqiang, China).

[0042] 4. In each immunohistochemically stained slide scan, randomly select 5 images of ATC tumor tissue areas.

[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. Further, the predicted value of ATC patients was calculated according to the formula: predicted value = TTK expression + M × 0.1. Among them, M was recorded as 1 if there was distant metastasis, and M was recorded as -1 if there was no distant metastasis. When the predicted value > 0.7, the predicted result was that the OS of ATC patients was < 10 months; when the predicted value ≦ 0.7, the predicted result was that the OS of ATC patients was ≧ 10 months.

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

[0046] The results are as follows:

[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: The quantitative data in the table are expressed as median (25% and 75% quantiles); the qualitative data are expressed as n value (percentage).

[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. The results of COX regression analysis showed that TTK expression >0.6 or ≤0.6 in ATC patients and the presence of distant metastasis were two independent risk factors affecting OS in ATC patients ( Figure 3 ). Among them, the hazard ratio (HR) of TTK expression was 4.09, and its 95% confidence interval (CI) was 1.17-14.28 (P=0.027). The HR of the factor 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 is less than 10 months based on whether the tumor has 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 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, indicating 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 TTK expression in tumor tissue, whether the patient had distant metastasis, and the predicted value of the present invention. The areas under the ROC curves (AUC) 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 use of whether the patient has distant metastasis to predict the OS of ATC patients, the AUC predicted by TTK expression increased but had no significant difference (0.881 vs 0.804, P = 0.551); the AUC predicted by the prediction value of the present invention increased further and had a significant difference (0.929 vs 0.881, P = 0.015). This result suggests that among the three prediction methods, the prediction effect of the present invention is the best.

[0066] 7. The age of ATC patients, primary tumor condition (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 prediction 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 is an example ( 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, the patient has a 95% probability of having an OS of less than 10 months.

[0067] The above results show that the method described in the present invention can be used to quickly and effectively evaluate the prognosis of ATC patients. Specifically, the expression of TTK protein in tumor tissue is obtained by immunohistochemistry and image analysis technology, and the predicted value of ATC patients is calculated by the formula: "Prediction value = TTK expression + M × 0.1" (M is calculated as 1 for distant metastasis and -1 for no distant metastasis). When the prediction value > 0.7, the prediction 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, especially introduces TTK, i.e., threonine and tyrosine kinase, for the first time, and evaluates the prognosis of anaplastic thyroid cancer in combination with whether the patient has distant metastasis, thereby constructing a more accurate prognosis prediction system. The patented technology of the present invention is easy to operate and can provide a basis for whether more active measures should be taken in the clinic to treat ATC patients, thereby helping to improve the prognosis of ATC and avoid unnecessary medical expenses, making up for the shortcomings of the existing technology.

[0069] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, 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, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0070] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of TTK 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 the critical value, the patient with anaplastic thyroid cancer is evaluated as having a highly poor prognosis. The 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 anaplastic thyroid cancer patient; and M is the distant metastasis coefficient. When the anaplastic thyroid cancer patient has distant metastasis, M takes a value of 1, and when the anaplastic thyroid cancer patient 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 prediction 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 the 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.

6. A model for predicting the prognosis of anaplastic thyroid cancer, characterized in that: The model calculates the predicted value of the prognosis of anaplastic thyroid cancer patients by formula 1 based on the expression of TTK in the tumor tissue of anaplastic thyroid cancer patients and whether the anaplastic thyroid cancer patients have distant metastasis. If the predicted value is higher than the critical value, the anaplastic thyroid cancer patient is evaluated as having a highly poor prognosis. The 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; the M is the distant metastasis coefficient, when the anaplastic thyroid cancer patient has distant metastasis, M takes a value of 1, and when the anaplastic thyroid cancer patient does not have distant metastasis, M takes a value of -1; The critical value of the predicted value is 0.7.

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