KR101557183B1 - Prognostic Marker for Diagnosis of Bladder Cancer - Google Patents

Abstract

The present invention relates to a novel biomarker for the diagnosis of bladder cancer and a use thereof. More specifically, the present invention relates to a novel biomarker for the diagnosis of bladder cancer, ) Non-muscle Invasive Bladder Cancer (NMIBC) and its use as a marker for bladder cancer prognosis.

Description

≪ RTI ID = 0.0 > Prognostic Marker for Diagnosis of Bladder Cancer &

The present invention relates to a novel biomarker for diagnosis of bladder cancer and a use thereof, and more particularly to a biomarker for diagnosing bladder cancer, which comprises at least one kind of genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3, (Non-muscle Invasive Bladder Cancer) (NMIBC) prognostic markers.

Bladder cancer is the most frequently occurring cancer in the urinary tract, with 16.5 cases per 100,000 population in the West, compared with 4.5 cases in Korea. In addition, the incidence rate is higher than in the Western world, and the incidence rate is increasing year by year. In Korea, it is known to be the most common cancer among urological cancers (Lee C, et al., 1992)

Bladder cancer is classified into non-muscle invasive bladder cancer and invasive bladder cancer depending on the degree of invasion. Non-invasive bladder cancer is a relatively mucosal lesion without involvement of the muscle layer, and can be treated with transurethral resection of bladder tumor or intravesical chemoradiotherapy or BCG. However, recurrence of cancer and invasive cancer Is a problem. On the other hand, invasive bladder cancer refers to a state in which cancer has penetrated into the muscle layer. In order to treat the bladder cancer, complicated urinary diversion should be performed along with radical cystectomy, and the result may be fatal to the patient. Therefore, prediction and early detection and prevention of relapse and progression after primary treatment is very important

Recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. DNA methylation mainly occurs in the cytosine of the CpG island (CpG island) of the promoter region of a specific gene, thereby preventing the binding of the transcription factor And the methylation of the CpG islands of the promoter of the tumor suppressor gene is highly useful for cancer research. The methylation specific PCR (hereinafter referred to as "MSP ") or the methylation specific PCR An attempt has been actively made for the diagnosis and screening of cancer by an automatic base analysis or the like.

Promoter methylation of tumor-related genes is an important indicator of cancer, and it can be used in many aspects such as diagnosis and early diagnosis of cancer, prediction of carcinogenesis risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to chemotherapy. Recently, attempts have been made to investigate the promoter methylation of tumor-associated genes in blood, sputum, saliva, stool, urine and the like and to use them in various cancer treatments (Esteller, M. et al., Cancer Res., 59:67 , 1999: Sanchez-Cespedez, M. et al., Cancer Res., 60: 892, 2000; Ahlquist, DA et al., Gastroenterol., 119: 1219, 2000).

Thus, the present inventors have found that HOXA9, ISL1, and ALDH1A3 genes that are specifically methylated in bladder cancer cells are hypermethylated through microarray analysis from non-muscle invasive bladder cancer (NMIBC) tissue, Has been down-regulated.

The present invention relates to novel uses of the specific genes HOXA9, ISL1 and ALDH1A3,

It is an object of the present invention to provide a composition for markers for diagnosis of bladder cancer which contains at least one of the above genes.

Another object of the present invention is to provide various uses of the genes for the diagnosis of bladder cancer and bladder cancer.

It is another object of the present invention to provide a method for screening a substance having a therapeutic function for bladder cancer using the genes.

In order to solve the above problems, the present invention provides a biomarker of at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3, and a marker composition for diagnosing bladder cancer, which contains them. In particular, it is effective for the diagnosis of non-muscle invasive bladder cancer (NMIBC).

Since the genes are hypermethylated in bladder cancer and expression is decreased, diagnosis of bladder cancer is possible. If possible, the above genes are preferably used in combination of two or more, and the more the number of genes used in combination, the more preferable. Most preferably, the combination of all three genes is used.

In addition, the diagnosis of bladder cancer according to the present invention may include a diagnosis of bladder cancer. The bladder cancer prognosis may include progression and recurrence of bladder cancer. In particular, regarding the combination of genes, Is most preferably a combination of HOXA9 and ISL1, and in the case of recurrence it is most preferably used in combination of HOXA9, ISL1 and ALDH1A3.

Based on the function of the gene, the present invention provides, in one embodiment, a composition for diagnosing bladder cancer, which comprises an agent for measuring at least one gene expression level selected from the group consisting of HOXA9, ISL1 and ALDH1A3.

Determining the level of expression of said gene comprises measuring the level of methylation, mRNA or protein, and most preferably measuring the level of methylation of said genes.

In addition, in the present invention, the expression level of the gene

The methylation level can be measured by known PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing, And bisulfite sequencing may be used.

The agent for measuring the level of gene mRNA may include a primer that specifically binds to at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3, and the agent for measuring the level of the protein may be HOXA9, ISL1, Lt; RTI ID = 0.0 > ALDH1A3. ≪ / RTI >

Further, the present invention provides, as another embodiment, a kit for the prognosis of bladder cancer comprising the diagnostic composition. At this time, the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit.

The present invention also provides, as yet another embodiment, a method for providing information for the diagnosis of non-muscle invasive bladder cancer prognosis, comprising:

Measuring the methylation level of at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3 from the biological sample isolated 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 corresponding gene of the normal control sample.

At this time, the genes are preferably used in combination of two or more, and the sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.

The present invention also provides a screening method for bladder cancer, particularly non-neuronal (superficial) bladder cancer treatment, comprising the following steps as another embodiment utilizing the gene function of the biomarker.

(a) methylating at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3 in the presence of a candidate substance; And

(b) selecting a candidate substance for treating bladder cancer when the methylation of the genes is inhibited, as compared to when one or more genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3 are methylated without candidate substances.

At this time, it is also preferable that the genes are used in combination of two or more.

As described above, the present invention is applicable to a variety of uses based on the novel functions of the HOXA9, ISL1 and ALDH1A3 genes as bladder cancer, most preferably non-muscle invasive bladder cancer (NMIBC) Lt; / RTI >

As described above, the present invention relates to a novel function of a specific gene, and is effective for providing a composition and a method for diagnosing bladder cancer, particularly Non-muscle Invasive Bladder Cancer (NMIBC) prognosis . The present invention may also provide a method for screening a therapeutic agent for bladder cancer by screening a substance that inhibits hypermethylation of HOXA9, ISL1 and ALDH1A3 genes.

Therefore, the present invention relates to the gene function of HOXA9, ISL1 and ALDH1A3 in bladder cancer and may be useful for the early diagnosis and treatment of bladder cancer.

Figure 1 is a simplified schematic representation of an experimental design and strategy for this embodiment.
FIGS. 2 and 3 are the results of deriving the gene methylation and expression patterns in NMIBC and NC as microarray and PSQ values.
FIGS. 4 to 10 are graphs showing Kaplan-Meier curves for predicting recurrence and progression according to the methylation status in NMIBC patients (HOXA9 (FIG. 4), ISL1 (FIG. 5), ALDH1A3 And M score (Figure 7); (FIG. 8), ALDH1A3 (FIG. 9), Kl and M score (FIG. 10)

The terms used in the present invention are defined as follows.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to predict or confirm a bladder cancer prognosis. In particular, it is useful for the diagnosis of non-muscle invasive bladder cancer (NMIBC).

"Diagnostic markers or diagnostic markers are substances that can distinguish bladder cancer cells from normal cells and include polypeptides or nucleic acids (eg, mRNA) that show increased patterns in bladder cancer cells compared to normal cells, (Monosaccharide, disaccharide, polysaccharide, etc.), etc. For the purpose of the present invention, the bladder cancer prognostic marker is HOXA9, ISL1 and ALDH1A3, which are hypermethylated in bladder cancer tissues, Genes whose expression is decreased.

"Prognosis" refers to the progression of disease and prediction of outcome. In the present invention, it includes predicting the progression of recurrence of bladder cancer. Therefore, it is very important to accurately predict the recurrence and progression of bladder cancer. Factors that can predict the response of the treatment while complementing clinical indicators such as the degree of differentiation and stage of cancer are required. The genes of HOXA9, ISL1, Since ALDH1A3 has such an indicator function, it can be used as a prognostic factor for bladder cancer. In other words, the measurement of the expression of these genes can be used as a prognostic marker (diagnostic marker) for predicting the differentiation, stage, and progression of bladder cancer.

"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 island.

"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 methylation, mRNA expression, and protein expression of a gene promoter 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 .

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

The term "down-regulation" refers to the expression of a specific gene into mRNA or a marked reduction in the amount of protein expressed by gene transcription or gene translation, as compared to normal tissue cells it means.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

"Epigenetics" refers to the transfer of genes to the offspring without altering the DNA sequence. A study of the concept that abnormal genetic information due to changes in four base sequences (deletion, substitution, amplification, etc.) is accumulated in tumorigenic genes or tumor suppressor genes and their function is amplified or lost, , But this alone did not explain the development, growth and metastasis of cancer. In recent years, hosiology, which regulates only the expression pattern of genes without mutations, is developing into a new field of cancer-related research. Welfare changes occur through processes such as DNA methylation, histone modification and genomic imprinting.

"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.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The disease to be diagnosed in the present invention is a disease associated with bladder cancer, particularly non-muscle invasive bladder cancer (NMIBC).

It is known that bladder cancer is the most frequent urological tumor in Korea. It is known that the mechanism and progression of bladder cancer occur through various causes and stages. Recent studies on chromosomes and gene abnormalities, The prognostic factors that can predict the prognosis are actively researched.

Bladder cancer can be classified as superficial cancer and invasive cancer at the time of initial diagnosis, and it is superficial in about 75% of patients. In the case of superficial tumors, 30% of recurrent superficial tumors are more malignant or progressing to stage than malignant tumors, and 10% of them are involved in muscle layer. Recurrence in 50-70%, progression to invasive bladder cancer in 5%, and recurrence in more than 80% of highly malignant T1 lesions and invasive bladder cancer in 50% of patients within 3 years. Among the factors that are considered to affect the progression of these tumors are bladder cancer, multiple intraepithelial cancer, high recurrence rate, residual tumor after BCG therapy in bladder, and expression of p53 gene in T1 staging and G3 malignant pathway (T1G3) .

In patients with invasive bladder cancer, bladder ablation is the standard treatment, and the prognosis after invasive bladder cancer is T stage, N stage, lymph node density, malignancy, tumor size, number, morphology, lymphatic / And the like. Approximately 80-90% of myoclonic bladder cancers are those of primary, near-invasive bladder cancer without a history of previous superficial bladder cancer, and about 15% of cases have previously progressed from superficial bladder cancer to invasive bladder cancer. The first diagnosis is invasive bladder cancer (primary invasive cancer), and the first diagnosis is superficial bladder cancer. However, recurrence of invasive bladder cancer (progressive invasive cancer) is also present.

Therefore, these frequent recurrences and stage progression are problems frequently encountered in bladder cancer, and it is necessary to develop an indicator for finding an effective marker for recurrence and invasiveness of bladder cancer

As a result of the study on gene abnormality, it has been reported that gene amplification such as 1q21-24, 3p24, 6p22, 8q21-22, 10q22-23, 12q15-21, 17q11-21, 17q22 is frequently found in bladder cancer (Int J Oncol 2000 ; HOXA9, ISL1, and ALDH1A3 genes and bladder cancer prognosis; and the prognosis of bladder cancer. There is no information about the relevance of

The present inventors have found that HOXA9, ISL1 and ALDH1A3 genes are hypermethylated in Bladder Cancer cancer tissues. That is, the expression of bladder cancer and the specific genes was first identified.

Therefore, the present invention relates to the use of at least one gene selected from among HOXA9, ISL1 and ALDH1A3 as a marker for prognosis of bladder cancer, and it is possible to predict and diagnose bladder cancer prognosis through confirmation of hypomethylation of the gene and reduction in expression thereof . In particular, it is very useful for the prognosis of non-muscle invasive bladder cancer (NMIBC).

The prognosis of bladder cancer includes recurrence and progression.

Recurrence is defined herein as a recurrence of primary NMIBC at a lower or equivalent pathological stage and progression is defined as muscle infiltration (TNM stage T2 or higher) or metastatic disease.

[Bladder cancer prognostic marker use]

Accordingly, the present invention relates to the use of at least one gene selected from among HOXA9, ISL1 and ALDH1A3 as a marker for prognosis of bladder cancer and bladder cancer.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The bladder cancer prognostic markers of the present invention show the same results in repeated experiments with genes whose expression changes directly or indirectly with the onset of bladder cancer and the difference in expression level is very large when compared with the control group, They are highly reliable markers with little probability. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The specific genes of the present invention are hypermethylated and expressed down-regulated in bladder cancer tissues.

Thus, bladder cancer can be predicted by methylation and / or down-regulation confirmation of HOXA9, ISL1 and ALDH1A3 genes.

In particular, the present invention can be used as a diagnostic or prediction marker, either individually or in combination with some marker genes in the form of a panel display, and some marker genes can be used as a diagnostic or predictive marker, And efficiency can be confirmed. The genes identified in the present invention can be used individually or as a set of genes in which the genes mentioned in the present embodiment are combined. Alternatively, the genes can be ranked, weighted, and the level of likelihood of developing cancer by the number and the significance of the methylated genes together.

Preferably a combination of two or more of the genes, most preferably all three genes.

In one preferred embodiment, the progression of bladder cancer can be measured by a combination of HOXA9 and ISL1, and in the case of recurrence by HOXA9, ISL1 and ALDH1A3.

From the same viewpoint, the present invention relates to a composition for the diagnosis of bladder cancer, which comprises an agent for measuring the level of expression of at least one gene selected from among HOXA9, ISL1 and ALDH1A3.

Here, the 'measurement of gene expression level' includes all of measuring the level of methylation, mRNA or its protein. Most preferably, the level of methylation is measured.

One. DNA  Methylation Methylation ) Level measurement

"DNA methylation" is a DNA methyl transferase (DNMT) that binds a methyl group (CH3) at the 5-carbon site of CpG, which occurs in the promoter CpG island, which causes cell cycle or apoptosis Regulates, restores DNA, and is involved in cell adhesion and intercellular interactions.

A CpG island is a region with a C + G content of 50% or more and a CpG ratio of 3.75% or more and a length of 0.2 to 3 kb. The human genome has about 45,000 CpG islands, most of which are found in promoter regions that regulate gene expression. Indeed, the CpG isoform is found in the promoter of housekeeping genes that amount to about 50% of the human gene (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5: 309, . The CpG is a site where most vestigial changes occur frequently in mammalian cells.

In particular, the methylation of the CpG isoform of the promoters HOXA9, ISL1 and / or ALDH1A3 of the present invention inhibits the expression and function of the gene in the same manner as the mutation of the coding sequence, .

Therefore, as a specific example, a method for detecting whether the gene is promoter methylated may comprise the steps of:

(a) separating sample DNA from a clinical sample;

(b) amplifying the isolated DNA with a primer capable of amplifying a fragment comprising a CpG island of a gene promoter selected from the group consisting of HOXA9, ISL1 and ALDH1A3; And

(c) determining whether the promoter is methylated based on whether or not the amplified product is produced in step (b).

The DNA methylation level can be determined by various known methods. For example, the methylation measurement method can be performed by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing, and bi- And sulfite sequencing.

Methylation specificity PCR  ( 메틸레이션 specific PCR ) Way

When the genomic DNA is treated with bisulfite, the cytosine in the 5'-CpG'-3 region is methylated and remains cytosine as it is, and when it is unmethylated, it changes into uracil. Therefore, a PCR primer corresponding to the 5'-CpG-3 'nucleotide sequence was prepared from the base sequence after bisulfite treatment. At this time, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated primers were prepared. When pyroDNA is converted into bisulfite and then PCR is performed using the above two types of primers, PCR products are produced by using primers corresponding to the methylated nucleotide sequence when methylated, and in the case of unmethylated PCR products are produced using primers corresponding to unmethylated sequences. The methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real-time methylation specificity PCR  ( real time 메틸레이션 specific PCR )

Real-time methylation specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. The PCR primer corresponding to methylation of bisulfite treated with genomic DNA is designed and real-time PCR is performed using these primers . At this time, there are two methods of detecting using a TaqMan probe complementary to the amplified nucleotide sequence and using Sybergreen. Therefore, real-time methylation-specific PCR can quantitatively analyze only methylated DNA. At this time, a standard curve can be prepared using an in vitro methylated DNA sample, and the methylation degree can be quantitatively analyzed by amplifying a gene having no 5'-CpG-3 'sequence in the nucleotide sequence as a negative control for standardization.

Pyrosequencing

The pyrosequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. Similar to bisulfite sequencing, the genomic DNA was transformed by treatment with bisulfite, and then a PCR primer corresponding to the region lacking the 5'-CpG-3 'nucleotide sequence was prepared. The genomic DNA was treated with bisulfite, amplified with the PCR primer, and subjected to real-time sequencing using a sequencing primer. The amount of cytosine and thymine in the 5'-CpG-3 'region was quantitatively analyzed and the degree of methylation can be expressed as a methylation index.

Methylation DNA  Using specific binding proteins PCR  Or quantification PCR  And DNA  chip

In PCR or DNA chip method using methylated DNA-specific binding protein, when a protein specifically binding to methylated DNA is mixed with DNA, only the methylated DNA can be selectively isolated because the protein specifically binds to the methylated DNA . Genomic DNA was mixed with methylated DNA-specific binding protein and only methylated DNA was selectively isolated. These separated DNAs can be amplified using a PCR primer corresponding to the promoter site, and then methylated by agarose electrophoresis.

In addition, methylation can be measured using a quantitative PCR method. The methylated DNA separated by a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a complementary probe-integrated DNA chip to measure methylation .

Detection of differential methylation - Bisulfite  How to Sequence

Another method for detecting a nucleic acid containing methylated CpG comprises contacting a sample containing nucleic acid with an agent for modifying unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using a CpG-specific oligonucleotide primer . Here, the oligonucleotide primer may be characterized in that methylated nucleic acid is detected by distinguishing modified methylated and unmethylated nucleic acids. The amplification step is optional, but is not necessary. The method relies on PCR reactions to distinguish between modified (e. G., Chemically modified) methylated and unmethylated DNA. Such a method is disclosed in U.S. Patent 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

That is, in a specific embodiment of the present invention, the DNA methylation level of the HOXA9, ISL1 and ALDH1A3 genes can be measured using the above-described method or the like to provide an application for diagnosis of bladder cancer if it is hypermethylated.

2. mRNA  Measurement of expression level

"Measurement of mRNA expression level" refers to the measurement of the amount of mRNA in the biological sample to identify the presence or absence of mRNA and the expression level of the marker genes in order to diagnose bladder cancer.

RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) Northern blotting, DNA chips, and the like, but are not limited thereto.

At this time, the primers used can initiate DNA synthesis in the presence of reagents for the polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at the appropriate buffer solution and temperature. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. The primers can be chemically synthesized using other well known methods and can be modified using many means known in the art. The nucleic acid sequence may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

Thus, in one specific embodiment of the present invention, a bladder cancer diagnostic marker composition comprising a primer sequence specific to at least one gene selected from among HOXA9, ISL1 and ALDH1A3 can be provided. At this time, by measuring the mRNA expression level of the genes, down-regulation can be used to diagnose the prognosis of bladder cancer.

3. Measurement of protein expression level

In addition, the measurement of the protein expression level is a process for confirming the presence and the expression level of a protein expressed from a bladder cancer marker gene in a biological sample in order to diagnose bladder cancer. Preferably, the antibody is an antibody Can be used to confirm the amount of protein.

Antibody means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, But are not limited to, immunoelectrophoresis, immunohistochemistry, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. .

Accordingly, in another aspect, the present invention provides a bladder cancer diagnostic marker composition comprising an antibody specific for one or more proteins selected from among HOXA9, ISL1 and ALDH1A3. At this time, by measuring the level of protein expression of the genes, down-regulation can be used to diagnose that it represents the prognosis of bladder cancer.

[Sample]

The following protocol relating to the detection of a specific marker in the sample is presented for illustrative purposes.

For sample preparation, tissues or cell samples from mammals (typically human patients) can be used. Samples may be obtained by a variety of procedures known in the art including, but not limited to surgical excision, aspiration, or biopsy. The tissue can be fresh or frozen.

The tissue sample can be fixed (i.e., preserved) by conventional methods. Those skilled in the art will appreciate that the fixative may be selected for purposes in which the sample is histologically stained or otherwise analyzed. Those skilled in the art will also appreciate that the fixed length is determined by the size of the tissue sample and the fixative used.

The method further comprises a protocol for examining methylation level, mRNA or protein expression in a tissue or cell sample. As described above, methods for evaluating intracellular methylation, mRNA, and protein are well known in the art.

The method may also include a protocol to probe or detect methylation and mRNA in tissue or cell samples by microarray technology.

The microarray method allows researchers to simultaneously study the expression of RNA in thousands or even tens of thousands of genes in tumors, enabling more comprehensive insights into the molecular basis of human disease. It can also be used to assess gene expression patterns, clinical outcomes, and response to chemotherapy regimens in tumor classifications.

Thus, the prognosis of bladder cancer can be diagnosed by measuring the expression of at least one gene selected from among HOXA9, ISL1 and ALDH1A3 by the above-mentioned protocol using a sample. As noted above, it is preferred that the gene is measured in a number of different combinations.

Important probes can be labeled for detection and can be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.

In a similar aspect, the present invention relates to a kit for the diagnosis of human bladder cancer prognosis.

The kit is used to determine the prognosis of bladder cancer, preferably non-muscle invasive bladder cancer (NMIBC), by analyzing the expression of at least one gene selected from among HOXA9, ISL1 and ALDH1A3, This can be done in the following ways: genetic analysis and immunoassay.

To this end, the kit may comprise, for example, a primer or a probe that specifically binds to at least one gene sequence selected from HOXA9, ISL1 and ALDH1A3; Or an antibody which specifically binds to at least one protein selected from HOXA9, ISL1 and ALDH1A3.

That is, when the human bladder cancer diagnostic kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (for example, Thermus aquaticus thermostable DNA polymerases obtained from Thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. And, when the human bladder cancer diagnostic kit of the present invention is applied to immunoassay, the kit of the present invention may optionally include a secondary antibody and a labeling substrate.

The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

As described above, another aspect of the present invention provides a bladder cancer diagnostic kit comprising the composition for diagnosing bladder cancer according to the present invention. Preferably, the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

In one embodiment, the kit of the present invention comprises a first container containing a partitioned carrier means for containing a sample, a preparation delicately cleaving unmethylated cytosine, a second container containing a primer for amplifying a CpG containing nucleic acid, And a third container containing a means for detecting the presence of the uncut nucleic acid.

[Bladder cancer diagnosis method]

In another aspect, the present invention provides a method for diagnosing bladder cancer or a method for diagnosing bladder cancer comprising measuring the expression level of at least one gene selected from among HOXA9, ISL1 and ALDH1A3 based on the finding.

Since the above-mentioned " gene expression level measurement " includes all the measures of methylation, mRNA or its protein level, the information on the prognosis of bladder cancer can be predicted through methylation and / or down-regulation confirmation of HOXA9, ISL1 and ALDH1A3 genes .

Preferably, the combination of HOXA9 and ISL1 can provide information on the progression of the prognosis of bladder cancer to the case of Recurrence with a combination of HOXA9, ISL1 and ALDH1A3.

In one embodiment, the method of the present invention comprises

Measuring the expression level of at least one gene selected from HOXA9, ISL1 and ALDH1A3 from the biological sample separated from the patient; And

And comparing the expression level of the gene or the level of the protein encoded by the gene with the expression of the gene of interest in a normal control sample. In particular, it is characterized by measuring the methylation level of the genes. Other details are as described above.

That is, expression of the HOXA9, ISL1 and ALDH1A3 genes is an indicator for bladder cancer.

In other words, the method for diagnosing bladder cancer prognosis relates to the investigation of the expression of specific markers, particularly according to non-muscle invasive bladder cancer (NMIBC), and the methods disclosed herein are useful for the treatment of bladder cancer patients Effective, and cost-effective means of obtaining useful data and information when evaluating appropriate or effective therapies for the treatment of cancer.

[Screening method]

On the other hand, in another aspect, the present invention relates to a method for screening a substance for treating bladder cancer that inhibits methylation of HOXA9, ISL1, and ALDH1A3 based on the facts described above.

The method comprises, in one embodiment,

(a) expressing one or more genes selected from HOXA9, ISL1 and ALDH1A3 in the presence of a candidate substance; And

(b) selecting a candidate substance for treating bladder cancer when methylation of the genes is suppressed, as compared to when at least one gene selected from HOXA9, ISL1 and ALDH1A3 is expressed without a candidate substance have.

The present invention extends to a genetic approach to the upregulation or downregulation of the expression of genes associated with methylation of HOXA9, ISL1 and ALDH1A3.

As described above, the present invention shows the increase in the methylation and the decrease in the expression profile of at least one gene selected from among HOXA9, ISL1 and ALDH1A3 in bladder cancer, preferably non-muscle invasive bladder cancer (NMIBC) These results suggest that the gene, which has been characterized by the present invention, can be used as a target gene for developing or screening a bladder cancer diagnosis and treatment agent, utilizing the use of the genes as a marker for bladder cancer prognosis.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, IL). P <0.05 was considered statistically significant.

Sample collection

A total of 187 human bladder samples were used for methylation array or pyrosequencing (PSQ) analysis: 6 normal controls (NC) and 181 NMIBC. NMIBC samples were obtained from 181 primary NMIBC patients who underwent transurethral resection (TUR) between 1995 and 2010 for histologically-diagnosed transitional cell carcinomas.

To exclude the possibility of confounding variables that could have an impact on incomplete resection or analysis, patients were excluded from the study for follow-up for less than 6 months or patients who experienced disease recurrence within 6 months. Samples of normal urinary bladder epithelium were obtained from individuals with prostatic hyperplasia, bladder injury, and bladder stones were used as controls. Microarray gene expression data for 89 samples (NC = 6, NMIBC = 83) were obtained from our previous study Mol Cancer. 2010; 9: 3 and J Clin Oncol. 2010; 28: 2660-7.

Experiment plan

The experimental design and strategy are schematically shown in FIG.

All tumors were macro-incised within 15 min of surgical resection. Each NMIBC sample was identified by pathological analysis of the tissue sample portion (i.e., the sections from the TUR specimen were snap-frozen in liquid nitrogen and stored at -80 ° C). The samples used in the experiment were received from Chungbuk National University Hospital. The collection and analysis of all samples was approved by the Chungbuk National University Hospital Clinical Examination Committee and was also approved by each patient.

Tumors were graded according to the 2002 TNM classification and the 1973 WHO grading system. When the bladder cancer sample did not contain adequate muscle or a high level of tumor was detected, a second TUR was performed 2-4 weeks after the first resection. Intermediate - or high-risk NMIBC patients received one cycle of urinary bladder intravesical therapy. Each patient was followed up and managed according to standard recommendations.

Recurrence was defined as recurrence of primary NMIBC at a lower or equivalent pathologic stage and progression was defined as TNM stage T2 or higher or metastatic disease.

DNA  Methylation profile

89 bladder samples for microarray gene expression data were available and 24 matched DNA samples (NC = 6, NMIBC = 18) were used for DNA methylation profiling. A methylation pattern was analyzed using a genome-wide Infinium array (Illumina Inc., San Diego, Calif.) Capable of interrogating 27,578 CpG dinucleotides covering 14,495 genes.

β values showed quantitative measurements of the level of DNA methylation of the specific CpG island from 0 (complete unmethylation) to 1 (complete methylation).

PSQ  analysis

DNA methylation positions of NMIBC-specific hypermethylated CpG sites were analyzed by PSQ using PyroMark Q96 ID (Qiagen, Valencia, Calif.).

 The PSQ primer was designed to contain the CpG site analyzed on the Illumina Infinium array. The primer sequences and amplification conditions are described below.

Primers were designed using NCBI Reference Sequences build version 36.1. PCR reactions included 0.01 [mu] M primer, Bioneer Taq (Bioneer, Daejeon, Korea), and 20 ng bisulfite-treated DNA. The thermal cycling parameters are as follows: denaturation at 94 DEG C for 5 minutes; Annealing at 94 캜 for 30 seconds, 52 캜 for 30 seconds, and 45 cycles at 72 캜 for 30 seconds; And a final height of 5 minutes at 72 ° C.

bp, base pairs; TSS, transcription start site

Statistical analysis

Standardization of DNA methylation and gene expression profiles was standardized using variable site standardization in the R language environment (version 2.10.0, available at http://www.r-project.org/). A specific method of analysis is Mol Cancer. 2010; 9: 3; J Clin Oncol. 2010; 28: 2660-7; And Clin Cancer Res. 2011; 17: 4523-30.

Using the 24 matched DNA methylation and expression profiles, three criteria were used to detect methylation-silencing genes with significantly different methylation levels between NMIBC and NC. The criteria are as follows: (1) the difference in DNA methylation levels (Δβ value) between NMIBC and NC> 0.4; (2) average beta value for NC <0.15; And (3)> 3-fold difference in gene expression levels between NMIBC and NC.

To evaluate the genes identified in this experiment, microarray methylation data (Ta = 17, T1 = 5, T2 = 4) from 26 bladder cancer tissues and 6 NC tissues from Western clusters were used. Differences in group-to-group continuity were analyzed using ANOVA trend analysis using a 2-sample t-test or polynomial control. Pearson correlation coefficients were used to evaluate the intergroup relationships with continuous variance. Using a receiver operating characteristic (ROC) curve, an optimal cut-off point representing the highest combined sensitivity and specificity for prediction (recurrence or progression) was determined. The patients were then divided into subgroups (low methylation or hypermethylation).

To include the total amount of methylation, the methylation score (M score) of each patient was calculated as the sum of the methylation levels of the selected genes increased by the Cox regression-derived regression coefficient used to estimate the predictive value of each gene . For the multivariate Cox proportional hazards regression models, the prognostic value of the methylation location was assessed separately and the well-known clinicopathologic features (sex, age, tumor size, number of tumors, Degree, and step) elements in the bladder.

Example  One: NC  And NMIBC  Patient characteristics

Baseline characteristics of patients with NC and NMIBC are listed in Table 1 below.

Thus, the median survival and progression-free survival of patients with NMIBC was 47.2 ± 40.4 months (median 35.8, range 6.1 to 183.3) and 61.1 ± 41.7 months (median 50.9, range 6.6 to 183.3), respectively.

Example  2 : NMIBC  And NC Lt; RTI ID = 0.0 &gt; methylated &lt; / RTI &gt;

Genomic-wide methylation and expression profiles from 18 NMIBC patients were compared to those of six NCs. A complete set of microarray data from human bladder tissue was used on-line (http://www.ncbi.nlm.nih.gov/geo/) under data series session number GSE37817.

We identified 42 unique CpG island loci in 39 methylated methylated NMIBC compared to NC using very strict selection criteria (Δ β value> 0.4 and mean β value in NC <0.15) To screen for silencing genes, the corresponding gene expression levels of 24 human bladder samples were compared.

As a result, 42 distinct, hypermethylated loci and 7 loci in 6 genes showed at least a 3-fold decrease in gene expression in NMIBC compared to NC.

Genes as methylation markers for NMIBC were evaluated using an independent set of Western-derived Infinium microarray methylation data (Clin Cancer Res. 2011; 17: 5582-92). The value of △ β almost coincided with that of other experiments. This is shown in Table 2 below.

Example  3: PSQ  analysis

PSQ analysis was performed using the bisulfite-modified genomic DNA obtained from 187 human bladder samples (NC = 6, NMIBC = 181) to assess the level of DNA methylation of candidate genes using alternative methods.

PSQ analysis was technically feasible with four of the six candidate genes [HOXA9 (Homeobox A9), ISL1 (ISL LIM homeobox 1), ALDH1A3 (Aldehyde dehydrogenase 1 family, member A3), EOMES Respectively. To test the reliability of the bisulfite PSQ technique, 24 bladder samples obtained from Infinium arrays and PSQ were compared.

As a result, as shown in Table 3, the Pearson correlation coefficient showed an allowable range of 0.715 to 0.940.

P-values are calculated using Pearson correlation coefficient.

Example  4 : NMIBC  And NC Of methylation and gene expression patterns

To confirm DNA methylation-induced gene silencing, the correlation between DNA methylation and gene expression levels was calculated using microarray expression data and the PSQ values were matched from 89 subjects.

As a result, as shown in Figs. 2 and 3, HOXA9 (r = -0.453, P <0.001), ISL1 (r = -0.501, P <0.001), and ALDH1A3 (r = -0.150 , P <0.049) showed a significant negative correlation. In addition, NMIBC patients showed significantly higher methylation and lower expression levels than NC (P <0.001)

.

Example  5: Association between methylation level and clinicopathologic variables

To assess the correlation between the methylation pattern and the clinicopathologic factors, methylation levels were observed in terms of known prognostic factors such as tumor number, tumor size and tumor grade and stage.

As a result, increased methylation values for HOXA9, ISL1, ALDH1A3, and EOMES were generally correlated with increases in tumor number, size, extent and stage, as shown in Table 4 below.

^a P-values are calculated using Student t-test

^b P-values are calculated using the ANOVA trend analysis test.

Example  6: prognosis As a predictor  Methylation status

To determine whether the selected methylation markers are associated with prognosis (recurrence or progression), the methylation value of each gene was divided into two with an optimal cut-off point based on ROC curve analysis.

The results are shown in Table 5 below.

In addition, univariate and multivariate Cox regression analysis confirmed that relapses (HOXA9, ISL1 and ALDH1A3) and progression (ISL1 and ALDH1A3) were closely related to the methylation markers of the present invention.

The M scores for recurrence (HOXA9, ISL1 and ALDH1A3) and progression (ISL1 and ALDH1A3) were calculated, respectively, to incorporate the methylation positions of prognostic methylation markers. The patients were classified into two groups according to the patients. According to multivariate Cox regression analysis, the M-score classifier was an independent predictor of recurrence (Hazard ratio [HR], 1.93; P = 0.033) and progression (HR, 10.86; P = 0.004, Table 6).

^a HR (hazard ratios) predicts disease outcomes following methylation (low methylation vs. hypermethylation).

^b HR is defined as the degree of clinical pathology (sex, age, tumor size (<3 cm vs. ≥ 3 cm), single versus multiple, intravesical therapy (no vs. yes) (G1 vs. G2 vs. G3), and T stage (Ta vs. T1)]. In multivariate analysis, HR is determined by the respective gene methylation status and clinicopathologic factors.

And, as shown in FIGS. 4 to 10, similarly, the Kaplan-Meier evaluation identified a significant difference in time to recurrence or progression according to the methylation status (log-rank test, P < 0.05).

The present invention has been described with reference to the preferred embodiments. Those skilled in the art will appreciate that the present invention is not limited to the embodiments described herein,

It will be understood that the invention may be embodied in various other forms without departing from the spirit or essential characteristics thereof. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (14)

ISL1 &lt; / RTI &gt; and &lt; RTI ID = 0.0 &gt; ALDH1A3. &Lt; / RTI &gt; The method according to claim 1,
Wherein said genes are hypermethylated and under-expressed in bladder cancer. The method according to claim 1,
Wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC). The method according to claim 1,
Wherein the diagnosis of bladder cancer includes progression and recurrence of bladder cancer. delete A composition for diagnosing bladder cancer, which comprises a probe, an antibody or a primer which measures the expression level of a gene consisting of ISL1 and ALDH1A3. delete The method according to claim 6,
Wherein the expression level of the gene is measured by measuring the level of methylation, mRNA or protein. The method according to claim 6,
Wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC). delete A kit for the diagnosis of bladder cancer comprising the composition according to claim 6. Measuring the methylation level of the gene consisting of ISL1 and ALDH1A3 from the biological sample isolated 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. 13. The method of claim 12,
Wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC). 13. The method of claim 12,
Wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.

Images