KR20200049647A - Biomarker Test Method for Ancillary Diagnosis of Companion Tumor Diseases - Google Patents

Abstract

The present invention provides a tumor disease examination method of a companion animal, which provides a method using a combination of three biomarkers for auxiliary diagnosis of tumor diseases in the companion animal. According to the present invention, by using the method of using the combination of three biomarkers for auxiliary diagnosis of tumor diseases in the companion animal, the positive predictive rate of tumor disease, which detects various tumor diseases in companion animals, increases from 73 to 90%.

Description

Biomarker Test Method for Ancillary Diagnosis of Companion Tumor Diseases

The present invention relates to a method for examining a malignant tumor in a companion animal, and more particularly, to a method used for diagnosis through a combination of various biomarkers.

Unlike humans, tumor diseases in animals do not have an assistive diagnostic means, such as detection of appropriate biomarkers for tumor diseases, such as regular examinations. In animals, there are limitations because it is not possible to routinely perform detailed examinations such as formative examinations and biopsies in general animal hospitals. In general, even in humans, biomarkers, which are indicators that can detect changes in the body using proteins, DNA, RNA (re-nucleic acid), and metabolites, can be used to determine the normal or pathological conditions of living organisms and the degree of reaction to drugs. Measure objectively. These biomarkers can be applied to companion animals, and in companion animals, tumor diseases generally involve various inflammations and changes in blood vessels, so these biomarkers can help in the diagnosis and prognosis of tumor diseases.

However, the diagnosis, prevention, and management of companion animal tumor diseases have limitations in terms of cost and system that can not be used for routine examinations, unlike human tumor disease management. No way of detecting the tumor has been suggested.

In the case of humans, the regular check-up system provides health care for the elderly population by preventing various diseases and tumor diseases, but in the case of companion animals, the regular check-up system despite the proportion of aged dogs over 6 years old exceeding 35%. It is a reality that this is not common and lacks checkup items.

Therefore, in the present invention, by using a biomarker in combination with the above, the tumor disease of a companion animal and the accompanying inflammation and protein markers that increase in the blood during changes in blood vessels confirm the effectiveness in the blood sample of the companion animal tumor disease and check the companion animal. It is intended to present an invention that can be used as an auxiliary system for the diagnosis of tumor disease, but simpler and more accurate than existing methods for diagnosing malignant tumors.

An object of the present invention is to propose a test method for improving the accuracy in detecting various tumor diseases occurring in a companion animal by performing three types of marker tests on the companion animal's blood.

In order to achieve the above object, the present invention is to generate a measurement value for each biomarker by measuring a plurality of biomarkers in a sample collected from a companion animal, comparing the biomarker measurement value with a corresponding reference value, and It provides a method for examining a tumor disease of a companion animal, comprising the step of determining the possibility of a tumor disease of the companion animal according to the number of biomarkers in which the measured biomarker value exceeds the reference value.

According to the present invention as described above, by using a combination of the three biomarkers presented in the biomarker test method for the secondary diagnosis of tumor disease in companion animals, the positive predictive rate of tumor disease detecting various tumor diseases in companion animals This effect has been increased from 73% to 90%.

1 is a view for explaining a cCRP inspection method according to an embodiment of the present invention.
2 is a view for explaining a D-dimer inspection method according to an embodiment of the present invention.
3 is a diagram illustrating an AFP inspection method according to an embodiment of the present invention.
4 shows a screen of a dedicated device according to an embodiment of the present invention.
5 is a view for explaining the principle of immunofluorescence of cCRP, D-dimer, AFP cartridge according to an embodiment of the present invention.
6 is a view for explaining the housing of the cartridge used in the embodiment of the present invention.
7 is a view for explaining the structure of the strip used in the embodiment of the present invention.
8 is a view illustrating a reagent applied according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

Provided is a method for examining a tumor disease in a companion animal using a biomarker test method for an auxiliary diagnosis of a tumor disease in a companion animal according to one embodiment of the present invention.

The method for examining a tumor disease of a companion animal includes measuring a plurality of biomarkers in a sample collected from a companion animal to generate a measurement value for each biomarker, comparing the measured biomarker value with a corresponding reference value, and And determining a possibility of tumor disease of the companion animal according to the number of biomarkers whose biomarker measurement value exceeds the reference value.

The companion animal is a mammal, and preferably a dog.

The sample may be selected from one or more of a companion animal's cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.

The biomarker measurement uses a fluorescence reader, which is an optical analysis device, and can preferably be performed through fluorescence LFI (Lateral flow immunoassay). The fluorescence LFI (Lateral flow immunoassay, lateral flow immunoassay) uses an immunological method to detect a target using an antibody of the same principle as ELISA, but a general ELISA detects by labeling an enzyme with an antibody while fluorescence LFI ( Lateral flow immunoassay (lateral flow immunoassay) is a device that quantifies the protein concentration by reading the fluorescence attached to the sample and converting it into a numerical method by labeling fluorescence. More preferably, Bet chroma may be used in the fluorescent LFI (Lateral flow immunoassay) device.

When performing the analysis through the fluorescent LFI (Lateral flow immunoassay, lateral flow immunoassay), it is detected by applying a sample to the lateral flow analysis strip.

The lateral flow analysis strip 100 shows its configuration in FIG. 6. As shown, the lateral flow analysis strip 100 is formed of a porous matrix 110 allowing capillary flow in the housing 102 formed by combining up and down. The sample pad 120 allows a mixture of a sample and a detection buffer to be applied from the sample port 104 to flow to the porous matrix 110. On the porous matrix 110, a test line 140 and a control line 160 are positioned downstream from the sample pad 120. A capture agent is fixed to the test line 140 and the control line 160 in a physical adsorption method. The capture agent may be a specific antibody, antigen, or oligonucleotide that hybridizes to a certain sequence of nucleic acids in response to the analyte. Preferably, the capture agent may be a D-dimer, AFP or cCRP protein. In addition, an absorption pad 170 is disposed at a downstream end of the porous matrix 110. The absorption pad 170 absorbs the excess liquid sample applied from the sample port 104 and maintains lateral flow along the porous matrix 110.

The control line 160 may be located upstream or downstream of the test line 140. Positive or negative control reagents or capture agents are fixed to the control line 160.

Fluorescent labels capable of detecting fluorescent labeling in the fluorescent LFI (Lateral flow immunoassay, lateral flow immunoassay) include gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up It can be selected from the group consisting of up converting phosphors and organic fluorophores. Preferably, the up-converting phosphors (up converting phosphors) of the lanthanide-based phosphors Europium (Europium, EU (Eu ₂ O ₃ )), gadolinium (Gadolinium, Gd (Gd ₂ O ₃ )), terbium (Terbium, Tb (Tb ₄₎ O ₇ )) or Ytterbium (Yb (Yb ₂ O ₃ )) atoms may be a fluorescent conjugated antibody.

The biomarker may be one or more selected from the group consisting of D-dimer, AFP or cCRP protein, and preferably, all three biomarkers are selected and used.

The reference value is a reference value for tumor diagnosis of a companion animal, which is a value set based on the cut off concentration value of the biomarker. At this time, the cut-off concentration value that has been set as a reference is a biomarker concentration value set based on when the sensitivity of the biomarker is 80%.

The cut-off concentration value is different for each biomarker, D-dimer is 66.46 ng / ml or more, AFP is 39.24 ng / ml or more, and cCRP is based on 2.81 ng / ml or more. At this time, when the measured value of the biomarker is greater than or equal to the respective cut off concentration value, it is possible to diagnose whether or not the companion animal has tumor diagnosis.

The tumors capable of diagnosing tumors include epithelial cell carcinoma (SCC, transitional cell carcinoma (TCC)), anal sac adenocarcinoma (Perianal adeno carcinoma), lung tumor (Lung adenocarcinoma, Pulmonary adenosquamous cell carcinima) , Hepatocellular carcinoma (HCC), Multicentric lymphoma (lymphoma), Carotid body tumor, Nasal cell carcinoma, Ovarian carcinoma, Oral melanoma / melanoma in oral, Mast cell tumor, bronchiectasis / Thyroglosa duct cyst with bronchiectasis, gallbladder mucocele, basal cell carcinoma, and mammary tumor.

The step of classifying the risk group may be classified according to the number of biomarkers in which the biomarker measurement value exceeds a reference value.

The classification is classified as a risk group when the number of biomarkers is one, a high risk group when two, and a very high risk group when three.

The present invention proposes a method for providing supplementary diagnostic information of various tumor diseases through examination of the three biomarkers as described above in a companion animal. In humans, various biomarkers, such as AFP, CEA, PSA, and CA-12, are used for screening or prognostic testing in order to diagnose malignant tumors. Examination of these tumor-specific biomarkers has been proven as an auxiliary diagnostic means by many literatures.

In humans, primary diagnosis is performed through imaging, endoscopy, and biopsy, and tumors are identified, and diagnosis is performed by obtaining supplementary information through biomarker tests specific to the tumor.

However, in animals, unlike humans, it is difficult to perform direct tumor tests such as imaging and biopsy due to cost, technical problems, and social problems. In view of this, the present inventor proposes a method of providing supplementary diagnostic information through a combination of three general biomarker tests and test results, although the tumor disease of the companion animal is not complete.

The biomarker applied in the embodiment of the present invention is as follows.

1. Canine C-reactive protein (cCRP)

cCRP is an Acute phase Protein (APP) protein produced in the liver at the beginning of an acute inflammatory reaction, and rapidly increases in blood when an inflammatory reaction or infection occurs. Therefore, it can be used for monitoring before and after infection or surgery by rapidly responding to inflammatory reactions or tissue damage in the body. Since inflammation is a basic response to many abnormal conditions, CRP testing is relatively specific for detecting the presence of systemic inflammation, but it does not necessarily provide information about the cause of systemic inflammation. It is known that CRP levels are elevated in dogs with viral infections, dogs with active bacterial or fungal infections, dogs with chronic inflammatory diseases such as some types of arthritis, and sometimes dogs with cancer. Therefore, if the inflammation caused by cancer occurs, it is thought that it will increase in companion animal cancer.

2. D-dimer

As an important test method for diagnosing intravascular coagulation (DIC), DIC activates intravascular coagulation, followed by fibrinolysis, where D-dimer increases. D-dimer is also used in the diagnosis of deep vein thrombosis (DVT) or pulmonary embolism because it increases in thrombogenic diseases. D-dimer is also likely to increase when cancer cells cause thrombosis.

3. AFP [α-fetoprotein, α-FP]

α-Petoprotein appeared in clinical trials after it was known that it was detected in the blood of primary liver cancer patients. It is confirmed that AFP is made from liver cancer cells, and is called a fetal fetal protein (carcinofetal protein) in the fact that it is a common protein between fetal liver and tumor. It is said that 90% of primary liver cancers are positive for AFP, and primary liver cancers that cannot detect AFP in the blood are made in liver cancer cells but are not secreted. In addition, it is also made in a part of gastric cancer, and hepatitis and cirrhosis transducers sometimes show high levels in the blood due to hot flashes. Fetal cancer of the testicles or ovaries also produces AFP. Therefore, if the inflammation caused by cancer occurs, it is thought that it will increase in companion animal cancer.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1.

Prepare blood samples from healthy control group and control group with malignant tumor. The prepared blood is separated by centrifugation to prepare plasma. Using the cap portion of the capillary cap, make a hole in the cap of the test solution, and then take 50 µl of the sample (plasma) or quality control material (control sample to maintain the quality of the vet chroma) with a pipette and d-Dimer test solution. Put it in the containing tube. Insert the capillary cap completely into the test tube and shake the sample and test solution more than 10 times. Remove the cap of the capillary cap of the sample and the sample test tube, discard the 2 drops by pressing the test tube, and add the next 2 drops to the sample inlet of the cartridge. After installing the dispensed test cartridge in the dedicated equipment, start the inspection. After 12 minutes, it is measured automatically. Confirm the measurement result (screening test result) of Bet chroma, a device dedicated to fluorescent LFI (Lateral flow immunoassay).

Example 2.

The same procedure as in Example 1 was performed, but instead of the d-Dimer test solution, a tube containing AFP test solution was used. After installing the dispensed test cartridge in the dedicated equipment, start the inspection. Measured automatically after 15 minutes

Example 3.

Performed in the same manner as in Example 1, but taking 10 µl of the amount of the sample, using a tube containing the cCRP test solution instead of the d-Dimer test solution, and mounting the dispensed test cartridge on a dedicated device for inspection Start, but after 3 minutes, check the result.

The measurement results performed in Examples 1 to 3 were as shown in Table 1 below.

■ Measurement results of three types of markers in companion animal samples Diagnostic Decision Screening test results (mean concentration, average value) D-dimer [ng / ml] AFP [ng / ml] cCRP [mg / L] No. of Samples Epithelial cell cancer SCC (Squamous-cell carcinoma), TCC (transitional cell carcinoma) 1575.01 81.05 51.6 8 Anal tumor Anal sac adenocarcinoma, perianal adeno carcinoma 1607.09 119.68 44.58 6 Lung tumor Lung adenocarcinoma, Pulmonary adenosquamous cell carcinima 4744.23 59.36 119.03 4 Liver cancer Hepatocellular carcinoma (HCC) 3081.97 876.12 43.29 14 Lymphoma Multicentric lymphoma, lymphoma 179.77 141.18 4.74 24 Carotid cancer Carotid body tumor 169.69 46.45 5.58 4 Nasal cell cancer Nasal cell carcinoma 931.64 66.7 12.63 5 Ovarian cancer Ovarian carcinoma 3058.38 74.56 59.67 3 Oral melanoma, mastocytoma melanoma in oral / Mast cell tumor 143.34 31.57 26.16 3 Bronchiectasis, thyroid cyst Thyroglosa duct cyst with bronchiectasis 1077.66 164.25 299.3 2 Gallbladder mucous cyst gallbladder mucocele 8738 51.63 123.61 5 Basal cell tumor basal cell carcinoma 66.67 98.54 16.54 One Tumor samples Cancer group 2114.45 150.93 67.23 79 Healthy individuals Healthy control 149.69 71.09 4.97 30 Reference value Human 250 70 10 -

At this time, the tumor test reference concentration, which is a standard for comparing the measured values of Examples 1 to 3, is cut off for each marker of a standard having a sensitivity of 80% or more, and the corresponding concentrations are shown in Table 2 below.

Biomarker unit Detection range Normal value Tumor diagnosis P value AUC responsiveness Specificity D-Dimer ng / mL 50-10.000 <250 > = 66.46 0.004 0.693 80% 50% AFP ng / mL 5-350 <20 > = 39.24 0.706 0.527 80% 27% cCRP mg / L 5-500 <10 > = 2.81 0.245 0.583 80% 23%

The population of the normal group measured in Table 2 was 30 dogs, and the population of the disease group (tumor disease) was 79 dogs, and MediCalc. Was used for the statistical program using the ROC curve analysis.

Calculation example 1.

As described above, cut off concentration based on 80% sensitivity in D-dimer canine tumor disease is 66.46 ng / ml, sensitivity in AFP canine tumor disease is 39.24 ng / ml in cutoff concentration based on 80%, cCRP sensitivity in canine tumor disease. It was confirmed that the cut off concentration based on 80% was 2.81 ng / ml.

The average value of each biomarker measured based on this is shown in Table 3 below.

D-dimer AFP cCRP Diagnostic Decision Diagnostic Decision Diagnostic Decision Vet chroma measurement Positive Negative Positive Negative Positive Negative Positive 64 15 64 23 63 23 Negative 15 15 15 7 16 7 Sensitivity (Sen.) 81% 81% 80% Specificity (Sep.) 50% 23% 23% Accuracy 72% 65% 64% Positive predictive rate (PPV)
= TP / (TP + FP) 81% 74% 73%

 Referring to Table 3, items that can be confirmed are as follows.

The positive item means the number of individuals determined to be tumor disease. In order to explain the meaning of positive in the results of vet chroma measurement, AFP, D-dimer. After measuring the cCRP, the cut-off concentration based on 80% sensitivity is calculated by the Medcalc.statistics program, and it means the individual higher than the concentration, and the negative item means the lower individual than the selected cut-off concentration, which is the upper positive judgment criterion. .

The sensitivity is a probability of being positively judged in a population with tumor disease, that is, a probability of being diagnosed as positive as a result of measuring Vet chroma in a patient with tumor disease.

The specificity is a probability of receiving a negative judgment in a normal group of individuals, that is, a probability of being diagnosed as a negative in the normal group of individuals as a result of measuring Vet chroma.

The above accuracy (Accuracy) is a probability that the population with tumor disease is judged as positive in the entire population, and the probability that the normal group is judged as Negative, that is, the probability that the diagnosis name and the Vet chroma positive, and the negative judgment rate match. Means

The positive predictive rate is a probability that there is a tumor disease of a diagnosis in a positive individual group, and the calculation formula and meaning will be described as follows.

Diagnostic Decision
Positive Diagnostic Decision
Negative Sub Total Vet chroma Positive True positive (TP) False Positive (FN) TP + FP Vet chroma Negative False Negative (FN) False Negative (FN) FN + TN Sub Total TP + FN FP + TN TP + FP + FN + TN Sensitivity Probability of being positive in the diagnostic positive group as a result of measuring Vet chroma TP / (TP + FN) * 100 Specificity Probability of negative judgment as a result of Vet chroma measurement in diagnostic negative TN / (FP + TN) * 100 Accuracy Probability of negatively determining normal disease with positive tumor disease in all subjects TP + TN / (TP + FP + FN + TN) * 100 Positive predictive value
(PPV, positive prediction rate) Probability of tumor disease (Positive) in diagnosis in positive subject group by Vet chroma test TP / (TP + FP) * 100

When the positive prediction rate is confirmed with reference to Table 3, the 81% oncology positive estimation rate can be diagnosed through the test of the D-dimer marker, and the 74% oncology positive estimation rate can be diagnosed through the test of the AFP marker. And, through the examination of the cCRP marker, it was confirmed that the positive estimation rate of 73% tumor disease can be diagnosed. As described above, individuals who are recognized as positive in each marker and can diagnose a positive estimation rate can be classified and managed as a benign tumor risk group as the probability of a benign tumor increases.

Calculation Example 2.

After detecting the markers measured in Examples 1 to 3 by a single test and classifying the results of the two classifications, if any one of the three markers matches the results in the tumor diagnosis, it is recognized as a positive or negative, as shown in Table 4. As a result of analyzing the sensitivity, specificity, and positive positive prediction rate, the positive prediction rate as shown in Table 5 below was obtained.

D-dimer-AFP D-dimer-cCRP AFP-cCRP Diagnostic Decision Diagnostic Decision Diagnostic Decision Vet chroma measurement Positive Negative Positive Negative Positive Negative Positive 74 14 75 10 75 19 Negative 5 16 4 20 4 11 Sensitivity (Sen.) 94% 95% 95% Specificity (Sep.) 53% 67% 37% Accuracy 83% 87% 79% Positive predictive rate (PPV) = TP / (TP + FP) 84% 88% 80%

If the positive predictive rate is confirmed with reference to Table 5, 84% of the tumor disease positive estimation rate can be diagnosed through screening of D-dimer and AFP (single marker D-dimer, more positive than AFP (81, 74%)). Diagnosis rate is increased), 88-% tumor disease positive predictive rate can be diagnosed through screening tests of D-dimer and cCRP (positive diagnosis rate is higher than that of single marker D-dimer, cCRP (81, 73%)), and screening of AFP and cCRP Through this, it can be confirmed that 80% tumor disease positive predictive rate can be diagnosed (positive diagnosis rate is higher than that of the single marker AFP and cCRP (74, 73%) tests). The individuals recognized as positive in the two markers can be managed as classified as a high-risk group of benign tumors as the probability of a weak tumor is higher than the individuals recognized as positive in one marker.

Calculation example 3.

Based on the results obtained by detecting and measuring the markers measured in Examples 1 to 3 with a single test, if all three markers match the results in the tumor diagnosis, they are recognized as Potential and Negative, and sensitivity, specificity, and positive are shown in Table 4. As a result of analyzing the positive prediction rate, the positive prediction rate as shown in Table 6 below was obtained.

D-dimer-AFP-cCRP Diagnostic Decision Vet chroma Positive Negative Positive 77 9 Negative 2 21 Sensitivity (Sen.) 97% Specificity (Sep.) 70% Accuracy 90% Positive Positive Prediction Rate (PPV) = TP / (TP + FP) 90%

Referring to Table 6, the predictive rate of positive for tumor disease of companion animals is 90% when simultaneously testing three markers containing D-dimer, AFP, and cCRP, which is significantly higher than that of a single marker or two markers combined testing. You can check. Thus, individuals recognized as positive in three markers can be managed as classified as benign tumor ultra-high risk groups as the probability of being a weak tumor is higher than those recognized as positive in two or fewer markers.

As described above, since a specific part of the present invention has been described in detail, it is obvious to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

100 Lateral flow analysis strip 102 Housing 104 Sample port
110 Porous Matrix 120 Sample Pad 140 Test Line
160 Control line 170 Absorption pad

Claims (5)

In the biomarker test method for the diagnosis of tumor diseases in companion animals,
Generating a measurement value for each biomarker by measuring a plurality of biomarkers in a sample collected from a companion animal;
Comparing the biomarker measurement value with a corresponding reference value; And
And determining the likelihood of the tumor disease of the companion animal according to the number of biomarkers where the biomarker measurement value exceeds the reference value.
According to claim 1,
The companion animal,
Biomarker test method for the secondary diagnosis of tumor disease in a companion animal, characterized in that the mammal.
According to claim 1,
The sample,
Biomarker test method for the secondary diagnosis of tumor disease in companion animals, characterized by comprising cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.
According to claim 1,
The biomarker measurement,
Biomarker test method for the secondary diagnosis of tumor disease in companion animals, characterized in that is performed through a fluorescent LFI (Lateral flow immunoassay, lateral flow immunoassay).
According to claim 1,
The biomarker,
D-dimer, AFP or cCRP protein biomarker test method for the secondary diagnosis of tumor disease in a companion animal, characterized in that at least one selected from the group consisting of.

Images