KR20220101919A - Method for diagnosing tumor prognosis in companion dogs using the measurement of cell-free DNA - Google Patents

Abstract

The present invention relates to a method for diagnosing tumor prognosis in a companion dog by using measurement of a cell-free nucleic acid and, more specifically, to a method for predicting tumor prognosis in a companion dog, which comprises: a step (a) of measuring a level of cell-free DNA (cfDNA) in a biological sample from a subject; and a step (b) of comparing the measured level of the cfDNA with a cut-off value set by analysis of an ROC curve. In addition, the present invention relates to a method for diagnosing a tumor of a companion dog, which comprises: a step (a) of measuring a level of cell-free DNA (cfDNA) in a biological sample from the subject; and a step (b) of comparing the measured level with a level of cfDNA of a normal control. The method for diagnosing a tumor of a companion dog and predicting tumor prognosis according to the present invention is a method for simply measuring a concentration of cfDNA present in blood of a companion dog, thereby allowing a user to rapidly and precisely diagnose tumor prognosis including determining whether a tumor develops or not and predicting a survival rate of the companion dog with a tumor.

Description

Method for diagnosing tumor prognosis in companion dogs through measurement of cell-free nucleic acid {Method for diagnosing tumor prognosis in companion dogs using the measurement of cell-free DNA}

The present invention relates to a method for predicting or diagnosing a tumor prognosis in a dog through measurement of cell-free nucleic acid.

Currently, in Korea, one-to-one households are moving beyond nuclear families, and the number of people raising companion animals is increasing. Dogs account for the largest portion of companion animals, and dogs are known to live on average 11 to 13 years, but recently, their lifespan has been extended to 15 to 20 years due to improvements in public health and medical care. In addition, as the level of public awareness has improved, companion animals are often regarded as human beings on a level similar to that of humans.

However, since most companion animals, including many dogs, live in houses, they tend to be easily exposed to diseases due to lack of exercise and a lot of stress. In particular, in companion animals, tumor diseases, unlike humans, do not have diagnostic means such as appropriate biomarkers for tumors, such as regular checkups, and there is a limitation in that detailed examinations such as plastic examinations and biopsies cannot be routinely performed at veterinary hospitals.

Therefore, it is necessary to develop a technology that can quickly and accurately diagnose tumor diseases in companion animals.

On the other hand, cell-free nucleic acid (cell-free DNA; cfDNA) refers to a piece of DNA that does not exist in a cell and is floating in the blood. In particular, it has been reported that cfDNA is increased in the blood of cancer-stricken individuals in humans and animals, because tumor DNA is derived from tumor cell death and necrosis, secretion from active cells, and lysis and circulating cells. In addition, recently, in the field of liquid biopsy, non-invasive analysis of cancer cell-derived nucleic acids present in body fluids is drawing attention as a plasma biomarker for early diagnosis of cancer, evaluation of stages and prognosis, and monitoring of response to anticancer therapy. .

However, veterinary research on cfDNA as a tumor biomarker has not yet been progressed.

Republic of Korea Patent Publication 10-2020-0049647

Accordingly, the present inventors extracted cfDNA from the blood of healthy dogs, dogs with non-neoplastic diseases and dogs with tumors, respectively, and measured the concentration of cfDNA in the blood for each experimental group to analyze the correlation between tumor and cfDNA in dogs, Furthermore, in the case of a dog with a tumor, the present invention was completed by establishing a new diagnostic method that can quantitatively determine the diagnosis of the dog's tumor prognosis by analyzing the correlation between the response to tumor treatment and the change in cfDNA concentration.

Therefore, an object of the present invention, (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; and (b) comparing the measured cfDNA level with a cut-off value set by ROC curve analysis, to provide a method for predicting tumor prognosis in dogs.

Another object of the present invention, (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; And (b) to provide a method for diagnosing a canine tumor, including the step of comparing it with the cfDNA level of a normal control.

In order to achieve the above object of the present invention, the present invention (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; and (b) comparing the measured cfDNA level with a cut-off value set by ROC curve analysis.

In one embodiment of the present invention, the biological sample may be whole blood, plasma or serum.

In one embodiment of the present invention, if the measured cfDNA level is lower than the cut-off value, it is determined that the survival probability of the dog is high, and the measured cfDNA level is cut-off (cut-off) off) may further include the step of determining that the survival probability of the dog is low when it is higher than the value.

In an embodiment of the present invention, the set cut-off value may be 1,247.5 ug/L.

In one embodiment of the present invention, when the measured cfDNA level is lower than the cut-off value of 1,247.5 ug/L, the survival probability of the dog can be predicted to be 80% or more.

In one embodiment of the present invention, when the measured cfDNA level is higher than the cut-off value of 1,247.5 ug/L, the survival probability of the dog can be predicted to be less than 30%.

In one embodiment of the present invention, the tumor is hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell carcinoma, renal cell carcinoma, transitional epithelial cancer, intestinal adenocarcinoma, lymphoma, leukemia, angiosarcoma, stromal sarcoma, It may be selected from the group consisting of oral leiomyosarcoma, gastrointestinal stromal tumor (GIST), multilobed bone tumor, mast cell tumor, pheochromocytoma, and peripheral nerve sheath tumor.

In addition, the present invention, (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from the subject; And (b) provides a method for diagnosing a canine tumor, including the step of comparing it with the cfDNA level of a normal control.

In one embodiment of the present invention, the biological sample may be whole blood, plasma or serum.

In one embodiment of the present invention, when the cfDNA level derived from the subject is higher than the cfDNA level of the normal control, the step of determining that a tumor has occurred may be further included.

In one embodiment of the present invention, when the cfDNA level derived from the subject is higher than the concentration of 1,553.9 ug/L, it can be determined that a tumor has occurred.

In one embodiment of the present invention, the tumor is hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell carcinoma, renal cell carcinoma, transitional epithelial cancer, intestinal adenocarcinoma, lymphoma, leukemia, angiosarcoma, stromal sarcoma, It may be selected from the group consisting of oral leiomyosarcoma, gastrointestinal stromal tumor (GIST), multilobed bone tumor, mast cell tumor, pheochromocytoma, and peripheral nerve sheath tumor.

The present invention confirmed that the concentration of cfDNA (cell-free DNA) in the blood of tumor-inducing dogs was increased compared to healthy dogs. As it is confirmed that it is significantly higher, it has the effect of diagnosing whether a dog develops a tumor by measuring the concentration of cfDNA. Furthermore, the present invention identified a cut-off value that can predict the prognosis and survival rate of a tumor by analyzing the change tendency between the response to treatment and cfDNA during the tumor treatment period. Compared with the off value of 1,247.5 ug/L, it is possible to objectively and accurately predict and diagnose the prognosis and survival rate for tumor treatment.

1 is a result of measuring the average parameters of clinical data for a healthy group, a non-neoplastic disease group and a tumor disease group in an embodiment of the present invention, (A) is a white blood cell (WBC), neutrophil (NEU), C- It is the result of measuring the average concentration of reactive protein (CRP) and albumin, (B) is the result of measuring the average concentration of red blood cells (RBC), hemoglobin (Hgb) and hematocrit (Hct), (C) is alanine aminotransfer The results of measuring the average liver enzyme activity of rase (ALT), alkaline phosphatase (ALKP) and gamma-glutamyl transpeptidase (GGT) are shown. All data are expressed as mean±SD.
Figure 2 shows the results of measuring the average concentration of cfDNA between the healthy group and the clinically diseased group, (A) is a comparison of the average cfDNA concentration of the healthy group and the disease group (composed of a non-neoplastic underlying disease group and a tumor disease group) and (B) compares the mean cfDNA concentrations for the healthy group, non-neoplastic disease group, and tumor disease group.
3 shows the results of measuring cfDNA concentrations in the healthy group, non-neoplastic underlying disease group, and various types of tumor disease group.
Figure 4 shows the ROC (receiver operating characteristic) analysis curve that identifies the diagnostic sensitivity and 1-specificity for cfDNA as a prognostic marker for dogs with tumor disease, and the 95% confidence interval for AUC is 0.701 (0.523-) for cfDNA. 0.880) and was statistically significant (P <0.05).
5 is a result of analyzing the correlation between cfDNA and survival rate in dogs with oncology. The Kaplan-Meier plot using a cfDNA concentration of 1,247.5 μg/L as a cut-off showed a statistically significant difference (n = 38). , P < 0.05).
6 is a result of comparing the cfDNA concentration according to the treatment response. The cfDNA concentration is indicated by a solid line, and the sum of LDs at various time points is indicated by a dotted line. In (A)-(C), the left vertical axis of each graph represents the cfDNA concentration, and the right vertical axis represents the sum of LDs. In addition, in each of all graphs, the horizontal axis labels indicate the plasma sampling date and tumor response status, and (A) to (J) are analysis results for dogs having tumors corresponding to dogs no.1 to no.10, respectively.
7 is a result of analyzing the correlation between the sum of LD and cfDNA concentration in dogs with multicentric lymphoma.

The present invention is a technology that provides a new method for diagnosing a dog's tumor and predicting the prognosis of the tumor. Specifically, the present invention measures the level of cfDNA (Cell-free DNA) in a dog's biological sample, and the dog's tumor It is characterized in that it identified the cut-off value for the cfDNA level as an objective indicator that can predict the diagnosis or prognosis.

As mentioned in the prior art, there is still no method for objectively and accurately diagnosing a tumor in a companion animal.

Accordingly, the present inventors isolated cfDNA (Cell-free DNA) from blood from the experimental group classified into a healthy group, a non-neoplastic disease group, and a tumor disease group for dogs, and quantitatively measures the concentration thereof, A quantitative reference value, that is, a cut-off value, which is a criterion for predicting the prognosis of a tumor was identified.

In particular, in the present invention, cfDNA was used as a means for diagnosing canine tumors. The term “cfDNA (Cell-free DNA)” refers to DNA derived from cells present in plasma, and small cell-free DNA or cell-free DNA in blood. is mixed Such cell-free DNA is being used in a non-invasive test to check for abnormalities in fetal sex, genetic variation, or chromosome number. However, it has not yet been used for diagnosing tumors in dogs or predicting the prognosis of tumors.

On the other hand, according to an embodiment of the present invention, it was confirmed whether it was possible to diagnose tumor occurrence in dogs through analysis of the level (concentration) of cfDNA. After dividing into group and tumor disease group, and measuring the concentration of cfDNA in the blood from each dog, and analyzing the average concentration, the healthy group showed 877.8 ± 181.7 μg/L, the non-neoplastic disease group was 1,154.4 ± 326.5 μg/L, In the tumor disease group, an average cfDNA concentration of 1,553.9 ± 544.3 μg/L was measured.

Through this, the present inventors were able to confirm whether or not tumors occurred in dogs by measuring the level of cfDNA in the blood, and furthermore, it was found that non-neoplastic diseases and tumor diseases can be differentiated and diagnosed.

In another embodiment of the present invention, cfDNA concentrations were compared and analyzed for disease groups of various tumor types. For this purpose, lymphoma (n = 13), carcinoma (n = 15), sarcoma (n = 5), etc. cfDNA concentrations in tumors (n = 5) were analyzed.

As a result, the cfDNA concentration in the tumor disease group was significantly higher than in the healthy group, and the cfDNA concentration in the lymphoma was increased to the highest level among the disease groups of various tumor types.

Therefore, the present invention, (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; And (b) it may provide a method for diagnosing a canine tumor comprising the step of comparing it with the cfDNA level of a normal control.

In the present invention, the biological sample derived from the subject may be whole blood (blood), plasma or serum.

Measuring the level of cfDNA (Cell-free DNA) in the biological sample derived from the subject is performed by isolating cfDNA from the biological sample and measuring the concentration. Separation of cfDNA involves extracting nucleic acids from the sample , separation and purification can be carried out through methods known in the art. In an embodiment of the present invention, a plasma or serum sample was centrifuged at 16,000 × g and 4 °C for 10 minutes to obtain cell-free plasma or serum, and the upper layer containing purified cfDNA after centrifugation After obtaining a liquid, it was used for quantitative analysis.

In addition, the canine tumors that can be diagnosed in the present invention include, but are not limited to, hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell carcinoma, renal cell carcinoma, transitional epithelial carcinoma, intestinal adenocarcinoma, lymphoma , leukemia, angiosarcoma, stromal sarcoma, oral leiomyosarcoma, gastrointestinal stromal tumor (GIST), multilobed bone tumor, mast cell tumor, pheochromocytoma, and peripheral nerve sheath tumor. Preferably it may be a lymphoma.

In the present invention, the term “diagnosis” in a broad sense means judging the actual condition of a diseased individual in all aspects. The content of the judgment includes the disease name, etiology, disease type, severity, detailed mode of the disease, and the presence or absence of complications.

Furthermore, the present inventors conducted an experiment to determine whether the dog's tumor prognosis can be predicted by measuring the level of cfDNA in the dog's blood. According to an embodiment of the present invention, plasma cfDNA for 38 tumor disease groups was obtained. Analysis was conducted during the observation period (up to 60 weeks).

As a result, an ROC curve was generated based on the cfDNA level of dogs in each experimental group, and as a result of analyzing the cut-off value for the cfDNA concentration, the cut-off value was measured to be 1,247.5 μg/L. As a result of analyzing the survival curve for dogs diagnosed with tumors using the cut-off value, the group with a cfDNA concentration lower than the cut-off value of 1,247.5 μg/L had a survival probability of 82.1%, indicating a relatively high survival rate. On the other hand, the group with a cfDNA concentration higher than 1,247.5 μg/L showed a low survival rate with a survival probability of 26.5%.

Therefore, the present inventors found that it is possible to predict the tumor prognosis of the dog by measuring the cfDNA concentration in the blood even for a dog with a tumor.

Accordingly, the present invention comprises the steps of: (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; and (b) comparing the measured cfDNA level with a cut-off value set by Receiver Operation Characteristic (ROC) curve analysis.

In the present invention, the “prognosis” refers to an object for a specific disease or condition, that is, determining whether a test subject will have a disease in the future, determining the responsiveness of a test subject to treatment, or determining the efficacy of a treatment It includes both monitoring the condition of an entity to provide information.

In addition, the “tumor prognosis” refers to determining whether the subject develops, metastases, drug reactivity, resistance, survival rate, etc. after treatment of the tumor.

In addition, the method determines that the survival probability of the dog is high when the measured cfDNA level is lower than the cut-off value, and the measured cfDNA level is higher than the cut-off value In this case, the method may further include determining that the survival probability of the dog is low.

In this case, the set cut-off value may be 1,247.5 ug/L.

More specifically, when the measured cfDNA level is lower than the cut-off value of 1,247.5 ug/L, the survival probability of the dog can be predicted to be 80% or more, and the measured cfDNA level is cut- If it is higher than the cut-off value of 1,247.5 ug/L, the survival probability of the dog can be predicted to be less than 30%.

In addition, the above tumor diseases whose prognosis can be predicted by measuring cfDNA levels include, but are not limited to, hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell cancer, renal cell cancer, transitional epithelial cancer, intestinal adenocarcinoma, It may be selected from the group consisting of lymphoma, leukemia, angiosarcoma, stromal sarcoma, oral leiomyosarcoma, gastrointestinal stromal tumor (GIST), multilobed bone tumor, mast cell tumor, pheochromocytoma, and peripheral nerve sheath tumor.

Hereinafter, the components and technical features of the present invention will be described in more detail through the following examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples.

<Test materials and methods>

1. Experimental design

The experiment in the following example was a randomized in vitro experiment performed on dogs who visited Gyeongsang Animal Medical Center, and the plasma cfDNA concentrations of all dogs were measured and clinical data were analyzed according to disease classification. Institutional Animal Management of Gyeongsang National University (GNU) Experiments were performed with the approval of the Committee (IACUC).

<1> Select case

This experiment was analyzed using blood samples collected from random selection of 120 animals who visited Gyeongsang Animal Medical Center from 2019 to 2020, and control samples obtained from local hospitals in Jeonju and Gwangju were used. Dog breeds, sex, and age were all diverse, and the diagnosis was classified into a tumor disease group, a non-neoplastic disease group, and a normal group (healthy dog group). In addition, clinical data and medical records of all dogs were collected and evaluated together. Data on samples include age, sex, breed, complete blood count (CBC), serum biochemistry, venous blood gas analysis and diagnostic imaging results, underlying disease progression and clinical results. It was defined as dead or euthanized.

<1-1> healthy group

Dogs from healthy owners were used as controls for comparison with dogs with underlying disease. Blood samples were obtained from 35 clinically normal customer-owned dogs delivered to veterinary hospitals in Jeonju and Gwangju. In addition, dogs with a history of current disease, abnormal findings found in regular clinical examinations, or abnormal symptoms in whole blood (CBC), serum biochemistry, or urine analysis were excluded.

<1-2> Non-neoplastic disease group (group with underlying disease)

In the non-tumor group, all dogs who showed clinical symptoms and were diagnosed with an underlying disease other than tumor were included. All non-neoplastic groups underwent physical tests, laboratory tests, and diagnostic imaging analyses. Medical records of the non-neoplastic group included medical records, medical history, physical examination, clinical signs, routine blood analysis, complete urinalysis, and diagnostic images.

Based on these screening tests, several cases were evaluated through additional tests for an accurate diagnosis. Endocarditis was further diagnosed through echocardiography and blood culture when the same causative bacteria were cultured in at least three different sites through venipuncture at 1 hour intervals. Blood parasites such as Mycoplasma haemocanis and Babesia canis were diagnosed by polymerase chain reaction (PCR) analysis. All dogs with immune-mediated hemolytic anemia (IMHA) were identified on the basis of hemolysis by saline agglutination test and blood film test. Canine hyperadrenocorticism (HAC) was diagnosed as pituitary-dependent hyperadrenocorticism (PDH) or adrenal tumor (AT) based on the low-dose dexamethasone inhibition test (LDDST). Dogs with a history of exogenous steroid treatment were examined at least 2 weeks after discontinuation of treatment. Pancreatitis can be diagnosed with abdominal pain, vomiting, diarrhea, A comprehensive diagnosis was made based on clinical signs such as fever and abdominal ultrasound. For acute pancreatitis, response to treatment was monitored via canine pancreatic specific lipase (Spec cPL; canine spec cPL® test, IDEXX Laboratories Inc., Westbrook, ME, USA). Lymphangiectasia and vascular ectasia were confirmed by gastrointestinal endoscopy based on gross lesions and histopathology of biopsy samples. Dogs with calcinosis cutis were confirmed to have a lesion around the foreskin, and a definitive diagnosis was confirmed based on a skin biopsy of the lesion. Finally, bronchomalacia and atherosclerosis were diagnosed based on computed tomography (CT) scans (Canon Aquilion Lightning 160; Canon Medical Systems Corporation, Otawara, Japan).

<1-3> tumor disease group

Although the types of tumors in the tumor group were diverse, we analyzed a single tumor group consisting of 38 dogs with malignant tumors. It was performed based on cytology through needle aspiration). Histopathologically, a definitive diagnosis was made by biopsy or surgical excision. If the dog's tumor was in an area where surgical resection or biopsy was difficult, the diagnosis was made by comprehensively evaluating previous diagnostic methods. Malignant tumors were staged according to tumor type according to the World Health Organization staging system (OwenLN. 1980), including computed tomography, abdominal ultrasound, and blood tests. Clinical staging of canine lymphoma (OwenLN.1980) was based on clinical examination and microneedle aspiration analysis of superficial lymph node, liver and spleen involvement. Bone marrow aspiration was used to evaluate terminal infiltration. All peripheral nodes and other tumor masses were measured in two dimensions, possibly in three dimensions. Observation time was calculated immediately after diagnosis. Blood was repeatedly drawn from 10 dogs with malignant tumors over time to determine if cfDNA levels were associated with disease progression. Medical records of the tumor population were reviewed, including medical records, fertility, sex, medical history, physical examination, clinical signs, routine blood analysis, and diagnostic imaging. Radiation therapy was performed at S Animal Hospital in Yangsan, and Elekta Synergy® (Elekta, Stockholm, Sweden) was used. All radiotherapy protocols were to be applied individually for each dog.

<2> Blood sample collection and processing

All samples from clinically normal dogs and diseased dogs were collected by jugular venipuncture and stored in EDTA tubes or lithium heparin tubes or serum separation tubes. EDTA blood samples were analyzed using an automated hematology analyzer. Heparinized plasma or serum was separated from peripheral blood by centrifugation at 2,000 × g for 5 minutes within 30 minutes of sample collection. The supernatant of plasma or serum was transferred to a plastic cryo tube. Samples from the local veterinary hospital were stored on dry ice and transported to GNU. All samples were stored frozen at a temperature of -80 °C in a cryogenic freezer prior to DNA analysis. Heparinized plasma or serum samples were thawed at room temperature (RT) and centrifuged immediately prior to analysis. Blood samples from multiple oncological dogs were sampled before chemotherapy was administered.

2. Comparative analysis of clinical data

Data collected from medical records included evaluation of all clinical pathologic data including breed, age, sex, weight, history, clinical signs, physical examination results, complete blood count, and serum biochemistry.

<2-1> Clinicopathological parameters

The clinical condition of 120 dogs was performed using a ProCyteDx complete blood count (IDEXX, Westbrook, ME, USA), a blood gas analyzer (Nova pHOX analyzer, Nova Biomedical, Waltham, MA, USA), blood urea nitrogen (BUN), creatinine, phosphorus, glucose, total protein, albumin, globulin, total bilirubin, ALKP, alanine aminotransferase (ALT), GGT, cholesterol , amylase, and lipase were analyzed with a Catalyst Dx chemical analyzer (IDEXX, Westbrook, ME, USA) and a urine analyzer (IDEXX, Westbrook, ME, USA).

<2-2> Evaluation criteria for nodal lymphoma

The length of superficial lymph nodes in dogs with multicentric lymphoma was evaluated as the total number of lymph nodes with a diameter greater than 20 mm compared to the longest diameter of the lymph nodes based on the existing consensus criteria (Vail et al., 2010). Lymph node diameters in dogs with multicentral lymphoma were measured during each physical examination immediately prior to chemotherapy. The raters included two veterinarians. Results were recorded when the difference in the sum of the lymph node diameter (LD) values of each rater was less than 20% according to the existing consensus criteria.

<2-3> Criteria for chemotherapy response evaluation

Response to treatment and recurrence criteria is defined according to the criteria description according to art-known lymphomas ((Vail et al., 2010) and other solid tumors ((Nguyen et al., 2015)) based on physical examination and further examination. The lesion response was evaluated at all visits.

In the evaluation of treatment response for nodular lymphoma, the target lesion was a peripheral lymph node (PLN) with an LD greater than or equal to 20 mm at baseline. Target lesions were selected based on size (PLN with LD) and suitability for accurate repeat measurements (ie, clinically without imaging techniques). LD was measured for each target lesion. The sum of the LDs for all target lesions was calculated and reported as the baseline sum LD. All other lesions were defined as non-target lesions and the length of the lesion was recorded.

The minimum size of the target lesion in the evaluation of the treatment response of solid tumors was defined as follows.

i) Clinical examination: 10 mm based on caliper measurements and digital photos;

ii) CT scan, magnetic resonance imaging (MRI): 10 mm; Maximum slice thickness 5mm,

iii) chest radiograph: 20 mm, and

iv) LN: short central axis > 15mm

All other lesions were considered non-measurable. For example, lesions based on caliper measurements are <10 mm; pathological LN <15 mm shortening; ascites, pleural and pericardial effusions; It was defined as an unmeasurable lesion under conditions such as organ hypertrophy found by physical examination (Nguyen et al., 2015).

3. cfDNA Isolation and Quantitation

Plasma or serum samples were thawed at room temperature for 10 min, and then all samples were centrifuged at 16,000 × g and 4 °C for 10 min to obtain cell-free plasma. Only the supernatant purified after centrifugation was used for cfDNA analysis. cfDNA was quantified with a Qubit dsDNA HS Assay Kit and a Qubit 1.0 fluorometer (Life Sciences, Carlsbad, CA, USA) according to the manufacturer's specifications. Qubit analysis uses a fluorescent dye having high intensity when binding to dsDNA, and the amount of the measured fluorescent dye is proportional to the amount of dsDNA in the sample. The reaction volume used per analysis was 200 µL including 10 µL of plasma or serum samples. The concentration of cfDNA was calculated using the dilution algorithm provided by the manufacturer in Qubit 1.0 (Life Science, Carlsbad, CA, USA). Qubit 1.0 was calibrated with a designated standard before each analysis, and all samples were analyzed twice.

4. Statistical processing

Mean, standard deviation, and significant difference were calculated. The concentrations of cfDNA data were not normally distributed in the Shapiro-Wilk test, so the data were inversely transformed for normalization. Comparisons between categorical variables (eg, disease status, presence of tumors) were performed using the Mann-Whitney U test and comparisons between tumor types were performed using the Kruskal-Wallis test and Dunnett T3 test. The diagnostic value of cfDNA for survival time prediction was evaluated using a receiver operating characteristic (ROC) curve. Kaplan-Meier curve analysis was used to analyze survival curves to determine survival time of tumor groups with cut-off values obtained from ROC. Spearman's correlation test was used to analyze the correlation between the sum of superficial lymph nodes and the DNA concentration of polycentric lymphoma. Correlations between cfDNA values and hematological parameters in subjects with continuously monitored disease progression were analyzed using a linear mixed model. The association between cfDNA concentration and all clinical pathologic variables was analyzed by modifying a multiple linear regression model with stepwise exclusion.

Statistical significance was set at P < 0.05 and P < 0.01, and all statistical evaluations were performed using the statistical software package SPSS (SPSS 25.0, SPSS Inc, Chicago, IL, USA). All graphs were plotted using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA), and all results were expressed as mean ± standard deviation (SD).

<Example 1>

Classification and Characteristics of Dogs

A total of 120 dogs of various breeds, weights and ages were used in the study. These animals were divided into 85 diseased animals and 35 healthy controls according to the presence or absence of underlying disease. According to the presence or absence of oncological disease, 38 animals were classified into oncological disease group and 47 animals into non-tumor disease group.

Clinical data including total blood cell count, blood gas analysis, biochemical analysis, and diagnostic images were analyzed for a total of 85 animals. For 35 healthy controls, clinical records, complete blood cell count and biochemical methods were analyzed. Blood gas and electrolyte analysis was performed in part because blood gas and electrolyte changes may occur during transport and transport of animals.

Specifically, the classification and characteristics of the dogs used in this experiment will be described as follows.

The 35 healthy dogs included 21 males (4 unneutered males) and 14 females (4 unneutered females), with an average age of 5.4 years (range: 1-19 years). The 47 dogs with non-neoplastic disease were 26 males (4 intact) and 21 females (9 intact), with a mean age of 9.5 years (range: 1-17 years). 38 dogs with neoplastic disease had an average age of 11 years (range: 3-20 years), including 11 males and 27 females (4 intact). The age of dogs with neoplastic disease and non-neoplastic disease was significantly higher than that of the healthy control group (mean 11 years for the oncological disease group, 9.5 years for the non-neoplastic disease group, and 5.4 years for the healthy group, respectively, Po0.01 and Po0.05). The most common malignant tumor types according to tumor origin were carcinoma (n=15; 3 hepatocellular carcinomas, 3 ectopic thyroid carcinomas, 2 lung adenocarcinomas, 2 mammary carcinomas, 1 C-cell thyroid carcinoma, 1 squamous cell carcinoma, and renal 1 cell carcinoma, 1 transitional cell carcinoma, 1 intestinal adenocarcinoma), followed by lymphoma or leukemia (n = 13), sarcoma (n = 5, hemangiosarcoma 1 patient, , stromal sarcoma 1 patient, oral 1 leiomyosarcoma, 1 gastrointestinal stromal tumor (GIST) and 1 multilobed bone tumor), mast cell tumor (n = 2), and pheochromocytoma and peripheral nerve sheath tumor in 1 patient, respectively. Characteristic confirmation for each experimental group classified as described above is summarized in Table 1 below (characteristic confirmation for the healthy group, non-neoplastic underlying disease group, and tumor disease group used in the experiment of the present invention).

[Table 1]

F, female; FS, female spayed; M, male; NM, neutral male

<Example 2>

Comparison of clinical data

Clinical data for each experimental group of Example 1 were analyzed, including hematology, biochemistry and electrolyte parameters. For comparison of clinical pathological data, experimental animal groups were subdivided according to the presence or absence of tumors, significant differences were confirmed between the tumor disease group and the healthy group, and hematological data of all 120 animals were analyzed.

Mean variables for clinical data in the healthy group, non-neoplastic disease group, and tumor disease group

Data are expressed as mean ± SD, Kruskal-Wallis test, P <0.05; Mann-Whitney U test, * indicates P <0.05 and ** indicates P < 0.01.

BUN: blood urea nitrogen, iCa: ionized calcium, iMg: ionized magnesium, HCO _3: bicarbonate.

The results of hematological analysis are shown in Table 2, and according to the analysis results, the average number of neutrophils in both the tumor disease group and the non-neoplastic disease group (tumor disease group, 12.50 ± 8.95 × 10 ⁹ /L, non-neoplastic group, 11.54 ± 8.29 × 10 ⁹ /L) was significantly higher (P < 0.01), and the levels of red blood cells, hematocrit and hemoglobin concentration were significantly lower than in the healthy group (tumor disease group: 5.72 ± 1.46 × 10 ¹² /L). , 36.45 ± 9.68%, 12.50 ± 3.33 g/dL, respectively, non-neoplastic disease group: 5.84 ± 1.62 × 10 ¹² /L, 38.36 ± 11.19%, and 13.18 ± 3.73 g/dL, respectively) ( FIGS. 1A and 1B ). In addition, the tumor disease group (63.58 ± 6.05 fL) showed reduced MCV compared to the healthy group (66.31 ± 2.45 fL), whereas the non-neoplastic disease group (73.07 ± 74.80 × 10 ³ /L, 0.30 ± 0.34 × 10 ⁹ ) /L, respectively) showed reduced reticulocyte and eosinophil counts (P <0.05) compared to the healthy group (80.60 ± 31.45 × 10 ³ /L and 0.41 ± 0.24 × 10 ⁹ /L, respectively).

In addition, ALKP, GGT and amylase levels were measured in the tumor group and non-neoplastic group (tumor group: 555.15 ± 798.71 U/L, 10.65 ± 15.64 U/L and 888.96 ± 446.48 U/L, respectively, in the non-neoplastic disease group: 738.70 ± 1370.21 U/L, 33.97 ± 72.35 U/L and 770.44 ± 300.54 U/L, respectively) in the healthy group (92.34 ± 119.31 U/L, 1.13 ± 3.24 U/L, 606.40 ± 187.96 U/L, respectively) ) was significantly increased compared to ( FIGS. 1A and 1C ). Also, the creatinine level of the non-neoplastic disease group (0.88 ± 0.99 mg/dL) was significantly increased compared to that of the healthy group (0.82 ± 0.18 mg/dL) (P <0.05). However, albumin levels were significantly decreased in the tumor group and non-neoplastic group (2.94 ± 0.51 g/dL and 3.01 ± 0.48 g/dL, respectively) compared to the healthy group (3.40 ± 0.32 g) (P < 0.01). . (Fig. 1A).

In addition, as a result of electrolyte and blood gas analysis, both the tumor disease group and the non-neoplastic disease group showed decreased sodium, potassium and bicarbonate concentrations compared to the healthy group (tumor disease group: 146.14 ± 3.50 mmol/L, 4.18 ± 0.50 mmol/L) and 22.07 ± 4.01 mmol/L, respectively, non-neoplastic disease group: 144.99 ± 5.69 mmol/L, 4.21 ± 0.62 mmol/L, 21.17 ± 4.04 mmol/L, respectively, healthy group: 148.30 ± 2.65 mmol/L, 4.71 ± 0.49 mmol/L and 24.03 ± 2.59 mmol/L, respectively) (P <0.05). However, the ionized calcium concentration was significantly increased in the tumor and non-neoplastic disease groups (1.27 ± 0.06 mmol/L and 1.26 ± 0.09 mmol/L, respectively) than in the healthy group (1.21 ± 0.07 mmol/L) (P <0.05). ). No other electrolyte differences were observed between the tumor disease group and the non-neoplastic disease group, except for the low lactate concentration in the tumor disease group.

<Example 3>

cfDNA concentration comparison result

<3-1> cfDNA analysis in healthy and diseased groups

The results of cfDNA concentration analysis for healthy dogs (healthy group) and clinically diseased dogs (disease group) are shown in FIG. 2A . Specifically, it was confirmed that the average concentration of cfDNA in the healthy group was 877.8 ± 181.7 μg/L. A total of 85 dogs diagnosed or treated at the clinic were classified as clinically diseased. The average concentration of cfDNA in the disease group was found to be 1,333 ± 478.3 μg/L, and this result was confirmed to be significantly higher than that in the healthy group (P <0.001).

Next, for the clinically diseased dogs, a group with a tumor and a group without a tumor were classified. The number of dogs with tumor disease was 38, and 47 dogs were diagnosed with other diseases. The mean cfDNA concentrations in the healthy group, non-neoplastic disease group and tumor disease group were 877.8 ± 181.7 µg/L, 1,154.4 ± 326.5 µg/L and 1,553.9 ± 544.3 µg/L, respectively. There was a significant difference in cfDNA concentration between the tumor disease group and the non-neoplastic disease group (P < 0.001), and there was also a significant difference between the tumor disease group and the healthy group (P < 0.001) ( FIG. 2B ).

<3-2> Comparison of cfDNA concentration in various tumor types

Furthermore, the present inventors compared and analyzed the cfDNA concentration in various tumor types according to the cause of the tumor. To this end, cfDNA concentrations in lymphoma (n = 13), carcinoma (n = 15), sarcoma (n = 5), and other tumors (n = 5) were analyzed, respectively, and compared with the healthy group.

As a result, as shown in FIG. 3 , plasma DNA in the tumor disease group was significantly higher than in the healthy group (P <0.001). On the other hand, when the concentration of cfDNA was compared according to tumor origin, it was found that lymphoma was present at the highest concentration.

Based on the fact that the concentration of cfDNA in the blood increases when a tumor develops in a dog compared to a case in which no tumor occurs, the present inventors can predict or diagnose whether a dog develops a tumor by analyzing the concentration of cfDNA. knew that it could be

<Example 4>

Evaluation of the prognosis of tumor-bearing dogs by cfDNA analysis

To analyze whether the change in the concentration of cfDNA can be used as an important indicator in the prognostic evaluation of disease, plasma cfDNA for 38 tumor disease groups was analyzed during the observation period.

As a result, an ROC curve was generated based on the cfDNA level, and as a result of analyzing the cut-off value for the cfDNA concentration, the cut-off value was found to be 1,247.5 μg/L, and the area under the curve (AUC) was 0.701 ( 95% confidence interval, 0.523-0.880; Po0.05), 86.7% sensitivity and 52.2% specificity (Fig. 4).

Most of the dogs were divided into two groups: a group with cfDNA of 1,247.5 μg/L or less (14 dogs) and a group with cfDNA of 1,247.5 μg/L or more (24 dogs). As a result of analyzing survival curves using cut-off values, Dogs lower than the cut-off value showed a survival probability of 82.1%, whereas dogs higher than the cut-off value showed a survival probability of 26.5% (Fig. 5). Therefore, as a result of monitoring the concentration of cfDNA in dogs of the tumor disease group for 60 weeks, it was found that the prognosis for the tumor disease group, specifically the survival probability, could be predicted through the concentration analysis of cfDNA. If the cut-off value is lower than 1,247.5 μg/L, a high probability of survival can be predicted or diagnosed, whereas when the concentration of cfDNA is higher than the cut-off value of 1,247.5 μg/L, the survival probability is predicted or diagnosed as low. knew that it could be

<Example 5>

Monitoring of tumor treatment response through cfDNA analysis

<5-1> Case Description

A total of 10 tumor-diagnosed dogs were continuously monitored for cfDNA concentrations during chemotherapy or radiation therapy. Of the 10 animals diagnosed with tumors, 6 dogs had lymphoma, 2 dogs had ectopic thyroid carcinoma, 1 animal had intestinal adenocarcinoma, and 1 dog had hemangiosarcoma. was diagnosed with Also out of 10 animals, 6 received chemotherapy, 1 underwent surgical resection, and 1 underwent palliative care. The remaining two animals were treated with radiation therapy. All four dogs diagnosed with multicentric B-cell lymphoma received chemotherapy treatment based on the CHOP (UW-Madison-19) protocol. L-asparaginase was used for induction in all four animals. 3 out of 10 animals were evaluated for complete remission, 1 for stable disease, 2 for progressive disease response, and 4 for death due to disease progression. The characteristics for each of these dogs are shown in Table 3 below, and the monitoring procedure for each experimental animal is described below and is shown in FIG. 6 .

10 Clinical Characteristics of Oncology Groups Evaluated During Follow-up Study

CR: complete remission, SD: stable disease, PD: progressive disease, NA: not available. * According to the WHO staging system (Liptak et al., 2004; Valli et al., 2011).

dog no. 1: It is a stage IVb multicentric B-cell lymphoma, and the cfDNA concentration was 1,070 μg/L just before receiving the second dose (cyclophosphamide) of L-CHOP chemotherapy (UW-Madison-19). The dog visited the hospital every week for chemotherapy. At the time of diagnosis, the sum of LD was 268 mm, but it gradually decreased to 0 mm after 2 months of chemotherapy. Abdominal lymph node size, liver and spleen were also monitored every 2 months. As a result, after chemotherapy, lymph node size gradually decreased every 2 months, and tumor cell infiltration into the liver and spleen was not observed at the last evaluation. At the same time, the concentration of cfDNA was found to be the lowest at 860 μg/L (Fig. 6A). During disease progression, the amount of cfDNA was significantly correlated with lymph node size (P < 0.01) (Fig. 7).

dog no. 2: With stage Vb multicentric B-cell lymphoma disease, the cfDNA concentration first measured before L-CHOP chemotherapy was 1,585 μg/L and the sum of LDs was 205 mm. Both variables appeared to decrease 2 weeks after initiation of chemotherapy ( FIG. 6B ). When the sum of cfDNA and LD was measured at approximately 2-week intervals for the next 2 months, partial remission (PR) was evaluated based on the sum of LD and cfDNA. After 166 days in partial remission, plasma cfDNA was found to be twice the previous concentration, and the sum of LD was also increased 6 months after initiation of chemotherapy. At the same time, a physical examination revealed fever and hypotension, and a blood test showed an increase in leukocytes (60.59 × 10 ⁹ /L) with left neutrophils (band cell, 10,809 cells/μl) and severe thrombocytopenia (27 × 10 ³ /μl). This was confirmed. After one week, clinical signs were similar to baseline and blood tests confirmed pancytopenia. Both LD and plasma DNA levels were lower than before. The dog died 5 days later. There was a significant correlation between cfDNA and lymph node size during disease progression (P < 0.01) (Fig. 7).

dog no. 3: I have stage IVb multicentric B-cell lymphoma with a plasma cfDNA level of 930 μg/L prior to initiation of chemotherapy. One month after initiation of L-CHOP chemotherapy (UW-Madison-19) treatment, the sum of LDs decreased, but cfDNA showed a similar concentration ( FIG. 6C ). but no. In 3 dogs, the experiment was suspended for a week due to neutropenia. One week later, on day 34, the cfDNA concentration was 1,705 μg/L and the sum of LDs was 137.5 mm, and a sharp increase in two variables was observed. These variables decreased one week after doxorubicin (4 cycles of L-CHOP protocol) injection. However, the dog died 3 months after initiation of chemotherapy and had severe clinical signs such as anorexia, depression, disease progression and fever with drug resistance. The sum of LD and cfDNA concentrations showed a significant correlation (P < 0.01) (Fig. 7).

dog no. 4: With stage Vb multicentral T-cell lymphoma disease, the cfDNA concentration at the first visit before chemotherapy was 1,860 μg/L and the sum of LDs was 59 mm. On day 4, lomustine-based chemotherapy (rescue protocol) was initiated with L-asparaginase injection. Finally, when the dog was treated for the second time after 3 weeks, it was found that on day 2 the superficial lymph nodes were barely palpable and the plasma DNA level dropped to 866.5 μg/L ( FIG. 6D ). After that, cfDNA was analyzed every 3 weeks, and the concentration of cfDNA was generally similar as shown in FIG. 6D. Exact numbers were not recorded, but it did not appear that the superficial lymph nodes developed after the second chemotherapy treatment. After the last 5 cycles, the dog showed complete remission (CR) and has maintained CR status until now.

dog no. 5: The dog was diagnosed with adenocarcinoma of the intestine after surgical resection of the small intestine. After surgery, he was transferred to a veterinary hospital for metastasis evaluation, and the initial cfDNA concentration measured at that time was 1,555 μg/L. Thereafter, clinical signs, blood parameters and CT scans were monitored for metastasis evaluation every 2 months, and response to treatment was evaluated by CR, and cfDNA concentrations were measured every 2 months, showing a gradual decrease in concentrations (Fig. 6E). ).

dog no. 6: We have stage IV retroperitoneal hemangiosarcoma disease. Abdominal mass was the main problem at the first visit, and the concentration of cfDNA was 1,400 μg/L. After surgical resection of the mass, piroxicam (Piroxicam; GL Pharma, Chungbuk, Korea) 0.3 mg/kg, cyclophosphamide (Alkyloxan; Ildong Pharm, Seoul, Korea) 15 mg/m ² , doxycycline (Doxycycline Hyclate; Kukje Pharm, Seongnam, Korea) was administered chemotherapy in which 5 mg/kg was orally administered every 24 hours until the biopsy result. After 3 weeks, the cfDNA level decreased to 1,120 μg/L ( FIG. 6F ). Before starting the VAC protocol (Alvarez et al., 2013), cfDNA levels further dropped to 989.5 μg/L. However, the dog DNA concentration gradually increased after the second round of VAC chemotherapy. Finally, on the last day of the highest cfDNA concentration, SDMA increased and blood tests confirmed azotemia. The dog owner decided to stop chemotherapy on day 74, the last cfDNA measurement.

dog no. 7: The dog was diagnosed with non-epithelial cutaneous T-cell lymphoma. The plasma DNA concentration at the first visit was 985.5 μg/L. However, chemotherapy was not performed because clinical signs such as anorexia, depression, and melanoma worsened. Instead, palliative care was administered. The owner decided to euthanize the dog on day 40, and the cfDNA concentration on the last day of the dog was 1,435 μg / L, which was slightly higher than before (Fig. 6G).

dog no. 8: Diagnosed with stage IVb multicentric T-cell lymphoma. Chemotherapy using the L-CHOP protocol was initiated and the cfDNA concentration appeared to increase the next day (Fig. 6H). Finally, the dog died of oncolytic syndrome 4 days after chemotherapy.

dog no. 9: The dog was histopathologically diagnosed with ectopic thyroid cancer of the heart. At the first visit, cfDNA was measured at 1,490 μg/L. Thereafter, cfDNA levels were analyzed every 3 weeks and peritoneal effusion was removed by peritoneal puncture in combination with pancreatitis treatment. Dogs underwent pericardiocystectomy on day 51 and radiation therapy for 3 days one month later ( FIG. 6I ). cfDNA was analyzed at 1-month and 3-month intervals, and the level was found to gradually increase. However, peritoneal effusion, which was one of the clinical signs of dogs, was continuously observed from the first visit to the present, and the tumor size was maintained constant when periodically monitored by CT.

dog no. 10: He was diagnosed with ectopic thyroid carcinoma in the same area as dog 9 above, and was treated with pericardiotomy and radiation therapy. The DNA concentration measured before the initial diagnosis was 1,200 μg/L. Two weeks later, the dog underwent pericardectomy and a total of 12 radiation treatments. The second cfDNA concentration measured about one month after the last radiation treatment was 1,860 μg/L, which was higher than the level two months before (Fig. 6J). They also treated the dog, but the mass at the base of the heart increased in size, causing organ dislocation and coughing.

<5-2> Epidemiologic analysis of plasma cfDNA in response to chemotherapy

The trend of change in cfDNA concentration was analyzed during the treatment period of 8 mice in the tumor disease group (FIG. 6). In dogs #1, #2 and #3 diagnosed with multicentric B-cell lymphoma, cfDNA was continuously monitored by one person during the treatment period. The sum of superficial lymph node LDs in each dog was found to be significantly related to cfDNA concentration (P < 0.01) (Fig. 7). In addition, graphical analysis of the sum of cfDNA and LD showed an increasing and decreasing trend over time. Also, in general, CR (complete remission) or PR (partial remission) responding to chemotherapy showed a tendency to decrease in both curves, and showed a tendency to increase in PD (partial disease) ( FIG. 6 ). On the other hand, dogs 2 and 3 died from progressive disease, but the sum of cfDNA and LD decreased compared to 5-7 days before 1 week before death. Also, pancytopenia (neutropenia (0.889 × 10 ³ /μl, baseline range, 2.95-11.64 × 10 ³ /μl), thrombocytopenia (12 × 10 ³ /μl, baseline range, 200-500 × 10 ³ /μl) , Non-regenerative mild anemia (PCV, 33 %; baseline range, 37.3 to 61.7 %) was confirmed in two blood tests concurrently with two variable reductions, and in dog three, pancytopenia was confirmed one week before expiration. And it was found that the sum of cfDNA and LD decreased at the same time ( FIGS. 6B and 6C ).

In addition, dogs 4 and 5 were both evaluated as CR (complete remission). Dog 4 showed a marked decrease in cfDNA concentration as chemotherapy progressed. However, dogs 6, 7 and 8 showed progressive disease or death. In case of dog 6, the DNA concentration decreased at the start of chemotherapy, but showed a tendency to increase again. In dog 8, the increase was increased 1 day after initiation of chemotherapy, but eventually died of tumor lysis syndrome 4 days after chemotherapy.

<5-3> Epidemiologic analysis of plasma cfDNA in response to radiotherapy

Furthermore, cfDNA trends after radiation treatment were analyzed for two dogs in the tumor disease group ( FIGS. 6I and 6J ). Dogs 9 and 10 were both assessed as stable disease (SD) based on radiation status, but cfDNA concentrations were found to increase until 2 months later. In addition, tumor size and clinical signs were similar to those before treatment. Dog No. 10 showed coughing symptoms for one month after radiation treatment, and the size of the mass was larger than before on ultrasound.

According to the above experimental results, it was confirmed that cfDNA can be used as a biomarker for diagnosing tumors in dogs by confirming that there is a difference in cfDNA concentration between the healthy group, non-neoplastic disease group, and tumor disease group in dogs. could In addition, the present inventors first provided a quantitative cut-off value for the survival rate of dogs according to tumors by measuring the concentration of cfDNA in the blood for dogs with oncological diseases. It was found that it can be usefully used for predicting or diagnosing disease progression.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

(a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; and
(b) comparing the measured cfDNA level with a cut-off value established by Receiver Operation Characteristic (ROC) curve analysis,
A method for predicting tumor prognosis in dogs. According to claim 1,
The biological sample is whole blood, plasma or serum, characterized in that the dog's tumor prognosis prediction method. According to claim 1,
When the measured cfDNA level is lower than the cut-off value, it is determined that the survival probability of the dog is high, and when the measured cfDNA level is higher than the cut-off value, the dog's A method for predicting tumor prognosis in dogs, characterized in that it further comprises the step of determining that the probability of survival is low. According to claim 1,
The set cut-off (cut-off) value is 1,247.5 ug / L, characterized in that, the dog's tumor prognosis prediction method. 4. The method of claim 3,
If the measured cfDNA level is lower than the cut-off value of 1,247.5 ug/L, the survival probability of the dog is 80% or more, characterized in that it is predicted to be more than 80%. 4. The method of claim 3,
When the measured cfDNA level is higher than the cut-off value of 1,247.5 ug/L, the survival probability of the dog is predicted to be less than 30%, a method for predicting tumor prognosis in dogs. According to claim 1,
The tumor is hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell carcinoma, renal cell carcinoma, transitional epithelial carcinoma, intestinal adenocarcinoma, lymphoma, leukemia, angiosarcoma, stromal sarcoma, oral leiomyosarcoma, gastrointestinal stromal tumor (GIST) ), multilobed bone tumor, mast cell tumor, pheochromocytoma and peripheral nerve sheath tumor, characterized in that selected from the group consisting of, tumor prognosis prediction method in dogs. (a) measuring the level of cfDNA (Cell-free DNA) in a biological sample derived from a subject; and
(b) comparing the cfDNA level of a normal control,
How to diagnose a canine tumor. 9. The method of claim 8,
The biological sample is whole blood, plasma or serum, characterized in that the dog tumor diagnosis method. 9. The method of claim 8,
When the cfDNA level derived from the subject is higher than the cfDNA level of the normal control group, the method for diagnosing tumors in dogs, characterized in that it further comprises the step of determining that a tumor has occurred. 11. The method of claim 10,
When the cfDNA level derived from the subject is higher than the concentration of 1,553.9 ug/L, it is characterized in that it is determined that a tumor has occurred, a method for diagnosing a dog's tumor. 9. The method of claim 8,
The tumor is hepatocellular carcinoma, ectopic thyroid cancer, lung adenocarcinoma, mammary gland cancer, cell thyroid cancer, squamous cell carcinoma, renal cell carcinoma, transitional epithelial carcinoma, intestinal adenocarcinoma, lymphoma, leukemia, angiosarcoma, stromal sarcoma, oral leiomyosarcoma, gastrointestinal stromal tumor (GIST) ), multilobed bone tumor, mast cell tumor, pheochromocytoma and peripheral nerve sheath tumor, characterized in that selected from the group consisting of, a dog tumor diagnosis method.

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