CN110373466B - Marker combination and application thereof in preparation of colorectal cancer diagnostic reagent - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to a marker combination, application thereof in preparing a colorectal cancer diagnostic reagent and a colorectal cancer diagnostic reagent/kit. The invention combines the markers RANTES and CEA in blood, and finds that the combination has extremely high association degree with colorectal cancer, the sensitivity can reach 98.9 percent, and the specificity is even as high as 100 percent; the kit also has high sensitivity and specificity for early colorectal cancer diagnosis, the sensitivity can even reach 100%, the kit is extremely rare in clinic, and the kit can be used for early detection of colorectal cancer, strives for time for patients, starts treatment as soon as possible, and improves the survival rate of the patients.

Description

A marker combination and its application in the preparation of colorectal cancer diagnostic reagents

technical field

The invention belongs to the field of biomedicine, and relates to a marker combination and its application in preparing a colorectal cancer diagnostic reagent and a colorectal tumor diagnostic reagent/kit.

Background technique

Global cancer statistics predict 18.1 million new cancer cases and 9.6 million deaths in 2018. Colorectal cancer (CRC) is a common malignant tumor of the digestive tract. The incidence and mortality of colorectal cancer in my country ranks third among all malignant tumors, and the incidence is still rising. Studies have shown that screening individuals with risk factors for colorectal cancer can diagnose cancer earlier, reduce mortality, and even reduce the incidence of colorectal cancer by detecting and removing adenomas. The United States began to promote early screening of colorectal cancer as early as the 1980s. At present, the 5-year survival rate of colorectal cancer patients in the United States has reached 90%, which shows that the development of colorectal cancer screening can reduce morbidity and mortality. have an important impact.

Colorectal cancer screening in China adopts a two-step screening method, that is, first screening to identify high-risk groups, and then performing diagnostic screening on high-risk groups. At present, screening methods commonly used in high-risk groups include fecal occult blood test (FOBT), medical history symptom high-risk factor questionnaire, etc., but these methods have low sensitivity. Colonoscopy remains the gold standard for diagnosing colorectal cancer, with the advantage of being accurate and biopsying for pathology. However, it has a series of shortcomings, such as the need to inject gas during the inspection process, which will increase the pressure in the intestine, easily cause perforation, and cause certain discomfort and pain to the patient during the whole process. Again, if it is anesthesia colonoscopy, there is still a greater risk of anesthesia. In addition, the inspection cost is high, which is time-consuming and cannot be promoted on a large scale. However, methods such as fecal occult blood are not sensitive and specific enough, and the methods for detecting methylation in fecal genes are relatively expensive and have not yet been popularized.

Currently, a variety of reporter markers are used in combination to screen for cancer. For example, Wang et al. report markers Tissue Polypeptide Specific Antigen (TPS) combined with CEA, CA199, CA125 and CA153 to diagnose metastatic breast cancer. The sensitivity predicted by TPS alone was 50.0%, and the combined sensitivity still did not reach 80.0% (W. Wang et al., The diagnostic value of serum tumor markers CEA, CA199, CA125, CA153, and TPS in metastatic breast cancer. Clinica chimica acta ; international journal of clinical chemistry 470, 51-55 (2017). In addition, Zhang et al. reported circulating CD14+CD204+M2-like-monocytes and plasma hyaluronan (HA) combined with CEA and CA153 to diagnose breast cancer, The sensitivity of M2-like-monocytes alone in predicting breast cancer was 71.6%, and the combined sensitivity was 81.5% without significant improvement (B. Zhang et al., Combination of plasma HA and circulating M2-likemonocytes may serve as a diagnostic marker for breast Cancer.Journal of Cancer; 8, 3522-30 (2017). For another example, Fu et al. reported that platelet distribution width and fibrinogen combined with CA153 were used to diagnose breast cancer. Although the specificity increased from 67.4 to 82.9% after the combination, the sensitivity increased from 94.5 % dropped to 82.5% (S. Fuet al., Cancer antigen 15-3, platelet distribution width, and fibrinogen incombination to distinguish breast cancer from benign breast disease in non-conclusive mammography patients. Oncotarget; 8, 678 29-36 (2017) Kuo et al. reported that the marker phospholipid scramblase 1 (PLSCR1) was combined with CEA to diagnose colorectal cancer, and the combined sensitivity increased from 80% to 85% without significant improvement (Y.Kuo et al. al., Identification of phospholipid scramblase1 as a biomarker and determination of its prognostic value for colorectal cancer. Molecular medicine; 17, 41-47 (2011).

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a marker combination for the diagnosis of colorectal tumors with high detection sensitivity and high specificity.

A further object of the present invention is to provide a diagnostic reagent/kit for detecting colorectal tumors with high sensitivity and specificity.

Another object of the present invention is to provide a diagnostic system for colorectal tumors.

In one aspect, the present invention provides a molecular marker combination for the first time, and the detection amount of the molecular marker combination in serum has a very high corresponding relationship with colorectal tumors. The sensitivity and specificity of its detection results can even reach 100%, respectively.

This particular combination of molecular markers provided by the present invention includes the combination of markers RANTES and CEA.

RANTES is one of the members of the CC chemokine family. It is a small molecule protein composed of 68 amino acids with a molecular weight of 8kD. It is a class of cytokines that regulate the expression and secretion of normal T cells. It mainly includes the corresponding receptors such as CCR1, CCR3, CCR4 and CCR5, which is a typical Inflammation-derived chemokines. The chemotactic effect of chemotactic cytokines on leukocytes is an important initial step in the process of inflammatory response, and it is also an important aspect of the innate immune function of the body's defense and removal of foreign bodies such as invading pathogens, participating in a variety of inflammatory and immune responses.

In one embodiment of the present invention, the colorectal tumor diagnostic marker combination further includes one or both of the markers CA199 and CA125.

In another aspect, the present invention provides an application of the detection reagent of the colorectal tumor diagnostic marker combination in the preparation of a colorectal tumor diagnostic reagent or kit.

In one embodiment, the detection reagent is to detect the gene expression level of the marker.

In some of these embodiments, the detection reagent is to detect the mRNA expression of the marker.

In some of these embodiments, the detection reagent is to detect the expression level of the marker protein.

In certain embodiments, the detection reagent is one or more of fluorescent quantitative PCR dyes, fluorescent quantitative PCR primers, fluorescent quantitative PCR probes, antibodies, antibody functional fragments and conjugated antibodies.

In some embodiments, the kit is selected from the group consisting of qPCR kits, western blot detection kits, immunochromatographic detection kits, flow cytometry kits, immunohistochemical detection kits, ELISA kits and electrochemiluminescence kits. One or more of the detection kits.

In some embodiments, the kit is selected from ELISA kits. In one embodiment, the diagnostic reagent/kit comprises detection reagents for the markers RANTES and CEA.

In certain embodiments, the diagnostic reagent/kit further comprises detection reagents for one or both of the markers CA199 and CA125.

In one embodiment, the detection reagent is to detect the gene expression level of the marker.

In one embodiment, the detection reagent is to detect the mRNA expression of the marker.

In one embodiment, the detection reagent is to detect the expression level of the marker protein.

In one embodiment, the detection reagent is one or more of fluorescent quantitative PCR dyes, fluorescent quantitative PCR primers, fluorescent quantitative PCR probes, antibodies, antibody functional fragments and conjugated antibodies.

In certain embodiments, the kit is selected from the group consisting of qPCR kits, western blot detection kits, immunochromatographic detection kits, flow cytometry kits, immunohistochemical detection kits, ELISA kits and electrochemical detection kits. One or more of the luminescence detection kits.

In one embodiment, the kit is selected from an ELISA kit.

In another aspect, the present invention provides a diagnostic system for colorectal tumors, the diagnostic system comprising: a detection component: the detection component is used to detect the expression level of each marker in the marker combination; the result judgment component : The result judging component is used to output the disease result of the tumor patient according to the result of the expression level of each marker in the marker combination detected by the detection component; the marker combination is the combination of the markers RANTES and CEA.

In certain embodiments, the expression level of the marker is one or more of gene expression level, mRNA expression level and/or protein expression level.

In certain embodiments, the expression level of the marker is the protein expression level.

As an optional embodiment, the result judging component includes an input module, an analysis module and an output module; the input module is used to input the expression level of the marker; the analysis module is used to input the expression level of the marker according to the expression level of the marker , to analyze the possibility of disease risk results of tumor patients; the output module is used to output the analysis results of the analysis module.

As an optional embodiment, the detection component contains qPCR kit, western blot detection kit, flow cytometry analysis kit, immunohistochemical detection kit, ELISA kit, qPCR instrument, western blot detection device, One or more of flow cytometer, immunohistochemical detection device, ELISA detection device, and electrochemiluminescence detection device.

As an optional embodiment, the detection component is an ELISA detection device.

In some of the embodiments, the thresholds for the expression levels of the markers RANTES and CEA in the marker combination are 10915.666-12539.254 ng/mL and 1.84-3 ng/mL, respectively.

As a preferred embodiment, the thresholds of the expression levels of the markers RANTES and CEA in the marker combination are 10958.308-12106.426ng/mL and 1.875-2.03ng/mL, respectively;

As a further preferred embodiment, the thresholds for the expression levels of the markers RANTES and CEA in the marker combination are 10994.238-11980.896.ng/mL and 1.91-1.945ng/mL, respectively.

In certain embodiments, the marker combination further includes one or both of the markers CA199 and CA125.

As an optional embodiment, the thresholds of the expression levels of the markers CA199 and CA125 in the marker combination are 15.115-23.11 U/mL and 5.8-10.25 U/mL, respectively.

As a preferred embodiment, the thresholds of the expression levels of the markers CA199 and CA125 in the marker combination are 18.42-22.115 U/mL and 5.8-9.4 U/mL, respectively.

As a more preferred embodiment, the thresholds of the expression levels of the markers CA199 and CA125 in the marker combination are 18.8-21.17 U/mL and 5.8-7.85 U/mL, respectively.

In one embodiment, in the result judging component, when the expression level of any marker in the marker combination is higher than a threshold, it is determined that the colorectal tumor is positive; when the marker combination contains When the expression levels of all markers were lower than the threshold, it was judged that the colorectal tumor was negative.

The detection reagent or kit of the present invention only needs one sampling to detect the content of each marker in the combination of markers in the blood, and the detection results are classified according to the method: for common colorectal tumors, when the detection When the expression of any marker in the marker combination is higher than the threshold, it is judged that the colorectal tumor is positive, and the detection sensitivity at this time can reach 98.9% (the detection sensitivity of early colorectal cancer can even reach 100%). %); when it is detected that the expression levels of all markers in the marker combination are lower than the threshold value, the colorectal tumor is judged to be negative, and the detection specificity at this time is 100%. Therefore, by using the detection reagent or kit of the present invention, only one sampling is required, so that the detection results of patients or healthy persons undergoing physical examination can be extremely accurate, and no missed diagnosis or misdiagnosis will occur, and there is no need to wait for the results to be tested before proceeding. Diagnosed.

At present, whether it is a tissue sample, a stool sample, or a serum sample in the present invention, markers with different sensitivities vary, but are generally low. If the specificity is low, the probability of misdiagnosing healthy people as sick patients is high, which will add great psychological burden and pressure to these healthy people; the sensitivity is low, which will lead to a high probability of missed diagnosis, which is not conducive to colorectal cancer. Timely detection, buy time for patients, start treatment as soon as possible, improve the survival rate of patients and reduce mortality.

The diagnostic reagent or kit for colorectal tumor formed by the detection reagent of the combination of markers discovered for the first time in the present invention can just solve the problem.

That is, the colorectal tumor diagnostic reagent or kit of the present invention has extremely high sensitivity and specificity at the same time; this is very rare in clinical practice. For example, in the prior art, colorectal cancer is screened by SEPT9 gene methylation detection, The sensitivity is 69% and the specificity is 86%; 81.1% of colorectal cancer patients can be screened by fecal SDC2 gene methylation detection, and it is expensive. The colorectal tumor diagnostic reagent or kit of the present invention can be used for early detection of colorectal cancer because of its extremely high sensitivity and specificity, buy time for patients, start treatment as soon as possible, improve the survival rate of patients, reduce mortality, and alleviate The medical burden in our country is of great significance.

In certain embodiments, the sample for which the diagnosis is made is tissue, body fluid or excreta.

In one embodiment, the tissue is intestinal tissue.

In certain embodiments, the body fluid is blood, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.

As a preferred embodiment, the body fluid is blood.

As a more preferred embodiment, the body fluid is plasma.

In certain embodiments, the excrement is sputum, saliva, urine or feces.

In the present invention, the colorectal tumor is stage I, II, III, and IV colorectal cancer and precancerous adenoma.

As a preferred embodiment, the colorectal tumor is stage I-II colorectal cancer, that is, early-stage colorectal cancer.

Compared with the prior art, one of the above-mentioned technical solutions has the following beneficial effects:

1. Blood marker detection has the advantages of convenient sampling and non-invasiveness, and provides a faster way for colorectal cancer screening, diagnosis, monitoring and prognosis judgment.

2. The present invention combines the markers RANTES and CEA in serum, and it is found that it has a very high correlation with colorectal cancer, its sensitivity can reach 98.9%, and its specificity can even be as high as 100%; for the diagnosis of early colorectal cancer It also has high sensitivity and specificity, and its sensitivity can even reach 100%, that is, the colorectal tumor diagnostic reagent or kit of the present invention has extremely high sensitivity and specificity; this is very rare in clinical practice and can be used for The early detection of colorectal cancer is of great significance to buy time for patients, start treatment as soon as possible, improve the survival rate of patients, reduce mortality, and reduce the medical burden in our country.

Description of drawings

Figure 1 shows the ROC curve of CEA combined with RANTES to predict CRC in the synergy model.

Figure 2 shows the ROC curve of CEA, CA199, and RANTES jointly predicting CRC in the synergistic model.

Figure 3 shows the ROC curve of CEA, CA125, and RANTES jointly predicting CRC in the synergy model.

Figure 4 shows the ROC curve of RANTES combined with CEA, CA125, and CA199 three tumor markers for predicting CRC in the synergistic model.

Figure 5 shows the ROC curve of CEA combined with RANTES to predict CRC in the synthetic model.

Figure 6 is the ROC curve of the combined prediction of CRC by CEA, CA199, and RANTES in the synthetic model.

Figure 7 is the ROC curve of the combined prediction of CRC by CEA, CA125, and RANTES in the synthetic model.

Figure 8 is the ROC curve of RANTES combined with three tumor markers CEA, CA125 and CA199 in the synthetic model to predict CRC.

Figure 9 shows the ROC curves of the combined RANTES/CEA, RANTES/CEA/CA199, RANTES/CEA/CA125, and RANTES/CEA/CA125/CA199 in the synergy model to predict early CRC, respectively.

Detailed ways

The technical solutions of the present invention are further described below through specific embodiments, which do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

TP (True Positive) True Positive: Predicted to be positive and actually positive.

FP (False Positive): Predicted to be positive, but actually negative.

FN (False Negative): Predicted to be negative, but actually positive.

TN (True Negative) True Negative: Predicted to be negative and actually negative.

In the present invention, the sensitivity is the same as the sensitivity.

The term "diagnostic reagent/kit" can be either a diagnostic reagent or a diagnostic kit.

Colorectal Cancer: Colorectal cancer, CRC.

Threshold of marker expression in the marker combination: the expression threshold of the marker in the marker combination used in the present invention defines whether the colorectal tumor is diseased or not, that is, if it is higher than the predetermined threshold, the colorectal tumor is positive; The predetermined threshold is negative for colorectal tumor; and the threshold in the present invention is determined corresponding to the Youden index in the above-mentioned embodiment.

Synergy model (A/B): Represents "A or B", and its sensitivity indicates the percentage of patients whose detection results for either marker A or B are higher than the cutoff value in the total number of patients; its specificity indicates the marker The percentage of healthy people whose detection result of any one of marker A or B is lower than the cutoff value in the total number of healthy people.

Synthetic model (A-B): Represents "A and B", and its sensitivity represents the percentage of patients with marker A and B detection results that are both higher than the cutoff value in the total number of patients; its specificity represents the detection results of markers A and B The percentage of healthy people whose value is lower than the cutoff value at the same time as a percentage of the total number of healthy people.

Youden index=sensitivity+specificity-1.

The clinical samples in Examples 3-7 of the present invention are the samples described in Example 1.

data analysis

Statistical analysis of the data was performed using the R language. All data were not subject to normal distribution, and medians and quartiles were chosen. The Spearman correlation test was used to detect whether each index had significant statistical difference between patients and healthy controls. The Mann-Whitney U test was used to analyze the differences in tumor marker expression between groups. In order to study the diagnostic performance of different tumor markers and RANTES, ROC curves were drawn to evaluate their predictive ability.

In addition, the level corresponding to the point with the largest Youden index is selected as its cutoff value, that is, the optimal probability division threshold is determined by the point with the largest Youden index. According to the determined probability division threshold, the sensitivity, specificity, positive predictive value, negative predictive value, etc. of each combined detection scheme in the training group and the validation group can be calculated.

The ROC curve of the combined detection of the indicators was obtained by binary logistic regression analysis, and the optimal combined detection scheme was obtained by comparing the area under the curve. Statistical difference at p<0.05.

The calculation methods for sensitivity, specificity, positive predictive value, and negative predictive value are shown in Table 3:

Table 1 Calculation methods of sensitivity, specificity, positive predictive value, and negative predictive value

The sensitivity of the marker is calculated as: the percentage of patients whose detection result value is higher than the cutoff value in the total number of patients.

The specificity calculation method of the marker is: the percentage of healthy persons whose detection result is lower than the cutoff value in the total number of healthy persons.

Example 1 Sample source and analysis

A total of 87 patients (I, II, III, IV) who were diagnosed with colorectal cancer in the Sixth Affiliated Hospital of Sun Yat-sen University from September 2018 to December 2018 were collected. All of them were confirmed by imaging and pathological examinations. All pathological results were obtained by tissue biopsy. The patient had no serious organ disease, and did not receive chemotherapy, radiotherapy, or surgery. A total of 90 cases were collected from the healthy physical examination population during the same period as controls. The inclusion criteria for healthy controls were that there were no obvious abnormalities in the results of routine blood and biochemical tests, and no infectious diseases such as hepatitis B. The basic information is shown in Table 2. All blood samples were collected using EDTA anticoagulation tubes. Plasma separation was completed within 6 hours at room temperature, centrifuged at 3000 rpm for 2 minutes, and the plasma was transferred into 1.7 mL EP tubes and immediately placed in a -80°C refrigerator for future use.

All tumor markers were detected before treatment, and were determined by chemiluminescence enzyme immunoassay (CLEIA). All experiments were completed in the Laboratory Department of the Sixth Affiliated Hospital of Sun Yat-sen University.

Table 2 Basic information of colorectal cancer patients

Embodiment 2 Determination method of tumor marker content

The content of RANTES in the present invention is detected by enzyme-linked immunosorbent assay (ELISA), and commercial kits such as RayBiotech Human RANTES ELISA Kit, Biotek Elx800 microplate reader 450nm, Biotek Elx50 plate washer, pipette, small centrifuge tube, Ionized water, Sigmaplot analysis software, etc. The contents of CEA, CA125 and CA199 were detected by chemiluminescence enzyme immunoassay (CLEIA) on Abbott I2000.

(1) Equilibrate the kit and sample to room temperature (18-25°C);

(2) The diluent is diluted 5 times with deionized water for subsequent use;

(3) The plasma sample is diluted 40 times for use;

(4) Standard preparation: Centrifuge the standard tube, then add 400 μL of 1× dilution solution to the standard tube, and mix it evenly to obtain 50 ng/mL standard stock solution; pipette 40 μL of standard stock solution and add 960 μL of 1× dilution solution 1.5mL centrifuge tubes were prepared, and 400μL of 1× diluent buffer was added to each, and then labeled as STD2, STD3, STD4, STD5, STD6, STD7, STD8, STD9 ; Then use 2000pg/mL of STD1 gradient dilution standard, take 200μL 2000pg/mL STD1 into STD2 small tube, after mixing, take 200μL solution in this tube and add it to STD3 small tube and mix well, prepare STD8 in turn, STD9 is 400μL 1× dilution solution is standard 0pg/mL;

(5) dilution of washing liquid: dilute the concentrated washing liquid 20 times with deionized water for subsequent use;

(6) Centrifuge the detection antibody vial, add 100 μL of 1× diluent to fully dissolve, gently pipette up and down with a pipette, and then use it after diluting 80 times with 1× diluent;

(7) Centrifuge HRP-streptavidin, and then use it after diluting 300 times with 1× diluent;

(8) After the antibody-coated ELISA plate is equilibrated to room temperature, add 100 μL of prepared standards and samples to the corresponding wells, seal the entire plate with a sealing film, and incubate at 4°C overnight;

(9) Add the prepared 1× washing solution to the plate washer, wash the strips 4 times with the plate washer, and add 300 μL of the washing solution to each well;

(10) After washing the plate, add 100 μL of the prepared detection antibody (biotin-labeled antibody) to each well, and incubate at room temperature for 1 h;

(11) cleaning step is the same as 9;

(12) Add 100 μL of prepared HRP-streptavidin to each well and incubate at room temperature for 45 minutes;

(13) cleaning step is the same as 9;

(14) Add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature for 30 minutes in the dark;

(15) Add 50 μL of stop solution to each well, and immediately read on the microplate reader at 450 nm;

(16) Calculate the concentration value with Sigmaplot 12.0 software.

Example 3 Results of the diagnostic efficacy test of tumor markers for colorectal cancer

1. With CEA, CA125, CA199 and RANTES as tumor markers, the corresponding Youden index and cutoff value obtained by detecting the samples in Example 1 by the measurement method of Example 2 are shown in Table 3.

Table 3 The maximum value and cutoff value of Youden index corresponding to CEA, CA125, CA199 and RANTES

2. Indicators combined to predict the diagnostic efficacy of CRC

Comparison of the diagnostic efficacy of CEA, CA125, CA199, and RANTES in the detection of colorectal cancer of the samples in Example 1 by various combination schemes. Wherein, the data are calculated for 87 colorectal cancer patients and 90 healthy controls in Example 1.

The ROC curve is obtained by Logistic regression, in which the independent variable is the corresponding index, and the dependent variable is the cancer situation. Through the fitted regression curve, the probability of each individual suffering from cancer can be calculated, and different probability division thresholds can be determined, namely Prediction results are available.

The optimal probability division threshold is determined by the point with the largest Youden exponent. According to the determined probability division threshold, the sensitivity, specificity, positive predictive value, negative predictive value, etc. of each combined detection scheme in the training group and the validation group can be calculated.

(1) The index predicts the diagnostic efficacy of CRC jointly with the synergistic model (A/B)

The calculation method of sensitivity is: the percentage of patients whose detection result of any marker is higher than the cutoff value in the total number of patients;

The specificity calculation method is as follows: the percentage of healthy persons whose detection result of any one marker is lower than the cutoff value in the total number of healthy persons.

The ROC curves are shown in Figure 1, Figure 2, Figure 3, and Figure 4, and the results are shown in Table 4.

Table 4 The diagnostic efficacy of the indicators combined with the synergistic model to predict CRC

Detection Indicator Sensitivity specificity positive predictive value negative predictive value RANTES/CEA 0.989 0.606 0.869 0.952 RANTES/CEA/CA199 0.989 0.581 0.869 0.947 RANTES/CEA/CA125 0.989 0.500 0.851 0.938 RANTES/CEA/CA125/CA199 0.989 0.500 0.851 0.938

From the experimental results in Table 4, it can be seen that the synergistic model containing RANTES and CEA jointly predicts CRC, and its sensitivity can be as high as 98.9%.

(2) The index predicts the diagnostic performance of CRC jointly with the synthetic model (A-B)

1) The sensitivity calculation method is: the percentage of patients whose detection value of each marker is higher than the cutoff value in the total number of patients;

2) The specificity calculation method is as follows: the percentage of the number of healthy persons whose detection value of each marker is lower than the cutoff value in the total number of healthy persons.

The ROC curves are shown in Figure 5, Figure 6, Figure 7, and Figure 8, and the results are shown in Table 5.

Table 5 The diagnostic performance of the indicators combined with the synthetic model to predict CRC

Detection Indicator Sensitivity specificity positive predictive value negative predictive value RANTES-CEA 0.701 1.000 1.000 0.776 RANTES-CEA-CA199 0.218 1.000 1.000 0.570 RANTES-CEA-CA125 0.310 1.000 1.000 0.600 RANTES-CEA-CA125-CA199 0.115 1.000 1.000 0.539

From the experimental results in Table 5, it can be seen that the synthetic model containing RANTES and CEA jointly predicts CRC, and its specificity can be as high as 100%.

Example 4 Results of the diagnostic efficacy test of tumor markers for colorectal cancer

The cutoff values of the selected indicators are shown in Table 6; except that the selected cutoff values are different, others are the same as in Example 3, and the corresponding detection results are shown in Tables 7 and 8.

Table 6 Cutoff values of CEA, CA125, CA199, RANTES

Table 7 The diagnostic performance of the indicators combined with the synergistic model to predict CRC

Detection Indicator Sensitivity specificity RANTES/CEA 0.977 0.697 RANTES/CEA/CA199 0.977 0.677 RANTES/CEA/CA125 0.977 0.700 RANTES/CEA/CA125/CA199 0.977 0.676

Table 8 The diagnostic performance of indicators combined with synthetic models to predict CRC

Detection Indicator Sensitivity specificity RANTES-CEA 0.690 1.000 RANTES-CEA-CA199 0.207 1.000 RANTES-CEA-CA125 0.299 1.000 RANTES-CEA-CA125-CA199 0.207 1.000

Example 5 Results of the diagnostic efficacy test of tumor markers for colorectal cancer

The cutoff values of the selected indicators are shown in Table 9; except that the selected cutoff values are different, others are the same as in Example 3, and the corresponding detection results are shown in Tables 10 and 11.

Table 9 Cutoff values of CEA, CA125, CA199, RANTES

Table 10 The diagnostic performance of the indicators in the joint prediction of CRC with the synergistic model

Detection Indicator Sensitivity specificity RANTES/CEA 0.966 0.697 RANTES/CEA/CA199 0.954 0.774 RANTES/CEA/CA125 0.966 0.700 RANTES/CEA/CA125/CA199 0.966 0.733

Table 11 The diagnostic performance of the indicators combined with the synthetic model to predict CRC

Detection Indicator Sensitivity specificity RANTES-CEA 0.678 1.000 RANTES-CEA-CA199 0.218 1.000 RANTES-CEA-CA125 0.287 1.000 RANTES-CEA-CA125-CA199 0.207 1.000

Example 6 Results of the diagnostic efficacy test of tumor markers for colorectal cancer

The cutoff values of the selected indicators are shown in Table 12; except that the selected cutoff values are different, others are the same as those in Example 3, and the corresponding detection results are shown in Tables 13 and 14.

Table 12 Cutoff values of CEA, CA125, CA199, RANTES

Table 13 The diagnostic performance of the indicators in the joint prediction of CRC with the synergistic model

Table 14. The diagnostic performance of the indicators combined with the synthetic model to predict CRC

Detection Indicator Sensitivity specificity RANTES-CEA 0.655 1.000 RANTES-CEA-CA199 0.207 1.000 RANTES-CEA-CA125 0.276 1.000 RANTES-CEA-CA125-CA199 0.115 1.000

Example 7 Results of the diagnostic efficacy test of tumor markers for early colorectal cancer

Early-stage colorectal cancer samples were selected from the samples in Example 1, and the test methods in Example 3 were used, and the test tumor markers had the same diagnostic performance for early-stage colorectal cancer. The test results are shown in Table 15.

Table 15. The diagnostic efficacy of the index synergy model jointly predicting early CRC

Detection Indicator Sensitivity specificity positive predictive value negative predictive value RANTES/CEA 1.000 0.606 0.649 1.000 RANTES/CEA/CA199 1.000 0.581 0.649 1.000 RANTES/CEA/CA125 1.000 0.500 0.615 1.000 RANTES/CEA/CA125/CA199 1.000 0.500 0.615 1.000

The ROC curve is shown in Figure 9, and the results are shown in Table 15. It can be seen from the experimental results that the combined model containing RANTES and CEA can predict CRC, and its sensitivity can be as high as 100%.

Claims (39)

1. A marker combination for colorectal neoplasm diagnosis, wherein said markers are RANTES and CEA.

2. The colorectal tumor diagnostic marker combination of claim 1, further comprising one or both of the markers CA199 and CA 125.

3. Use of a detection reagent of the colorectal tumor diagnostic marker combination according to claim 1 or 2 for preparing a colorectal tumor diagnostic reagent or kit.

4. The use of claim 3, wherein the detection reagent detects the gene expression level of the marker.

5. The use of claim 3, wherein the detection reagent detects the amount of mRNA expression of the marker.

6. The use of claim 3, wherein the detection reagent detects the amount of expression of the protein of the marker.

7. The use of claim 3, wherein the detection reagent is one or more of a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment, and a conjugated antibody.

8. The use of claim 3, wherein the kit is selected from one or more of the group consisting of a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometry kit, an immunohistochemical detection kit, an E L ISA kit, and an electrochemiluminescence detection kit.

9. The use of claim 3, wherein said kit is selected from the group consisting of E L ISA kits.

10. A diagnostic reagent/kit for colorectal neoplasm, which is characterized in that the diagnostic reagent/kit comprises detection reagents for markers RANTES and CEA.

11. The diagnostic reagent/kit of claim 10, further comprising a detection reagent for the marker CA199 and/or CA 125.

12. The diagnostic reagent/kit as claimed in claim 10, wherein the detection reagent is to detect the gene expression level of the marker.

13. The diagnostic reagent/kit of claim 10, wherein the detection reagent is to detect the mRNA expression level of the marker.

14. The diagnostic reagent/kit of claim 10, wherein the detection reagent is to detect the expression level of the protein of the marker.

15. The diagnostic reagent/kit of claim 10, wherein the detection reagent is one or more of a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment and a conjugated antibody.

16. The diagnostic reagent/kit of claim 10, wherein the kit is selected from one or more of qPCR kit, immunoblot detection kit, immunochromatography detection kit, flow cytometry kit, immunohistochemistry detection kit, E L ISA kit and electrochemiluminescence detection kit.

17. The diagnostic reagent/kit of claim 10, wherein said kit is selected from the group consisting of E L ISA kits.

18. A diagnostic system for colorectal neoplasms, said diagnostic system comprising:

a detection means: the detection component is used for detecting the expression level of each marker in the marker combination;

a result judgment means: the result judging component is used for outputting the disease result of the tumor patient according to the result of the expression quantity of each marker in the marker combination detected by the detecting component;

the marker combination is the combination of the markers RANTES and CEA.

19. The diagnostic system of claim 18, wherein the expression level of the marker is one or more of a gene expression level, an mRNA expression level, and a protein expression level.

20. The diagnostic system of claim 18, wherein the expression level of the marker is a protein expression level.

21. The diagnostic system of claim 18, wherein said result determination means comprises an input module, an analysis module, and an output module; the input module is used for inputting the expression quantity of the marker; the analysis module is used for analyzing the possibility of disease risk results of the tumor patients according to the expression quantity of the marker; the output module is used for outputting the analysis result of the analysis module.

22. The diagnostic system of claim 18, wherein the detection component comprises one or more of a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an E L ISA kit, an electrochemiluminescence detection kit, a qPCR apparatus, an immunoblot detection apparatus, a flow cytometer, an immunohistochemical detection apparatus, an E L ISA detection apparatus, and an electrochemiluminescence detection apparatus.

23. The diagnostic system of claim 18, wherein said detection means comprises an E L ISA kit and/or an E L ISA detection device.

24. The diagnostic system of claim 18, wherein the threshold values for the expression levels of markers RANTES and CEA in the marker combination are between 10915.666-12539.254ng/m L and 1.84-3ng/m L, respectively.

25. The diagnostic system of claim 18, wherein the threshold values for the expression levels of markers RANTES and CEA in the marker combination are values between 10958.308-12106.426ng/m L and 1.875-2.03ng/m L, respectively.

26. The diagnostic system of claim 18, wherein the threshold values for the expression levels of the markers RANTES and CEA in the marker combination are between 10994.238-11980.896.ng/m L and 1.91-1.945ng/m L, respectively.

27. The diagnostic system of claim 18, wherein said marker combination further comprises one or both of the markers CA199 and CA 125.

28. The diagnostic system of claim 18, wherein the threshold values for the expression levels of markers CA199 and CA125 in the marker combination are values between 15.115-23.11U/m L and 5.8-10.25U/m L, respectively.

29. The diagnostic system of claim 18, wherein the threshold values for the expression levels of markers CA199 and CA125 in the marker combination are values between 18.42-22.115U/m L and 5.8-9.4U/m L, respectively.

30. The diagnostic system of claim 18, wherein the threshold values for the expression levels of markers CA199 and CA125 in the marker combination are values between 18.8-21.17U/m L and 5.8-7.85U/m L, respectively.

31. The diagnostic system of claim 18, wherein the result determination component determines that the colorectal neoplasm is positive when the amount of expression of any one of the markers in the combination of markers is above a threshold; and when the expression quantity of all the markers in the marker combination is lower than a threshold value, judging that the colorectal tumor is negative.

32. The use according to claim 3 or the diagnostic reagent/kit according to any one of claims 10 to 17 or the diagnostic system according to any one of claims 18 to 31, wherein the sample for which the diagnosis is made is a tissue, a body fluid or an excreta.

33. The use or diagnostic reagent/kit or diagnostic system of claim 32, wherein the tissue is intestinal tissue.

34. The use or diagnostic reagent/kit or diagnostic system as claimed in claim 32, wherein said body fluid is blood, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid or aqueous humor.

35. The use or diagnostic reagent/kit or diagnostic system of claim 32, wherein the bodily fluid is blood.

36. The use or diagnostic reagent/kit or diagnostic system of claim 32, wherein the bodily fluid is plasma.

37. The use or diagnostic reagent/kit or diagnostic system according to claim 32 wherein the excreta is sputum, saliva, urine or feces.

38. The diagnostic marker combination for colorectal tumours according to any one of claims 1 to 2 or the use according to any one of claims 3 to 9 or the diagnostic reagent/kit according to any one of claims 10 to 17 or the diagnostic system for colorectal tumours according to any one of claims 18 to 31, wherein the colorectal tumours are stage I, II, III, IV colorectal carcinomas, pre-cancerous adenomas.

39. A diagnostic marker combination for colorectal tumours according to any of claims 1 to 2 or the use according to any of claims 3 to 9 or a diagnostic reagent/kit according to any of claims 10 to 17 or a diagnostic system for colorectal tumours according to any of claims 18 to 31, wherein the colorectal tumour is colorectal cancer stage I-II.

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