KR20190035539A - Composition or kit comprising PHD3 measuring agent for diagnosis of renal cell carcinoma and method for diagnosis using the same - Google Patents

Abstract

According to one aspect of a composition for diagnosing renal cancer or predicting prognosis thereof, and a method for diagnosing renal cancer using the same or a method for predicting prognosis thereof, renal cancer can be easily diagnosed, the prognosis of renal cancer after surgery can be predicted, and renal cancer can be diagnosed with high accuracy and specificity.

Description

The present invention relates to a composition for diagnosing renal cancer including a PHD3 measurement agent and a kit for diagnosing renal cell cancer using the kit.

A method for diagnosing renal cancer using the composition for diagnosing kidney cancer comprising the agent for measuring the expression level of PHD3 gene or the amount of PHD3 protein and an agent for measuring the expression level of HD3 gene or the amount of PHD3 protein And a method for predicting the prognosis of renal cancer using the composition.

Renal cancer is a renal cell carcinoma that usually occurs in the kidneys (the kidney is composed of the water and the cortex, where the urine collecting cells are gathered).

Kidney cancer has little symptoms when the size of the tumor is small, and it only occurs when the tumor grows to some extent and pushes out the organ. Therefore, diagnosis is delayed in many cases, and when first diagnosed, about 30% of the patients are already metastasized. The most common symptom is hematuria, but it also occurs in only 60% of patients. Rather, depending on the site of the transition, symptoms such as dyspnea, cough, headache, and so on, the diagnosis of kidney cancer due to such metastases in 30% of the total patients. Because kidney cancer can cause hypertension, hypercalcemia, liver dysfunction, and so on, due to the specific hormones produced by cancer cells, a tumor may be found while testing for these other symptoms. In recent years, however, there have been many cases of medical examination without any symptoms, which are usually found on imaging studies.

The incidence of kidney cancer is lower than that of dabinous cancer, like the 5th cancer, but it is on an increasing trend. The incidence occurs in an average of 3 per 100,000 people. In adults, the incidence is higher in males than in females, and is common in fifties to sixties.

Therefore, it is necessary to develop a biomarker capable of easily diagnosing renal cancer and having high diagnostic accuracy and specificity.

One aspect provides a composition for diagnosing renal cancer.

Another aspect provides a kidney cancer diagnostic kit.

Another aspect provides a method of diagnosing renal cancer.

Another aspect provides a composition for predicting renal cancer prognosis.

Other aspects provide a way to predict kidney cancer prognosis.

One aspect provides a diagnostic composition for the diagnosis of kidney cancer comprising an agent that measures the level of expression of PHD3 gene or the amount of PHD3 protein. Prolyl hydroxylase (PHD) As a protein involved in the degradation process, the PHD family shares a conserved C-terminal hydroxylase domain, but the respective PHD isoforms, PHD1, PHD2, and PHD3 differ in function and tissue specific expression patterns. PHD3 is one of the PHD family and its function has not yet been clarified, and it can be mixed with Egl9n homolog 3, or EGLN3. The PHD3 protein is 1.14.11.2. Or 1.14.11.29. The PHD3 protein may be a protein comprising the amino acid sequence of NCBI Accession No. CDS9646.1., NP_001295032.1., Or NP_071356.1. The PHD3 protein is NCBI Accession NM_001308103.1. Or a protein encoded by a nucleic acid comprising a nucleotide sequence of NM_022073.3. The PHD3 protein may be one comprising the entire sequence or a fragment thereof. The fragment of the PHD3 protein may be an immunogenic fragment. The fragments of the PHD3 protein may have one or more epitopes that can be recognized by antibodies to the PHD3 protein.

As used herein, the term " agent for measurement " means a substance capable of detecting or quantifying an object.

The agent for measuring the expression level of the PHD3 gene is an agent for directly or indirectly measuring the expression level of the PHD3 gene in a biological sample to diagnose kidney cancer and can be specifically bound to the PHD3 gene Lt; RTI ID = 0.0 > oligonucleotides, < / RTI >

The gene may be DNA, RNA, or a combination thereof.

As used herein, the term " primer " refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a complementary template and having a starting point for template strand copy Quot; means a nucleic acid sequence of 7 nt to 50 nt functioning as a nucleotide sequence. Primers are usually synthesized but can also be used in naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. Primers are designed to initiate DNA synthesis in the presence of a polymerization reaction (i. E., A reagent for DNA polymerase or reverse transcriptase and four different nucleoside triphosphates) at the appropriate buffer solution and temperature The PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art. Thus, PCR amplification can be performed using the sense and antisense primers at the nucleotide sequence of PHD3 Thereby diagnosing renal cancer.

As used herein, the term " probe " refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or several hundreds of nucleotides that can specifically bind to mRNA and is labeled so that the presence of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art. Thus, hybridization can be performed using a probe complementary to the nucleotide sequence of the PHD3 gene, thereby diagnosing kidney cancer through hybridization.

The primers or probes can be chemically synthesized using the phospharamidite solid support method, or other well-known methods. Such nucleic acid sequences may incorporate additional features that do not alter the basic properties. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution with one or more nucleic acid homologs, and modification between nucleic acids.

The term " antisense nucleic acid " in the present specification means a nucleic acid-based molecule having a complementary sequence to the target PHD3 gene and capable of forming a dimer with the PHD3 gene, and can be used for detecting the PHD3 gene.

The antisense nucleic acid may be a PHD3 gene or a fragment thereof, or complementary thereto. The fragment may have a nucleotide of 5 nt or more, 10 nt or more, 10 nt to 1000 nt, 10 nt to 500 nt, 15 nt to 500 nt, 15 nt to 300 nt, 15 nt to 200 nt or 15 nt to 150 nt, but an appropriate length may be selected to increase detection specificity .

The expression level of the gene may be determined by PCR, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, RNase protection assay (RPA) microarray, northern blotting, or a combination thereof.

The agent for measuring the amount of the PHD3 protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the PHD3 protein or fragment thereof.

As used herein, the term " antibody " refers to a specific protein molecule directed against an antigenic site and may be used in combination with an " immunoglobulin ". For the purpose of the present invention, the antibody refers to an antibody that specifically binds to the marker of the present invention, and the form of the antibody is not particularly limited, and if it is a polyclonal antibody, a monoclonal antibody or an antigen- Some of which may be included in the antibody of the present invention and include all immunoglobulin antibodies. Furthermore, the antibody also includes a special antibody such as a humanized antibody. The antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F (ab ') 2, F (ab') 2, Fv or a combination thereof.

The amount of the protein may be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis , Tissue immuno staining, immunoprecipitation assay, complete fixation assay, FACS, protein chip, or a combination thereof.

The kidney cancer may be one stage or two stages. Because kidney cancer can be treated differently depending on the stage, it is important to classify the stage. The stage of renal cancer can be classified according to a known classification method. The first group is a case where a small-sized cancer is present in the kidney, for example, it may be a tumor in the renal capsule. 2 is a case in which a slightly larger size of cancer is present in the kidney than in one cancer, for example, it may be a tumor infiltrated into the perirenal fat. 3 may be a case where the cancer is present outside the kidney, for example, a tumor spread to the renal vein and lymph nodes. The fourth group may be that the cancer has spread to other parts of the kidney, for example, to the bone, liver, heart, or lung.

Can be distinguished by recurrence, cellular appearance, systemic effects, therapy, or a combination thereof. The level of PHD3 in an individual can function as a biomarker to distinguish malignant kidney cancer from healthy kidney tissue or benign renal tumors.

The composition may further comprise an agent that measures the level of expression of endocan gene or the amount of endocan protein.

Endocan (endothelial cell-specific molecule-1 or ESM-1) is a 50 kDa dermatan sulphate proteoglycan, expressed in vascular endothelial cells. The endocane protein may be a protein comprising the amino acid sequence of NCBI Accession No. NP_001129076, or NP_008967. The endokane protein may be a protein encoded by a nucleic acid comprising the nucleotide sequence of NCBI Accession NM_007036 or NM_001135604. The endocan protein may be one comprising the entire sequence or a fragment thereof. The fragment of the endocan protein may be an immunogenic fragment. The fragment of the endocan protein may be one having at least one epitope that can be recognized by an antibody to an endokane protein.

The agent for measuring the expression level of the gene or the amount of the protein is as described above.

Another aspect provides a kit for the diagnosis of kidney cancer comprising an agent that measures the level of expression of PHD3 gene or the amount of PHD3 protein.

The kit may include an agent that measures the level of expression of endocan gene or the amount of endocan protein.

The kit can diagnose kidney cancer by checking the expression level of PHD3 gene or the amount of PHD3 protein. The kit for the diagnosis of kidney cancer may include a primer, a probe, or an antibody for selectively recognizing a marker for measuring the expression level of PHD3 gene or the amount of PHD3 protein specific for diagnosis of kidney cancer, as well as one or more other Component composition, solution, or device. In addition, it may further comprise an antibody, a composition, a solution or an apparatus recognizing a primer, a probe, or alternatively a marker for measuring the level of expression of the endocane gene or the amount of endocannin protein.

The kidney cancer diagnostic kit comprising the agent for measuring the expression level of the PHD3 gene may be a kit containing elements necessary for performing RT-PCR. The RT-PCR kit can be used to detect the presence of enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNases, RNase inhibitors , DEPC-water (DEPC-water), sterilized water, and the like. In addition, the kit may be a kidney cancer-specific gene diagnosis detection kit including essential elements necessary for performing a microarray chip. The microarray chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof. For example, the microarray chip may be a kidney cancer diagnostic kit comprising a DNA microarray chip for kidney cancer diagnosis in which oligonucleotides or complementary strand molecules corresponding to 10 nt or more genes selected from the PHD3 gene are integrated . The oligonucleotide or its complementary strand molecule may comprise 18 to 30 nt of the gene, or 20 nt to 25 nt of the nucleic acid. The microarray chip can be easily prepared by a method commonly used in the art using the gene for kidney cancer-specific expression of the present invention. In order to fabricate a microarray chip, a micropipetting or a pin-type using a piezo electric method is used to immobilize the above-mentioned detected marker gene on a substrate of a DNA chip using the DNA as a probe DNA molecule A spotter method may be used. The substrate of the microarray chip may be coated with an activator selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto . The substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

The kit for diagnosing kidney cancer, which comprises an agent for measuring the amount of the PHD3 protein, includes a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic or fluorescent substance, and a chromogenic substrate for immunological detection of the antibody . The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme may be peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline- ), Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

Another aspect is a method for diagnosing a kidney cancer, comprising the steps of measuring the expression level of PHD3 gene or the amount of PHD3 protein in a biological sample separated from a subject suspected of having kidney cancer; And comparing the expression level of the gene or the amount of the protein to the expression level of the gene or the amount of the protein of the control group.

The method comprising the steps of: measuring the level of expression of endocan gene or the amount of endocannan protein in a biological sample isolated from a subject suspected of having kidney cancer; And comparing the expression level of the measured gene or the amount of the protein with the expression level of the gene or the amount of the protein of the control group.

The subject may be a vertebrate animal, a mammal, a human ( Homo sapiens ), a mouse, a rat, a cow, a horse, a pig, a sheep, a goat, a dog, a cat and the like. For example, the human being may be Asian or Korean. &Quot; Subject " and " patient " are used interchangeably herein.

The biological sample may be serum, blood, plasma, bone marrow fluid, lymph fluid, saliva, leakage fluid, mucosal fluid, amniotic fluid, kidney tissue, kidney cells or a combination thereof.

The biological sample may comprise an intact PHD3 protein. An intact protein may be a protein separated from a biological sample without modification of the separate protein. An intact protein may be a protein separated from a biological sample, for example, without protein degradation by proteolytic enzymes.

The measuring step may comprise incubating the biological sample with an antibody that specifically binds to the PHD3 protein or fragment thereof. Incubation can be performed in vitro. The measurement method is as described above.

The measuring step may include mass analyzing the biological sample. The step of mass-analyzing the biological sample may be a mass analysis of the amount of the whole protein.

The method comprises comparing the amount of the measured protein to that of a control. The control group may be a healthy individual, a subject having a benign tumor, a non-kidney cancer subject, or a biological sample isolated from a subject at risk of developing kidney cancer.

When the amount of the protein of the PHD3 protein or fragment thereof measured in the biological sample separated from the subject suspected of being kidney cancer is higher than the amount of the PHD3 protein or fragment thereof measured in the sample separated from the control sample, Or kidney cancer can be determined to be high.

When the amount of endocan protein or fragment thereof measured in a biological sample isolated from a subject suspected of being kidney cancer is increased over the amount of endocan protein or fragment thereof measured in a sample isolated from a control sample, Cancer patients or kidney cancer.

The biological sample may be a genomic DNA, a cell free DNA or a fragment thereof. The biological sample may be one comprising a purely isolated nucleic acid, a crude nucleic acid, a cell lysate containing a nucleic acid, or a cell free nucleic acid. The method of separating the genomic DNA, the cell free DNA or a fragment thereof from the biological sample can be carried out by a conventional nucleic acid separation method.

The method comprises comparing the expression level of the measured gene to the expression level of the gene of the control. The control group may be a healthy individual, a non-kidney cancer subject, a subject having a benign tumor, or a biological sample isolated from a subject at risk of developing kidney cancer.

The measuring step may comprise incubating the biological sample with a polynucleotide that hybridizes to the PHD3 gene or a fragment thereof. Incubation can be performed in vitro. The measurement method is as described above.

When the level of expression of the PHD3 gene or fragment thereof measured in a biological sample isolated from a subject suspected of being kidney cancer is higher than the expression level of the PHD3 gene or fragment thereof measured in a sample isolated from a control sample, Or kidney cancer can be determined to be high.

When the level of expression of the endocan gene or fragment thereof measured in a biological sample isolated from a subject suspected of having kidney cancer is increased above the expression level of the endocan gene or fragment thereof measured in a sample isolated from a control sample, Cancer patients or kidney cancer.

The method includes a method of providing information for diagnosis of kidney cancer.

The kidney cancer may be one stage or two stages. The kidney cancer may be a malignant kidney cancer.

Another aspect provides a composition for predicting prognosis of renal cancer, comprising an agent that measures the level of expression of PHD3 gene or the amount of PHD3 protein.

The prognosis may be to predict postoperative prognosis.

The agent for measuring the expression level of the PHD3 gene may include a primer, a probe, or an antisense oligonucleotide that specifically binds to the PHD3 gene.

The agent for measuring the amount of the PHD3 protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the PHD3 protein or fragment thereof.

The kidney cancer may be one stage or two stages. The renal cancer may be one in which the kidney cancer is a malignant renal cancer.

The composition may further comprise an agent that measures the level of expression of endocan gene or the amount of endocan protein.

The prognosis may be to predict postoperative prognosis. Predicting the prognosis for renal cancer refers to prediction of progress and recovery of the disease, and refers to prospective or preliminary evaluation. The prognosis may be a recurrence of kidney cancer, overall survival, disease-free survival, or a combination thereof.

Another aspect is a method for diagnosing a kidney cancer, comprising the steps of measuring the expression level of PHD3 gene or the amount of PHD3 protein in a biological sample separated from a subject suspected of having kidney cancer; And comparing the expression level or the amount of the protein of the measured gene with the expression level of the gene or the amount of the protein of the control group.

The method comprising the steps of: measuring the level of expression of endocan gene or the amount of endocannan protein in a biological sample isolated from a subject suspected of having kidney cancer; And comparing the expression level of the measured gene or the amount of the protein with the expression level of the gene or the amount of the protein of the control group.

The method comprising the steps of: measuring the level of expression of endocan gene or the amount of endocannan protein in a biological sample isolated from a subject suspected of having kidney cancer; And comparing the expression level of the measured gene or the amount of the protein with the expression level of the gene or the amount of the protein of the control group.

The biological sample may be serum, blood, plasma, bone marrow fluid, lymph fluid, saliva, leakage fluid, mucosal fluid, amniotic fluid, kidney tissue, kidney cells or a combination thereof.

The kidney cancer may be one stage or two stages. The kidney cancer may be a malignant kidney cancer.

The prognosis may be to predict postoperative prognosis. The operation may be a surgical operation, for example, a total removal (curative nephrectomy) or a partial removal.

Wherein said control is a subject having an abnormality at a level of a biomarker other than a subject having a benign tumor, PHD3, endocane, or a combination thereof, PHD3, endocane, or a combination thereof, , A healthy individual, a non-kidney cancer patient, or a biological sample isolated from a subject at risk of developing kidney cancer.

Conventionally, computer tomography has been performed to distinguish between malignancy and benignity of renal cancer. However, when the biomarker is used, the expression level of PHD3 gene or the amount of PHD3 protein can be measured to clearly distinguish malignancy from benignity , It is possible to accurately determine malignant patients, and the prognosis of these patients can be expected to be positive during surgery.

According to one aspect of the present invention, there is provided a composition for diagnosing or prognosing kidney cancer, and a method for diagnosing renal cancer or a method for predicting prognosis using the same, wherein the renal cancer can be easily diagnosed, the prognosis of renal cancer can be predicted, Can be used to diagnose high accuracy and specificity.

FIG. 1 shows the results of confirming the level of serum PHD3 between the RCC patient group and the control group.
In FIG. 2, A is the result of confirming the change of post-operative serum PHD3 level in all patients. In FIG. 2, B is a result of confirming the change of post-operative serum PHD3 level in patients having preoperative serum PHD3 level higher than 0.761.
Figure 3 is a result of confirming the RNA level of PHD in RCC, stomach cancer, breast cancer, lung cancer, kidney cancer, and pancreatic cancer.
FIG. 4 shows the results of confirming the level of serum endocannon between the RCC patient group and the control group.
FIG. 5 shows the results of confirming the change in the level of the endocardium after surgery in all patients.
FIG. 6 shows the results of confirming changes in serum PHD3 and / or endocane level after surgery in all patients.
FIG. 7 shows the results of confirming changes in serum PHD3 and / or endocane levels after surgery in patients over 50 years of age.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example 1 Identification of PHD3 Proteins High in Expression Level in Serum of Patients with Renal Cancer

1-1. Preparation of Plasma from Kidney Cancer Patients and Normal Persons

From October 2013 to March 2015, after obtaining approval from the Severance Hospital Clinical Trials Committee (IRB No. 4-2013-0166), participants were recruited and agreed to provide information from all participants.

During the study period, 56 patients who underwent radical or partial nephrectomy were classified as RCC patients. 56 healthy renal donors and 13 benign renal masses were classified as controls. All RCCs and benign renal neoplasms were diagnosed surgically and pathologically confirmed. The benign renal neoplasms included 5 cases of angiomyolipoma, 4 cases of oncocytoma, 3 cases of adenoma, and 1 case of pyelonephritis.

Both RCC and control group were sampled for blood samples prior to surgery. Blood samples were collected 1 and 3 months after surgery to confirm the degree of change of the biomarker PHD3. Blood was collected and centrifuged at 1000 g for 15 minutes within 30 minutes, and the serum of the upper layer was collected and stored at -80 ° C.

Expression levels of PHD3 gene were confirmed in patients with other carcinomas and RCC patients with clear cell carcinoma. Figure 3 is a result of confirming the RNA level of PHD in RCC, stomach cancer, breast cancer, lung cancer, kidney cancer, and pancreatic cancer. As shown in FIG. 3, it can be seen that the RNA level of PHD3 is higher in RCC patients than in other gastric cancer patients, breast cancer patients, lung cancer patients, kidney cancer patients, and pancreatic cancer patients.

1-2. Measurement of the amount of PHD3 protein in plasma by ELISA analysis

The level of PHD3 in all the serum samples obtained in 1-1 above was quantified using ELISA. The anti-PHD3 antibody was purchased from AbFrontier (mouse monoclonal 28A1, Seoul, Korea). Serum was diluted 2-fold before analysis. Recombinant PHD3 protein and diluted sera were coated overnight at 4 DEG C in a 96-well ELISA plate containing 0.1 M sodium bicarbonate buffer (pH 9.6). The coated plates were blocked with TBS containing 5% skim milk at 37 [deg.] C for 1 hour to block non-specific binding. After washing with TBS, the plate was incubated with primary antibody diluted 1: 500 in blocking solution for 2 hours and then incubated with secondary antibody diluted 1: 3000 in blocking solution for 1 hour. After the incubation, the plate was washed, 50 μl of tetramethylbenzidine substrate was added to induce the reaction, and 1N H ₂ SO ₄ was added to stop the reaction. Absorbance was measured at 495 nm using a spectrophotometer. All assays were repeated twice.

Clinical and pathological characteristics of the participants are shown in Table 1 below. All RCC patients were significantly older than the control group. The levels of PHD3 in the control group were determined as follows: age (= 50 vs.> 50, 0.73 ± 0.09 vs. 0.73 ± 0.10, P = 0.838) and sex (male vs. female, 0.73 ± 0.09 vs. 0.74 ± 0.10, P = 0.760), respectively. In addition, PHD3 levels were not significantly different between healthy kidney donor controls and benign kidney tumor controls (healthy individual versus benign renal mass group), 0.73 ± 0.09 vs. 0.73 ± 0.09, P = 0.872). Of the 56 RCC patients, 43 (76.8%) were T1, and 44 (78.6%) showed clear cell RCC.

RCC Control group P value Age (years)   Mean ± SD 52.9 ± 12.6 48.5 ± 9.3 0.024   Median (Quadrant, IQR) 54 (44-62) 49 (42-57) Sex, n (%) 0.852   male 35 (62.5) 42 (60.9)   female 21 (37.5) 27 (39.1) Size (cm)   Mean ± SD 3.64 ± 3.36   Median (quadrant, IGR) 2.7 (2-3.8) Stage, n (%)   T1a 43 (76.8)   T1b 6 (10.7)   T2a 2 (3.6)   T2b 2 (3.6)   T3a 3 (5.4) Pureman rating, n (%)   I-II 36 (64.3)   III-IV 20 (35.7) Cell type, n (%)  Clear cells 44 (78.6) Non-clear cells 12 (21.4)

The level of serum PHD3 according to tumor characteristics was determined in the RCC patient group, and is shown in Table 2. The level of PHD3 tended to increase in patients with large-sized tumors, high grade, T2-stage, and clear cell histology, but the difference was not significant.

Serum PHD3 levels P value Tumor size (cm) 0.161   2 (n = 16) 0.75 + 0.21   > 2,? 4 (n = 27) 0.78 ± 0.17   > 4 (n = 13) 0.87 ± 0.06 ordnance 0.15   T1 (n = 49) 0.78 ± 0.17   > T2 (n = 7) 0.88 ± 0.05 Pureman rating 0.086   I-II (n = 36) 0.76 + 0.16   III-IV (n = 20) 0.84 + 0.17 Cell type 0.535   Transparent cells (n = 44) 0.80 + 0.18   Non-transparent cells (n = 12) 0.76 + - 0.12

1-3. Statistical analysis of the level of PHD3 in RCC patients

To determine the effect of age and sex on the level of serum PHD3, we compared the levels of PHD3 in the control group according to gender and age (> 50 years vs.> 50 years old).

In RCC patients, changes in serum PHD3 were analyzed by tumor size, pathologic stage, Furmann grade, and histological analysis.

To assess the diagnostic function of serum PHD3 levels, serum levels of PHD3 protein were compared between RCC and control. Receiver operating characteristics (ROC) curves were obtained to assess area under curves (AUC). The optimal cutoff value for maximizing sensitivity and specificity was calculated using the YUEN index. ROC curves were obtained in subgroups of RCCs including patients with tumor size greater than 2 cm or patients with clear cell histology.

To evaluate the monitoring function of serum PHD3 levels, the amounts of PHD3 protein before and after surgery were compared. Quantitative values were compared using Student's t-test or one-way ANOVAR. Qualitative variables were compared using a chi-square test or Fisher's exact test. The levels of PHD3 before and after surgery were compared using a paired t-test. Statistical analysis was performed using the Social Science Statistical Package for Windows, version 18.0 (SPSS, Chicago, IL, USA). P value <0.05 was considered significant, and all P values were shown up and down. ROC analysis and graphics were performed using R version 3.2.5 (http://www.r-project.org).

FIG. 1 shows the results of confirming the level of serum PHD3 between the RCC patient group and the control group. As shown in Figure 1 A, the level of serum PHD3 between the RCC patient and the control group was higher than that of the control group (0.79 ± 0.17 vs. 0.73 ± 0.09, P = 0.023). ROC curves were obtained for the RCC patient group (n = 56) and the control group (n = 69), as shown in Fig. The AUC for PHD3 was 0.668 (95% CI, 0.565-0.769). Sensitivity, specificity, positive predictive value, and negative predictive value were 66.1%, 68.1%, 28.8%, and 37.3%, respectively, at the cutoff value 0.761 calculated by the Youden index. ROC curves were obtained in subgroups of RCCs including patients with tumor size greater than 2 cm or patients with clear cell histology. For each patient with tumor size greater than 2 cm (n = 40) or histologically transparent cells (n = 44), the AUC values for PHD3 were 0.709 and 0.688.

In FIG. 2, A is the result of confirming the change of post-operative serum PHD3 level in all RCC patients. 2, B shows the change in serum PHD3 levels in 37 RCC patients with preoperative serum PHD3 levels greater than 0.761. The levels of PHD3 before and after surgery were compared using a paired t-test. As shown in FIG. 2 A, the level of PHD3 decreased after 1 month and 3 months after surgery in 56 RCC patients. Thirty-seven patients (89.2%) had decreased PHD3 levels at 1 month after surgery and 37 patients (83.8%) had cut-off values of 0.761 or more. The level of PHD3 decreased after 6 months. In the paired t-test, levels of PHD3 at 1 month and 3 months after surgery were significantly lower (P <0.001 and P <0.001, respectively) than preoperative levels of PHD3.

Example 2. Confirmation of protein endocannal expression level in serum of kidney cancer patients

2-1. Preparation of Plasma from Kidney Cancer Patients and Normal Persons

A sample was prepared in the same manner as in Example 1-1 except that 56 healthy renal donors were classified as a control group.

2-2. Determination of the amount of endocannan protein in plasma by ELISA analysis

The endocane levels in all the serum samples obtained in Example 2-1 were performed using an ELISA kit (Lunginnovas, Lille, France) according to the manufacturer's instructions. Serum was diluted 2-fold before analysis. The microwell plate was coated with 100 [mu] l of capture antibody (2 [mu] g / ml) and incubated at 4 [deg.] C overnight. The coated plates were blocked with TBS containing 5% skim milk at 37 [deg.] C for 1 hour to block non-specific binding. After washing with PBS, the plate was incubated with 100 혈 of serum sample for 1 hour, washed, and incubated with 100 2 of secondary antibody diluted 1: 10,000 for 1 hour at room temperature. After washing, 100 占 퐇 of streptavidin-hos radial peroxidase diluted 1: 10,000 was added and incubated for 30 minutes. After the incubation, the plate was washed, 50 μl of tetramethylbenzidine substrate was added to induce the reaction, and 1N H ₂ SO ₄ was added to stop the reaction. Absorbance was measured at 495 nm using a spectrophotometer. All assays were repeated twice.

Clinical and pathological characteristics of the participants are shown in Tables 3 and 4 below. Of the 56 RCC patients, 43 (78.6%) showed clear cell RCC. RCC patients were significantly older than the control group (P = 0.027). In the control group, the levels of serum endocane did not differ between sexes (male vs. female, 0.53 ± 0.08 ng / mL vs. 0.52 ± 0.77, P = 0.764). The serum endocarcinogen levels were higher (0.49 ± 0.08 ng / mL vs. 0.56 ± 0.06 ng / mL, P = 0.001) than those with relatively older age (> 50 years) ). In the RCC patient group, serum endocane levels did not show any significant differences between the patients showing clear cells and non-transparent cells. In patients with clear cell RCC, serum endocannal levels were significantly higher in patients with larger tumor sizes and those with upper stage disease. However, no statistical difference was found in the entire RCC group.

RCC (n = 56) Control group (n = 56) P value Age (years)  Mean ± SD 52.9 ± 12.6 48.2 ± 9.0 0.027   Median (IQR) 54 (44-62) 49 (40-57) Sex, n (%) 1,000   male 35 (62.5) 35 (62.5)   female 21 (37.5) 21 (37.5) Tumor size (cm)   Mean ± SD 3.64 ± 3.36   Median (IQR) 2.7 (2-3.8) Weapon, n (%)   T1a 43 (76.8)   T1b 6 (10.7)   T2a 2 (3.6)   T2b 2 (3.6)   T3a 3 (5.4) Pureman rating, n (%)   I-II 36 (64.3)   III-IV 20 (35.7) Cell type, n (%)  Transparent cell 44 (78.6)  Non-transparent cell 12 (21.4)

Level of serum endocane P value Level of serum endocane
(For transparent cell RCC alone) P value Tumor size (cm) 0.095 0.006   2 (n = 16) 0.56 + 0.08 0.54 + 0.08   > 2,? 4 (n = 27) 0.59 + 0.06 0.59 + 0.06   > 4 (n = 13) 0.61 ± 0.05 0.63 + 0.02 ordnance 0.625 0.024   T1 (n = 49) 0.59 + 0.07 0.58 + 0.07   > T2 (n = 7) 0.59 + 0.07 0.63 + 0.02 0.836 0.937   I-II (n = 36) 0.59 + 0.06 0.59 + 0.06   III-IV (n = 20) 0.58 ± 0.08 0.59 + 0.07 Cell type 0.904 -  Transparent cells (n = 44) 0.59 + 0.06 - Non-transparent cells (n = 12) 0.59 + 0.08 -

2-3. RCC  In patients Endokan  Level statistical analysis

The level of endocane was compared according to gender and age (50 vs. less than 50) in the control group to determine the effect of age and sex on serum PHD3 levels.

In RCC patients, changes in serum endocarcinomas were analyzed by tumor size, pathologic stage, Furmann grade, and histological analysis. The analysis was carried out in the same manner as in Example 1-3. In addition, multivariate linear regression analysis was performed to determine whether RCC affects the endocannal level regardless of age because the level of serum endocarcinogen was high at the age of the control group.

FIG. 4 shows the results of confirming the level of serum endocannon between the RCC patient group and the control group. As shown in FIG. 4A, the level of endocannin between the RCC patient group and the control group was higher than that of the control group (0.59 ± 0.07 vs. 0.52 ± 0.08, P <0.001 ). As shown in Fig. 4B, ROC curves were obtained for the RCC patient group (n = 56) and the control group (n = 56). The AUC for endocane was 0.721 (95% CI, 0.628-0.817). Sensitivity, specificity, positive predictive value, and negative predictive value were 67.9%, 73.2%, 30.5%, and 28.3% at the cutoff value 0.568 ng / mL calculated by the Youden index. ROC curves were obtained in subgroups of RCCs including patients with tumor size greater than 2 cm or patients with histologically clear cells. For each patient with tumor size greater than 2 cm (n = 40) and histologically transparent cells (n = 44), the AUC values for endocane were 0.771 and 0.721.

After controlling the effects of age, RCC vs. The control group significantly affected the level of serum endocannan (P <0.001). When the whole cohort was divided by age, the AUC for the endocane was 0.613, while the AUC for the endocane was 0.813 for the relatively low age group (age = 50).

FIG. 5 shows the results of confirming the change in the level of the endocardium after surgery in all patients. As shown in FIG. 5, endocannal levels were decreased (0.56 ± 0.07 ng / mL and 0.56 ± 0.06 ng / mL, respectively) at 1 month and 3 months after surgery in 56 RCC patients. In the paired t-test, endocane levels at 1 month and 3 months postoperatively were significantly lower than pre-operative endocan levels (P = 0.047 and P = 0.009, respectively).

The level of serum PHD3 can serve as a new biomarker for RCC with high accuracy and reliability. The level of serum PHD3 may function as a biomarker for the diagnosis of RCC, or as a biomarker for post-operative monitoring. In addition, the level of serum PHD3 may serve as a biomarker for distinguishing and diagnosing malignant RCC from other benign renal tumors.

Example  3. Of kidney cancer patient serum PHD3  And Endokan  Identification of diagnostic efficiency by expression level

3-1. Statistical analysis of PHD3 and endocane levels in RCC patients

Serum PHD3 and endocane levels of PHD3 or ENTOCAN were compared to determine the efficiency of diagnosis of renal cancer according to the combination of serum PHD3 and endocane.

FIG. 6 shows the results of confirming changes in serum PHD3 and / or endocane level after surgery in all patients. As shown in FIG. 6, the AUC (0.722, P < 0.001) for serum PHD3 and serum endocarbons in RCC patients was significantly higher than the AUC of each of serum PHD3 (0.668, P = 0.001) or endocane (0.681, P = 0.001) Which is higher than that of the others. Table 5 shows the cutoff values of the combination of PHD3, endocane, PHD3, and entankan calculated with the Youden index. Sensitivity and specificity were 67.9% and 78.3% at cutoff value 1.366 ng / mL calculated by Youden index. In other words, when used in combination with serum PHD3 and endocane levels, it can serve as a new biomarker for RCC with higher accuracy and reliability.

Cutoff value responsiveness Specificity PHD3 alone 0.762 66.1% 68.1% Endokan solo 0.679 67.9% 69.6% PHD3 + ENTKAN 1.366 67.9% 78.3%

3-2. Statistical analysis of PHD3 and endocane levels in patients older than 50 years

Patients older than 50 years whose endocannabial availability decreased were further analyzed in the same manner as in Example 3- 1.

FIG. 7 shows the results of confirming changes in serum PHD3 and / or endocane levels after surgery in patients over 50 years of age. As shown in FIG. 7, the endocane AUC was 0.621 (p = 0.88), while the AUC of PHD3 was 0.667 (p = 0.019, It was predictable. Sensitivity and specificity were 71.0% and 68.7% at the cutoff value of 0.748 of PHD3 calculated by Youden index. In other words, PHD3 can function as a new biomarker for RCC even in patients over 50 years of age whose endocane availability is diminished.

Claims (13)

A composition for diagnosing renal cancer, comprising an agent for measuring an expression level of PHD3 gene or an amount of PHD3 protein. The composition according to claim 1, wherein the agent for measuring the expression level of the PHD3 gene comprises a primer, a probe or an antisense oligonucleotide that specifically binds to the PHD3 gene. The composition according to claim 1, wherein the agent for measuring the amount of the PHD3 protein is an antibody or an antigen-binding fragment thereof that specifically binds to the PHD3 protein or fragment thereof. The composition according to claim 1, wherein said kidney cancer corresponds to one or two stage. The composition according to claim 1, wherein the kidney cancer is a malignant kidney cancer. The composition of claim 1, further comprising an agent that measures the level of expression of the endocan gene or the amount of endocan protein. Measuring the level of expression of PHD3 gene or the amount of PHD3 protein in a biological sample isolated from a subject suspected of having kidney cancer; And
Comparing the expression level or the amount of the protein of the measured gene with the expression level of the gene or the amount of the protein of the control group. The method of claim 7,
Measuring the level of expression of the endocan gene or the amount of endocannan protein in the biological sample separated from the subject suspected of having kidney cancer; And
Comparing the expression level of the measured gene or the amount of the protein with the expression level of the gene of the control or the amount of the protein. A composition for predicting renal cancer prognosis comprising an agent for measuring the expression level of PHD3 gene or the amount of PHD3 protein. The composition according to claim 9, further comprising an agent for measuring the level of expression of the endocan gene or the amount of endocan protein. Measuring the level of expression of PHD3 gene or the amount of PHD3 protein in a biological sample isolated from a subject suspected of having kidney cancer; And
Comparing the expression level or the amount of the protein of the measured gene with the expression level of the gene or the amount of the protein of the control group. The method of claim 11,
Measuring the level of expression of the endocan gene or the amount of endocannan protein in the biological sample separated from the subject suspected of having kidney cancer; And
Comparing the expression level of the measured gene or the amount of the protein with the expression level of the gene of the control or the amount of the protein. 12. The method of claim 11, wherein the prognosis predicts a postoperative outcome.

Images