KR101860238B1 - Use of ZFP28, FAM155A and DPP6 for Prognostic Marker Diagnosis of renal cell carcinoma - Google Patents

Abstract

The present invention relates to a use of ZFP28, FAM155A and DPP6 as a renal cancer prognostic diagnosis marker. A composition for markers for prognostic diagnosis of renal cancer including ZFP28, FAM155A or DPP6 gene can be used for early distant metastasis or distant tumor relapse after clear cell renal cell carcinoma (ccRCC) surgery, thereby being useful for treatment of renal cancer and prediction of prognosis.

Description

The use of ZFP28, FAM155A and DPP6 as diagnostic markers of kidney cancer prognosis {Use of ZFP28, FAM155A and DPP6 for Prognostic Marker Diagnosis of Renal Cell Carcinoma}

The present invention relates to a novel biomarker for diagnosing renal cancer prognosis and its use, and more particularly to a marker for diagnosing a prognosis after renal cancer surgery using the expression characteristics according to a methylation pattern of ZFP28, FAM155A or DPP6 gene .

Renal cell carcinoma (RCC) patients worldwide reach 270,000 people every year, and about 120,000 people die from kidney cancer each year. Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, accounting for about 70% of kidney cancer types. Small and localized (I to III) ccRCC patients have a good survival rate for 5 years after surgery, but recurrence occurs in about 30% of patients treated for local lesions. Like other tumor types, ccRCC exhibits a variety of clinical heterogeneities, ranging from painless to highly aggressive.

It is essential to identify the precise predictors of clinical outcomes in order to facilitate individual counseling about appropriate treatment strategy decisions and adjunctive therapies. Currently, tumor recurrence prediction in patients receiving topical ccRCC surgery is based on clinical and pathologic features, including TNM stage and nuclear grade, which are dependent on interobserver variability, It may not be fully explained, and the hitherto known histological or multivariate risk models do not show sufficient sensitivity and specificity for kidney cancer diagnosis and prognosis.

Because of these limitations, it is necessary to develop accurate molecular markers for the diagnosis of kidney cancer patients, and it is important to understand the genetic and epigenetic genetic variation as a way to effectively manage kidney cancer patients. Recently, Genome-based prognostic markers have been developed. However, there has been little research on clinical diagnosis, prognosis prediction, and biomarker diagnosis by DNA methylation analysis of kidney cancer.

≪ tb > Published patent application 10-2012-0007826

Accordingly, the present invention provides a marker composition for diagnosing renal cancer prognosis, which comprises ZFP28 (Zinc finger protein 28), FAM155A (Family with sequence similarity 155 member A) or DPP6 (Dipeptidyl peptidase 6) gene.

Another object of the present invention is to provide a composition for diagnosing renal cancer prognosis, which comprises an agent for measuring the methylation level of the ZFP28, FAM155A or DPP6 gene.

Another object of the present invention is to provide a kit for the prognosis of renal cancer, which comprises the above composition.

Another object of the present invention is to provide a method for providing information for diagnosing kidney cancer prognosis.

In order to achieve the above object, the present invention provides a marker composition for diagnosing renal cancer prognosis, which comprises ZFP28, FAM155A or DPP6 gene.

In one embodiment of the present invention, the genes are hypermethylated in kidney cancer, so that diagnosis of kidney cancer is possible, and in particular, clear cell renal cell cancer can be diagnosed.

In one embodiment of the present invention, the diagnosis of renal cancer according to the present invention may include a diagnosis of renal cancer prognosis. Distant metastasis or distant tumor relapse after renal cancer surgery, . ≪ / RTI >

In one embodiment of the present invention, the ZFP28 gene is represented by SEQ ID NO: 1, the FAM155A gene is represented by SEQ ID NO: 2, and the DPP6 gene is represented by SEQ ID NO: 3.

In addition, the present invention provides a composition for diagnosing renal cancer prognosis, which comprises an agent for measuring the methylation level of a ZFP28, FAM155A or DPP6 gene.

The present invention also provides a kit for the prognosis of renal cancer, which comprises the above composition.

At this time, the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit.

In addition, the present invention provides a method for detecting the methylation level of a ZFP28, FAM155A or DPP6 gene from a biological sample isolated from a patient; And

And comparing the expression level of the gene or the level of the protein encoded by the gene with the methylation level of the corresponding gene of the normal control sample, to provide information for diagnosing renal cancer prognosis.

In one embodiment of the present invention, the renal cancer may be clear cell renal cell carcinomas (ccRCC).

In one embodiment of the invention, the methylation level may be measured using pyrosequencing or bisulfite sequencing methods.

In one embodiment of the present invention, the sequencing method comprises a primer pair represented by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, a primer pair represented by SEQ ID NO: 7, SEQ ID NO: , SEQ ID NO: 11 and SEQ ID NO: 12.

In one embodiment of the present invention, the sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.

The composition for markers for the diagnosis of renal cancer prognosis, which comprises the ZFP28 (Zinc finger protein 28), FAM155A (Family with sequence similarity 155 member A) or DPP6 (Dipeptidyl peptidase 6) gene of the present invention is a clear cell renal cell carcinoma ) Distant metastasis or distant tumor relapse that may occur after surgery can be diagnosed at an early stage, so it can be used for kidney cancer treatment and prognosis prediction.

FIG. 1 is a schematic diagram illustrating a research design and validation strategy process of the present invention.
Figure 2 is a Kaplan-Meier curve for predicting the probability of metastasis following the methylation state (bisulfite sequencing) of ZFP28 in ccRCC patients.
FIG. 3 is a Kaplan-Meier curve for predicting the probability of metastasis following methylation status (bisulfite sequencing) of FAM155A in RCC patients.
FIG. 4 is a Kaplan-Meier curve for predicting the probability of metastasis following DPP6 methylation status (bisulfite sequencing) in ccRCC patients.
FIG. 5 is a Kaplan-Meier curve for predicting the probability of metastasis following methylation score (M score) in ccRCC patients.
FIG. 6 is a Kaplan-Meier curve for predicting the probability of metastasis following the -M score adjusted according to pathological T stage in ccRCC patients.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for markers for diagnosis of renal cancer prognosis, which comprises ZFP28 (Zinc finger protein 28), FAM155A (Family with sequence similarity 155 member A) or DPP6 (Dipeptidyl peptidase 6) gene.

In the present invention, clear cell renal cell carcinomas (ccRCCs) are known as cancellous cell type renal cell carcinomas and are the most frequently occurring cancer in renal cell carcinoma.

Epigenetic gene silencing is a molecular mechanism by which the promoter region is methylated to silence the gene. In previous epigenetic studies, DNA methylation in renal-cancer-associated methylated genes and kidney cancers, Although some studies have been reported on genome-wide analysis of DNA methylation analysis, few biomarker studies have been done to diagnose kidney cancer prognosis based on DNA methylation analysis.

Therefore, the present inventors performed DNA methylation sequencing analysis to detect specifically methylated genes in clear cell renal cell carcinoma (ccRCC) in renal cancer and to characterize prognostic methylation markers of ccRCC of the detected novel candidate genes And clinical results were associated with each other, thus completing the present invention.

The term "diagnosing" as used herein means identifying the presence or characteristic of a pathological condition. For purposes of the present invention, the diagnosis is to predict or confirm renal cancer prognosis.

A "diagnostic marker" or "diagnosis marker" is a substance capable of distinguishing a renal cancer cell from a normal cell, and is a polypeptide or nucleic acid showing an increase pattern in a cell having kidney cancer as compared to a normal cell (for example, : mRNA, etc.), lipids, glycolipids, glycoproteins, sugar (monosaccharides, disaccharides, polysaccharides, etc.) and the like. For the purposes of the present invention, the renal cancer prognostic markers are hypermethylated in kidney cancer tissues with the ZFP28, FAM155A or DPP6 gene.

"Prognosis" refers to predicting the course and outcome of a disease, which includes predicting distant metastasis or distant tumor relapse that may occur after renal cancer surgery. Therefore, it is very important to accurately predict the distant metastasis or distant tumor relapse after renal cancer surgery. It is very important that the clinical indices such as the degree of differentiation and stage of the tumor are complemented and the response of treatment Predictable factors are needed, since the ZFP28, FAM155A or DPP6 methylation levels of the present invention have such an indicator function and can be used as a kidney cancer prognostic factor. In other words, the measurement of expression of these genes can be used as a prognostic marker (diagnostic marker) for predicting the degree of differentiation and stage of renal cancer, distant metastasis or distant tumor relapse that may occur after surgery .

"Metastasis" means that the tumor spreads from the first organs (primary site) to other tissues (including lymph nodes) as the malignant tumor progresses. In the present invention, cancer diseases or tumors Metastasis, tumor relapse or recurrence from the primary site, kidney, to the lymph nodes and / or other organs (lung, brain, contat, Kd, etc.) Local recurrence is not included.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue" or "cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule. The nucleic acid used in the present invention is preferably a CpG-containing nucleic acid such as a CpG site.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "gene expression" generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. The present invention encompasses all cases of gene methylation, mRNA expression, and protein expression as a mode of gene expression.

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequences are placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

≪ Preparation Example 1 >

 All embodiments of the present invention were conducted in accordance with the applicable laws and regulations, the clinical trial management standards for medicines, and the ethical principles of the Helsinki Declaration, and were approved by the Chungbuk National Ethics Committee (IRB approval number 2010-01-001) I agreed with them. All samples were collected and analyzed with the approval of the institutional review board of Chungbuk National University and consent was obtained from each patient.

The tissue sample was an original array set of 12 kidney tissue samples obtained from surgically treated ccRCC patients and 12 matched normal kidney surrounding tissues as an independent validation set, 152 ccRCC kidney tissues and 25 matched normal kidney tissues were used. In the case of ccRCC samples, radical or partial renal resection was performed between September 1997 and December 2014. Partial nephrectomy was obtained from an early ccRCC patient. Pathological samples were independently reviewed and confirmed by pathologists who did not know whether the clinical data were tumor or normal tissue. In order to reduce the confounding factor that may affect the analysis, patients who met the following criteria were used as subjects in this study: 1) lymph-node (LN) clinically and pathologically, Or T1 to T4 without distant metastasis; 2) Histologically pure cytoplasmic RCC; 3) minimum follow-up period is 3 months; 4) In the case of simple tumor removal and final pathology, the tumor was excluded to avoid correcting the survival estimate when the tumor was not completely removed (positive surgical margin).

The surgical procedures were performed including pure laparoscopic, hand-assisted laparoscopic, robot-assisted and open approaches. Pathological staging was performed according to the 7th edition of the American Joint Committee on Cancer classification system, and tissue differentiation was graded according to the Fuhrman nuclear grading system. After surgery, each patient was monitored according to standard guidelines and distant metastasis was defined as metastasis to the lymph nodes and / or other organs (lung, brain, contat, Kd, etc.), but local recurrence It is defined as not being included.

≪ Example 1 > Experimental method

<1-1> DNA methylation profiling (DNA 메틸레이션  profiling)

Genomic DNA was extracted using standard methods of the Wizard Genomic DNA Purification System (Promega, Madison, Wis.). DNA methylation profiling was performed using a total of 24 matched DNA samples (12 matched normal-kidney tissues, 12 cc RCC), wherein the 12 ccRCC tissue samples were randomly arranged in the pathology step . The methylation status was analyzed using Illumina Infinium Human Methylation 450K Bead-Chips (Illumina Inc., San Diego, Calif.) Containing 27,578 very useful CpG sites (CpG sites) covering 14,000 or more human RefSeq genes. DNA and chip processing transformed with bisulfite and data analysis were performed according to the manufacturer's instructions.

<1-2> Selection of candidate methylation-silenced genes

β values showed quantitative measurements of the level of DNA methylation of the specific CpG site from 0 (not fully methylated) to 1 (fully methylated). The selection criteria applied to screen for silenced gene candidates by methylation are: 1) the difference in methylation level (Δβ value) between RCC and the matched control> 0.25; 2) the mean β value for the matched control <0.15.

<1-3> Analysis of PSQ

Using the PyroMark Q96 ID (Qiagen, Valencia, Calif.) According to the manufacturer's instructions, the DNA methylation status of the candidate CpG site in the inventor's 152 ccRCC samples was determined by PSQ. Each primer was designed using Pyrosequencing Assay Design Software v2.0 (Qiagen, Valencia, Calif.). The PSQ primer was designed to include the position occupied by the CpG site analyzed on the Illumina Infinium array. Primer sequences and amplification conditions Are shown in Tables 1 and 2 below. The methylation rate was calculated as the average degree of methylation at the position occupied by the candidate CpG site produced by pyroshocking or bisulfite.

gene name order SEQ ID NO: ZFP28 cg24152605-F Gt; 4 cg24152605-R 5'biotin-CCAAAATAACTAAAATCCCTACAAT-3 ' 5 cg24152605-S 5'-TGTGGGTTTGTGAGG-3 ' 6 FAM155A cg05383490-F 5'-GGGGTGTTGTTAATTAGATTTTTAAGT-3 ' 7 cg05383490-R 5'biotin-AAAATTAAACCCTTTAAATCAATACATAT-3 ' 8 cg05383490-S 5'-ATTTTGGGAAAGTATAGTTTTG-3 ' 9 DPP6 cg06495961-F 5'-TGGTTTTTGGTTTTTTGTTTAAAGTAATA-3 ' 10 cg06495961-R 5'biotin-ATCTTCCAAATCTTCAATTTTCTCAC-3 ' 11 cg06495961-S 5'-AGTTGATTGTTATTGGGAT-3 ' 12

ZFP28 PCR product cg24152605
Forward PCR Primer cg24152605
Reverse PCR Primer cg24152605
Sequencing Primer Length (nt) 170 18 25 15 Location 5'- 3 ' 538-555 707-683 539-553 Temperature (℃) 60.6 56.1 47.8 % GC 42.9 55.6 28.0 53.3 Sequence to Analyze GATYGGTYGG AAGGG FAM155A PCR product cg05383490
Forward PCR Primer cg05383490
Reverse PCR Primer cg05383490
Sequencing Primer Length (nt) 159 27 29 22 Location 5'- 3 ' 484-510 642-614 551-572 Temperature (℃) 58.8 56.4 41.7 % GC 24.5 29.6 17.2 27.3 Sequence to Analyze GTYGATTTTA TYGTGAGGG DPP6 PCR product cg06495961
Forward PCR Primer cg06495961
Reverse PCR Primer cg06495961
Sequencing Primer Length (nt) 165 29 26 19 Location 5'- 3 ' 456-484 620-595 541-559 Temperature (℃) 57.9 59.4 46.6 % GC 26.7 20.7 30.8 31.6 Sequence to Analyze TYGTTGAGTA AAGATA

<1-4> Statistical analysis

In the R language environment (version 2.10.0, available at http://www.r-project.org/), quantitative standardization of DNA methylation data was standardized.

Kaplan-Meier analysis (100 permutations and 80% sampling) was performed to investigate the prognostic relevance of the location of the candidate CpG site in the test set of TCGA data, and those having statistical significance more than 50 times were analyzed by the inventor's clinical sample In order to verify the validity of the results.

The difference between consecutive variables between groups was analyzed using a two sample t-test or ANOVA trend analysis using polynomial contrasts.

The receiver operating characteristic (ROC) curve was used to determine the optimal cut-off point to have the highest sensitivity and specificity for distant metastasis prediction of candidate markers.

To correlate the methylation status of the methylation markers with the prognosis prediction, Cox regression analysis was used to evaluate the predicted methylation level of each gene selected for remote metastasis prediction, and the methylation level of the genes obtained therefrom The methylation score (M score) of each patient was calculated by multiplying the values by the regression coefficients. The Kaplan-Meier curve was used to predict the transition time according to the methylation status, and the difference was analyzed using a log-rank test.

Proportional hazards regression models of multivariate Cox proportional hazards regression models were used to assess prognostic value for the methylation status of candidate markers and to compare well-known clinicopathologic features (sex, age, body mass index, Tumor stage and stage) factors. Statistical analysis was performed using SPSS 20.0 software (IBM Co., Armonk, NY, USA). A P value <0.05 was considered statistically significant.

Example 2: Profiling methylation and identification of candidate silent genes in primary ccRCC

In order to identify the ccRCC-specific methylation gene, the DNA methylation profiling analysis method of Example <1-1> was used.

The methylation profiles of 12 pairs of ccRCC and normal normal kidney were analyzed using Illumina Infinium Human Methylation 450K Bead-Chips. Using Zinc finger protein 28 (ZFP28), Family (ZFP28) and Family (ZFP28) as compared with the normal normal kidney, it was confirmed by using very strict selection criteria with sequence similarity 155 member A (FAM155A) and dipeptidyl peptidase 6 (DPP6) genes were hypermethylated in ccRCC (see Table 3 and Figure 1).

gene Gene name Normal Cancer Difference (△ ß) P-value ZFP28 Zinc finger protein 28 0.0654182 0.360547636 0.295129436 0.00000102447 FAM155A Family With Sequence Similarity 155, Member 0.132351049 0.418272862 0.285921813 0.02223947920 DPP6 Dipeptidyl peptidase 6 0.132958368 0.38787536 0.254916992 0.00007835980

Example 3: Identification of individual methylated array results in primary ccRCC

To confirm the methylation array results, an individual validation cohort of 25 surgically treated primary ccRCCs and 25 tissues corresponding to peripheral normal height was performed (see Table 4). Individual cohort validation studies showed promoter methylation of ZFP28, FAM155A and DPP6 more frequently in ccRCC than in normal kidney (P <0.001 for each).

variable Cohort array
(Array cohort) Cohort Feasibility
(Validation cohort) Patient number 12 152 Mean age - years (yr) ± SD 59.0 + - 12.4 Follow-up - months, median (IQR) 39.2 (15.479.1) Male Gender (%) 110 (72.4) BMI - kg / m ² , median (IQR) 24.6 (22.1-26.6) HTN (%) 56 (31.6) DM (%) 29 (16.4) Previous or current smokers (%) 42 (27.6) Pathologic T Stage - Number of patients (%) pT1a 81 (53.3) pT1b 31 (20.4) pT2 14 (9.2) pT3 23 (15.1) pT4 3 (2.0) Nuclear grade 1-2 102 (67.1) 3-4 50 (32.9) Number of patients with recurrence (%) 22 (14.5)

< Example  4> The relationship between methylation levels and cancer aggressiveness

In order to quantitatively measure the methylation status of each candidate gene, PSQ analysis of the above Example <1-3> was performed and the relationship between the degree of methylation of the candidate gene and significant clinical and pathological factors Respectively. The relationship between the individual clinical pathology of the primary ccRCC and the degree of methylation of the candidate gene is shown in Table 5 below. Hypermethylation of the three genes was associated with late tumor stage and higher tumor grade (see Table 5).

Variables ZFP28 FAM155A DPP6 Methylation ratio (%) P-value Methylation ratio (%) P-value Methylation ratio (%) P-value pT armor
(pT stage) 0.001 0.002 <0.001 T1 18.30 ± 11.95 15.69 ± 10.10 19.41 + - 11.24 T2 26.56 ± 15.53 24.40 ± 17.39 25.39 ± 14.96 T3-4 28.19 ± 15.27 24.20 ± 19.15 32.61 ± 17.57 Pulmon rating
(Fuhrman grade) 0.022 0.039 0.001 1-2 18.85 ± 12.09 16.15 ± 10.89 19.21 + - 10.35 3-4 24.63 + - 15.32 21.61 ± 16.67 28.35 ± 17.42 Relapse
(Recurrence) 0.002 <0.001 <0.001 NO 19.38 ± 13.21 16.29 ± 12.13 20.44 ± 12.98 YES 28.86 ± 12.36 27.72 ± 15.66 32.74 ± 13.61

Example 5: Methylation status as a prognostic factor

Analysis using Cox regression analysis, Kaplan-Meier estimates and log-rank test showed median follow-up of 39.2 months (range, 15.479.1) ), 22 patients (14.5%) had distant metastases. Kaplan-Meier estimates showed significant differences in metastatic survival rates according to the methylation status of the three candidate genes (log rank test, all P <0.001) (see FIGS. 2 to 4). The M score, which is the combined methylation level of the three candidate genes, and the -M score adjusted according to the pathologic T stage showed the same results (log rank test, all P <0.001) (see FIGS. 5 and 6) . According to multivariate statistical analysis, HR was determined by the methylation state of each gene and the clinical pathologic factor, respectively. The methylation status of each of the three candidate genes (ZFP28, Hazardratio (1), 7.012, P = 0.008; FAM155A, HR, 7.080, P = 0.008; DPP6, HR, 6.444, P = 0.011) as individual predictors of cancer metastasis The methylation status was confirmed by multivariate statistical analysis at the integrated methylation score (M score, HR, 3.804, P = 0.006) of the three candidate genes (see Table 6). Here, M socre was evaluated as the sum of the regression coefficients multiplied by the methylation level of the three genes obtained from the Cox regression analysis.

Variables Univariate Multivariate HR (95%, Cl) P-value HR (95%, Cl) P-value age 1.016 (0.981-1.052) 0.373 Sex (female) 0.779 (0.304-1.998) 0.603 HTN (yes) 0.780 (0.317-1.919) 0.588 DM (yes) 0.928 (0.314-2.744) 0.893 BMI (≥23 kg / m ² ) 0.371 (0.160-0.859) 0.021 0.270 (0.092-0.791) 0.017 Smoking history (yes) 0.760 (0.273-2.114) 0.598 TNM armory pT1 pT2 One One pT3-4 5.651 (1.711 - 18.662) 0.004 7.111 (1.776-28.464) 0.006 Perlman rating (G3-4) 10.331 (3.811-28.011) <0.001 6.567 (2.133-20.215) 0.001 M score (≥153.28) ^* 5.385 (2.192-13.229) <0.001 2.442 (0.841-7.091) 0.101

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Use of ZFP28, FAM155A and DPP6 for Prognostic Marker Diagnosis of          신소 세포 <130> PN1611-408 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 1122 <212> DNA <213> Unknown <220> &Lt; 223 > Human ZFP28 (cg24152605) <400> 1 ggtcccaccc ctcgccgcaa gccccgcctc acccggtctg ccgctgcgcc tgcgcgactc 60 ccagaagcct ttggggggtg ggtgtgtagt cagagcggcc tctgcttccg gccacaccca 120 ggcccagcgt tggccaccac ttatgcggcg gctgcggtcc agggacaagt gagagcgacc 180 cgcagagggc aggacggacg ggcgttgcat aggcccgggg ccaaaccgta tcttgcaacc 240 tgagctccga gggccgccgc atccgtccct gcttgggact acgcttccca gcgggcattg 300 cgcggaggcc acgcccggtt ccggcggcgc tgcctgcgca ctggaagtgg gaagcgccgg 360 gcttctcttg gctgagggga ggggcggggc ggggcggggc gnnnnnnnnn nggggtgggg 420 aggggcgggg cgcggccggg ggcggggcgg ctccgcggcg cggcccagtg gatgccgggc 480 catgggggag cgtgcgcggg gctgttcgct gggcgtggcg ggcgggtgtg gccaggggtg 540 tgggtctgtg agggaccggt cggaagggcg tcgcgcggcc tcgggtgaca tgcggggggc 600 ggcgagcgcg agtgtccgcg agccgacgcc gctcccgggt agaggcgccc cccgcacaaa 660 gccccgggcg ggccgaggcc cgactgtagg gactccagcc accttggccc tccctgcccg 720 gggaaggccg cgctcaagga atggcctcgc atccaaaggc cagcgaggag cggcccctac 780 ggggcctggg cacagaggtg agagtgacag gtgtttgggg ccgagcggac agggacgaat 840 tcatctcggg atgagatctg ggagggggtt gggctctcgg ggaccagttg ctggagaact 900 gtgcccctgg tctttgaagc tgttggagtg ggagaatctg aagatttgga agctcaactg 960 attcagagca gcaagccgcg gtaaaagggt gttgactttc ccagggtgac tctaactaga 1020 cacttttcct gttttcgtgc tcccagggag gaaaaaaacc tatctccacg ttgtggcagt 1080 ccctaggcag ggtaatgatc tgatcccacc ttttcatgca gg 1122 <210> 2 <211> 1122 <212> DNA <213> Unknown <220> <223> Human FAM155A (cg05383490) <400> 2 ccgtgaagca ctggcccgag gacgcgccct gggggtaaca agtctccagg cgccacacgg 60 gcttggcaga gtttcctaga aaaagagcct tgccccggtc gtctttgcct cggttgccct 120 tgccgccgcc gccgccgccg tctcccgggg aggggggcag ggtgggggac gaggaggcgg 180 agaggagtct gtgtgcctgg gcggcgggcg aggactcccc catgctcgcc aggagcgcgg 240 gccaggaggg ctcctgctgc cgccgctgct gctgctgctg ctgccgctgc ctctgctgct 300 gctgctgctg ctgctgctgc cgctgctgct gctggtgctc cttgtcccgg gcccgggtca 360 gcttggcctc ggcgcagaac cacaagtgat cagagagcag gactgtgaaa aacaagagag 420 atgccagaga cagtcgccat ttctgagccc tctcggaatc gatgaacggt ttctcgttct 480 ctcggggtgc tgccaaccag atttttaagc cgtcgtcata ctgccgacac atccaagcac 540 ccctggtcat attttgggaa cgcacagccc tggccgactc caccgtgagg gcgcctgtgc 600 cggtgtcacc acaatatgca ttgacttaaa gggtttaatt tccttatccc ctcctcccgt 660 ttcttctctc tcctctctct ctttctctct cttcccctct ccctctctcc tctctctctc 720 tctccctctc cctctctttt atctctctct gtctcttttg cctacataaa tattaccagg 780 ctttgaagga aggtctgact tgcttcctaa tcatcagccg accccatcct ctgtagagtg 840 ggaaacaata atggagagag agagagagag agagacggag gaggctggtg ctggtggccg 900 gcggcgaggc tgggtgcagg gggcgatggt ggaggtgaca gggtggctgg cgcggctccg 960 gtcacccagg cattgtcagc gcgcgggtcc ccatggcccc gcggagccca gcccgctctc 1020 ccctgccagg ggcatgccgc gcctccgctg cccactgacg gcgcccggag cgcctcccct 1080 cctcctcctc ctcctcctct tcttctcctc ctcttcctcc tc 1122 <210> 3 <211> 1122 <212> DNA <213> Unknown <220> <223> Human DPP6 (cg06495961) <400> 3 gtttgtggaa gccgagcgtg cgtgcgccgc gcgcgcaccc agtccagcgc ggagtgggcg 60 tctacccgag gaggggtgtc tggggagggg ctgccctcgt tacccaaaca gtttgcgctc 120 gcttaacctt gatgcagctc gaggcttccc agtccagctc agttcagaca gaaaacctgg 180 cgcgcgcgcg cgcacacaca cacgcctccc ctggcgtcgc cgcccggccg ggtccctgcc 240 cttagggacc agagcggcga ccgctgcacc ccgcaccgcc tgctggagga gcccccggag 300 ccggggccga gccgccggcg tccccgagtg cgccccctgt gcgtgccgcc gcgctgttgc 360 tcgcagtgtg ctggcgccga gctcggtgga cacgcgcgca gtcagagctg cctctcgccc 420 tcgctagctg ggctcgcagc ctcttcctcc ctccctggct cctggctttt tgtttaaagc 480 aacacccacc ctccatccag gctttttttc tttctttctt tattggtagc ggccaaaaag 540 agttgattgc tattgggatc cgctgagtaa agacacgggc aggggtgcgc ggaggtgaga 600 aaactgaaga cctggaagat ttttttttcc ttcaaaaacc cgtttccatc cagtcttcag 660 ccagtccagt ctactttaat cctcaccagg acaatggatt aagtttctct tccctggacc 720 agaagtcggg ttcggacttg gggcaaaatg aaggaaaagg ccatgatcaa gaccgctaag 780 ggggggaccc 840 accgtgagga gcgatgctgg gggaggtctg tccttctcag tcccgaacct ccctggaagg 900 acagcgaccc catgcccgcg cgcggcggcg cttctcccac ttcccacccg agcccaccca 960 gcggcagggg gatgcggagg agcaggcatt tctttgcaaa ttgcaacttt gcggctccgc 1020 ggcccctctc cttcgggcat gtggctttgt gttttgggcg cgggatggga ggaaggggct 1080 gcggggagcc ctcgctgacc gcgggtcggt ccgagcccca ag 1122 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ZFP28 (cg24152605) forward primer <400> 4 gtgtgggttt gtgaggga 18 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ZFP28 (cg24152605) reverse primer <400> 5 ccaaaataac taaaatccct acaat 25 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> ZFP28 (cg24152605) probe <400> 6 tgtgggtttg tgagg 15 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> FAM155A (cg05383490) forward primer <400> 7 ggggtgttgt taattagatt tttaagt 27 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> FAM155A (cg05383490) reverse primer <400> 8 aaaattaaac cctttaaatc aatacatat 29 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > FAM155A (cg05383490) probe <400> 9 attttgggaa agtatagttt tg 22 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DPP6 (cG06495961) forward primer <400> 10 tggtttttgg ttttttgttt aaagtaata 29 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > DPP6 (cG06495961) reverse primer <400> 11 atcttccaaa tcttcaattt tctcac 26 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DPP6 (cg06495961) probe <400> 12 agttgattgt tattgggat 19

Claims (15)

A Zinc finger protein (ZFP28) gene, and a Family with sequence similarity (FAM155A) gene. The method according to claim 1,
Wherein the at least one gene selected from the group consisting of ZFP28 gene and FAM155A gene is hypermethylated in renal cancer tissue. The method according to claim 1,
Wherein the kidney cancer is clear cell renal cell carcinomas (ccRCC). The method according to claim 1,
Wherein the kidney cancer prognosis includes distant metastasis or distant tumor relapse. The method according to claim 1,
Wherein the ZFP28 gene is represented by SEQ ID NO: 1, and the FAM155A gene comprises the sequence represented by SEQ ID NO: 2. A ZFP28 gene, and a FAM155A gene. The composition for diagnosing renal cancer prognosis according to claim 1, The method according to claim 6,
Wherein the renal cancer is clear cell renal cell carcinomas (ccRCC). The method according to claim 6,
Wherein the renal cancer prognosis includes distant metastasis or distant tumor relapse. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; A kit for the prognosis of kidney cancer, comprising the composition according to claim 1 or 6. Measuring the methylation level of at least one gene selected from the group consisting of the ZFP28 gene and the FAM155A gene from the biological sample separated from the patient; And
Comparing the expression level of the gene or the level of the protein encoded by the gene with the methylation level of the gene of interest in a normal control sample. 11. The method of claim 10,
Wherein the kidney cancer is clear cell renal cell carcinoma (ccRCC). 11. The method of claim 10,
Wherein at least one gene selected from the group consisting of the ZFP28 gene and the FAM155A gene is hypermethylated to provide information about the risk of distant metastasis or distant tumor relapse of renal cancer. 11. The method of claim 10,
Wherein the methylation level is measured using a pyrosequencing or bisulfite sequencing method. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; 14. The method of claim 13,
The sequencing method uses one or more primer pairs selected from the group consisting of primer pairs shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and primer pairs shown in SEQ ID NO: 7, SEQ ID NO: &Lt; / RTI &gt; 11. The method of claim 10,
Wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.

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