KR102407703B1 - Biomarker Test Method for Ancillary Diagnosis of Companion Tumor Diseases - Google Patents

Abstract

The present invention provides a method for examining tumor diseases in companion animals, which uses three biomarkers in combination for the auxiliary diagnosis of tumor diseases in companion animals. According to the present invention as described above, By using a method that uses a combination of three biomarkers for auxiliary diagnosis, there is an effect of increasing the positive predictive rate of tumor disease, which detects various tumor diseases in companion animals, from 73% to 90%.

Description

{Biomarker Test Method for Ancillary Diagnosis of Companion Tumor Diseases}

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

Unlike humans, oncological diseases in animals do not have auxiliary diagnostic means such as regular check-ups, such as detection and testing of biomarkers suitable for tumor diseases. Animals have limitations in that they cannot routinely perform detailed examinations such as plastic examinations and biopsies in general veterinary hospitals. In general, even in the case of humans, if biomarkers, which are indicators that can detect changes in the body using proteins, DNA, RNA (ribonucleic acid), metabolites, etc. Measure objectively. These biomarkers can also be applied to companion animals, and since tumor diseases generally accompany various inflammatory and vascular changes in companion animals, these biomarkers can help in the diagnosis and prognosis of tumor diseases.

However, the diagnosis, prevention, and management of companion animal tumor disease still have limitations in cost and system that cannot be used routinely, unlike human tumor disease management. A method for detecting tumors was not suggested.

In the case of humans, the regular check-up system provides health care for the elderly population by preventing various diseases and oncological diseases. The reality is that this is not common and the number of examination items is insufficient.

Accordingly, the present invention intends to present an auxiliary diagnostic method capable of diagnosing a tumor disease of companion animals with high accuracy by using biomarkers in combination.

SUMMARY OF THE INVENTION It is an object of the present invention to propose a diagnostic method capable of diagnosing various tumor diseases in companion animals with simple and high accuracy by performing three types of biomarker tests with the blood of companion animals.

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

According to the present invention as described above, by using three biomarkers for auxiliary diagnosis of tumor diseases in companion animals in combination, the positive predictive rate of tumor diseases for detecting various tumor diseases in companion animals is reduced from the existing 73%. It has the effect of increasing up 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 test method according to an embodiment of the present invention.
3 is a view for explaining an AFP inspection method according to an embodiment of the present invention.
4 shows a screen of dedicated equipment 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 for explaining a reagent applied according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

One aspect of the present invention provides a biomarker test method for auxiliary diagnosis of tumor diseases in companion animals.

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

The companion animal may be a mammal, preferably a dog.

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

The biomarker measurement may be performed using a fluorescence reader, which is an optical analysis device, and preferably, fluorescence LFI. The fluorescence LFI is an immunological method for detecting a target using an antibody in the same principle as ELISA. However, in general ELISA, an enzyme is labeled on the antibody to detect it, whereas the fluorescence LFI is a fluorescence labeling method to detect the fluorescence attached to the sample. Quantify the protein concentration by reading and converting to quantification. More preferably, Vet chroma may be used in the fluorescent LFI device.

When the analysis is performed through the fluorescence LFI, a sample is applied to the lateral flow analysis strip for detection.

The configuration of the lateral flow analysis strip 100 is shown in FIG. 6 . As shown, the lateral flow analysis strip 100 is formed with a porous matrix 110 allowing capillary flow in the housing 102 formed by being vertically coupled. A solution in which the sample and the detection buffer are mixed is applied to the sample pad 120 through the sample port 104 and flows into the porous matrix 110 . A test line 140 and a control line 160 are positioned on the porous matrix 110 in a downstream direction 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. A capture agent can be a specific antibody that is responsive to an analyte, an antigen, or an oligonucleotide that hybridizes to a nucleic acid of constant sequence. Preferably, the capture agent may be a D-dimer, AFP or cCRP protein. An absorbent pad 170 is disposed at the downstream end of the porous matrix 110 . Absorbent pad 170 absorbs excess liquid sample applied to sample port 104 and maintains lateral flow along porous matrix 110 .

The control line 160 may be located upstream or downstream of the test line 140 . A positive or negative control reagent or capture agent is immobilized on the control line 160 .

Fluorescent labels detectable in fluorescent LFI include gold nanoparticles, colored latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, and organic fluorophores. ) can be selected from the group consisting of Preferably, among the up-converting phosphors, 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 (Ytterbium, Yb(Yb ₂ O ₃ )) atoms may be conjugated to a fluorescent conjugated antibody.

One or more of the biomarkers may be selected from the group consisting of D-dimer, AFP, and cCRP proteins, and it is preferable to select and use all three types of the biomarkers.

The reference value is a value serving as a standard for diagnosing tumors in companion animals, and is a value set based on a cut-off concentration value of the biomarker. In this case, the cutoff concentration value serving as the setting standard is the biomarker concentration value set as the reference when the sensitivity of the biomarker is 80%.

The cutoff concentration value is different for each biomarker, so that D-dimer is 66.46 ng/ml or more, AFP is 39.24 ng/ml or more, and cCRP is 2.81 ng/ml or more. At this time, when the measured value of the biomarker is equal to or greater than each cutoff concentration value, it is possible to diagnose whether the companion animal is diagnosed with a tumor.

Tumors that can be diagnosed include epithelial cell carcinoma (SCC (Squamous-cell carcinoma), TCC (transitional cell carcinoma)), 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 gland tumor.

The classifying of the risk group may include classifying according to the number of biomarkers for which the measured value of the biomarker exceeds a reference value.

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

The present invention provides a method of providing auxiliary diagnostic information for various tumor diseases through the examination of the above three biomarkers in companion animals. In humans, various biomarkers such as AFP, CEA, PSA, and CA-12 are used for screening tests or prognostic tests depending on the type and stage of the tumor for diagnosis of malignancy. The effectiveness of these tumor-specific biomarker tests as an auxiliary diagnostic tool has already been proven by many literatures.

In humans, the primary diagnosis is performed through imaging, endoscopy, and biopsy, the tumor is specified, and the diagnosis is performed by acquiring auxiliary information through the corresponding tumor-specific biomarker test.

However, unlike humans, it is difficult to perform direct oncology tests such as imaging and biopsy in animals due to cost, technical problems, and social problems. The present inventors, paying attention to this point, propose a method of providing auxiliary diagnostic information through a combination of three general biomarker test results, although it is not perfect for a companion animal's tumor disease.

The biomarkers applied in the examples of the present invention are as follows.

1. Canine C-reactive protein (cCRP)

cCRP is an acute phase protein (APP) produced in the liver at the beginning of an acute inflammatory response, and it increases rapidly in the blood when an inflammatory response 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 fundamental response to many abnormal conditions, the CRP test, while relatively specific for detecting the presence of systemic inflammation, does not necessarily provide information about the cause of systemic inflammation. CRP levels are known to be 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, when cancer-induced inflammation occurs, it is expected to increase in companion animal cancer.

2. D-dimer

As one of the important tests for diagnosing panvascular coagulation (DIC), DIC is a state in which the intravascular coagulation mechanism is activated, followed by fibrinolysis, and at this time, D-dimer increases. Since D-dimer is also increased in thrombotic diseases, it is also used in the diagnosis of deep vein thrombosis (DVT) or pulmonary embolism. It is considered that D-dimer is also likely to increase when cancer cell-induced thrombosis occurs.

3. α-fetoprotein [α-fetoprotein: AFP]

After it was known that α-fetoprotein was detected in the blood of patients with primary liver cancer, it came to appear in clinical tests. It has been confirmed that AFP is produced in liver cancer cells, and it is called a carcinofetal protein from the fact that it is a common protein between fetal liver and tumors. It is said that AFP is positive in 90% of primary liver cancers, and even primary liver cancers in which AFP cannot be detected in the blood are produced but not secreted by liver cancer cells. In addition, it is made in some stomach cancers, and there are cases where high levels in the blood are transiently high in hepatitis and cirrhosis patients. Fetal cancer of the testes (睾丸) or ovary (胎兒性癌) also produces AFP. Therefore, when cancer-induced inflammation occurs, it is expected to 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, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1.

Blood is collected and prepared from the healthy experimental group, the control group, and the experimental group with malignant tumors. The prepared blood is separated by centrifugation to prepare plasma. After making a hole in the test solution cap using the stopper of the capillary cap, take 50 μl of the sample (plasma) or quality control material (control sample checked to maintain the quality of vet chroma) with a pipette, d -Put into the tube containing the dimer test solution. Fully insert the capillary cap into the test solution tube and shake it at least 10 times to ensure that the sample and test solution are well settled. Remove the cap of the capillary cap of the sample and test solution tube, press the test solution tube to discard 2 drops, and then put the next 2 drops into the sample inlet of the cartridge. After loading the dispensed test cartridge into the dedicated equipment, the test starts. After 12 minutes, it is automatically measured. Check the measurement result (screening test result) of Vet chroma, a fluorescence LFI-only equipment, on the screen.

Example 2.

It was carried out in the same manner as in Example 1, except that the tube containing the AFP test solution was used instead of the d-Dimer test solution. After loading the dispensed test cartridge into the dedicated equipment, the test starts. After 15 minutes, it is automatically measured

Example 3.

Perform the same as in Example 1, except that 10 μl of the amount of the sample is taken, a tube containing the cCRP test solution is used instead of the d-Dimer test solution, and the dispensed test cartridge is mounted on the dedicated equipment and then the test is performed. Start, but check the result after 3 minutes.

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

Diagnostic Decision Screening test results
(mean concentration, mean value) D-dimer [ng/ml] AFP [ng/ml] cCRP [mg/L] No. of Samples epithelial cell carcinoma Squamous-cell carcinoma (SCC), transitional cell carcinoma (TCC) 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 artery cancer carotid body tumor 169.69 46.45 5.58 4 nasal cell carcinoma 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 mucinous cyst gallbladder mucocele 8738 51.63 123.61 5 basal cell tumor basal cell carcinoma 66.67 98.54 16.54 One whole tumor specimen Cancer group 2114.45 150.93 67.23 79 healthy object healthy control 149.69 71.09 4.97 30 reference value Human 250 70 10 -

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

biomarker unit Detection range normal 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 number of dogs in the normal group measured in Table 2 was 30 dogs, and the number of dogs in the disease group (tumor disease) was 79 dogs, and MediCalc was used as a statistical program for ROC curve analysis.

Calculation example 1.

As described above, the 80% sensitivity cutoff concentration in D-dimer canine tumor disease is 66.46 ng/ml, the sensitivity 80% reference cutoff concentration in AFP dog tumor disease is 39.24 ng/ml, and the sensitivity 80% in cCRP dog tumor disease It was confirmed that the reference cutoff concentration 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, the items that can be checked are as follows.

The positive item means the number of individuals determined to be a tumor disease. To explain the meaning of positive in the vet chroma measurement result, AFP, D-dimer. Measuring cCRP and calculating the cutoff concentration, which is the standard of 80% sensitivity, with the Medcalc statistical program, means an object higher than that concentration, and the negative item means an object lower than the selected cutoff concentration, which is the upper positive judgment criterion.

The sensitivity is the probability of being positive in the population with oncological disease, that is, the probability of being diagnosed as Positive as the result of Vet chroma measurement in the patient group with oncological disease.

The specificity is the probability of being negative in the normal group, that is, the probability of being diagnosed as negative in the normal group as a result of bet chroma measurement.

The accuracy is the probability of judging the population with tumor disease as Positive (positive) in the entire population and judging the normal group as Negative, that is, the probability that the diagnosis name matches the positive and negative bet chroma means

The positive predictive rate is the probability of having a diagnosed tumor disease in a positive population, 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 as a result of Vet chroma measurement in the positive diagnosis group TP/(TP+FN)*100 Specificity Probability of being negative as a result of Vet chroma measurement in Diagnosis Negative TN/(FP+TN)*100 Accuracy
(accuracy) Probability of diagnosing a tumor disease as positive in the entire population and negative in the normal group TP+TN/(TP+FP+FN+TN)*100 positive predictive value
(PPV, positive predictive rate) Probability of positive tumor disease in the positive population based on Vet chroma test TP/(TP+FP)*100

If the positive predictive rate is confirmed with reference to Table 3, it is possible to diagnose 81% tumor disease positive estimated rate through the test of the D-dimer marker, and 74% can diagnose the positive estimated rate of tumor disease through the test for the AFP marker. and it was confirmed that a 73% tumor disease positive estimation rate could be diagnosed through the test of the cCRP marker. As described above, the individual recognized as positive in each marker and capable of diagnosing the estimated positive rate increases the probability of being a benign tumor and can be classified and managed as a benign tumor risk group.

Calculation example 2.

After detecting the markers measured in Examples 1 to 3 with a single test and classifying the confirmed results into two types, if any one of the three markers matches the tumor diagnosis result, it is recognized as positive or negative, and as shown in Table 4 As a result of analyzing the sensitivity, specificity, and positive-positive predictive rate, the positive predictive 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, an 84% tumor disease positive estimate rate can be diagnosed through the screen test of D-dimer and AFP (single marker D-dimer, (81, 74%) of the AFP test) Higher positive diagnosis rate), 88% tumor disease positive predictive rate can be diagnosed through D-dimer and cCRP screen tests (positive diagnosis rate is higher than (81, 73%) of single marker D-dimer and cCRP tests), AFP and It can be confirmed that 80% of the positive predictive rate of tumor disease can be diagnosed through the cCRP screen test (positive diagnosis rate is higher than (74, 73%) of single marker AFP and cCRP tests). Individuals recognized as positive by two markers are more likely to be malignant tumors than individuals recognized as positive by one marker, and thus can be classified and managed as a high-risk group for benign tumors.

Calculation example 3.

Based on the results of detecting and confirming the markers measured in Examples 1 to 3 with a single test, if all three markers match the results in tumor diagnosis, they are recognized as positive and negative, and as shown in Table 4, sensitivity, specificity, and positive As a result of analyzing the positive predictive rate, it was possible to obtain the positive predictive rate as shown in Table 6 below.

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 positive predictive rate for companion animal tumor disease is 90% when simultaneously testing three types of markers including D-dimer, AFP, and cCRP, which is significantly higher than that of a single marker or two marker combination tests. can check Individuals recognized as positive by these three markers have a higher probability of being a malignant tumor than those recognized as positive by two or fewer markers, so they can be classified and managed as a benign tumor very high-risk group.

Above, a specific part of the present invention has been described in detail, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

100 Lateral Flow Analysis Strips
102 housing
104 sample port
110 porous matrix
120 sample pads
140 test line
160 control line
170 absorbent pad

Claims (5)

In the biomarker test method for auxiliary diagnosis of tumor disease in companion animals,
measuring a concentration of each of D-dimer, AFP, and cCRP in the sample by applying a sample collected from a companion animal to a lateral flow analysis strip as biomarkers;
comparing the measured values of the D-dimer, AFP, and cCRP biomarkers with a unique reference value set according to the sensitivity of each biomarker according to the type of tumor; and
determining the possibility of a tumor disease of the companion animal based on the number of biomarkers in which the biomarker measurement value exceeds the reference value
A biomarker test method comprising:
According to claim 1,
The companion animal is a biomarker testing method, characterized in that it is a mammal.
According to claim 1,
The sample is a biomarker test method, characterized in that at least one of cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, and urine.
According to claim 1,
The biomarker measurement is a biomarker test method, characterized in that it is performed through fluorescence LFI. delete

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