WO2014027831A1 - Bladder cancer prognosis diagnostic marker - Google Patents

Description

Bladder Cancer Prognosis Diagnostic Markers

The present invention relates to a novel biomarker and its use for the diagnosis of bladder cancer, more specifically non-invasive (superficial) using the expression characteristics according to the methylation pattern of one or more genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3. Non-muscle Invasive Bladder Cancer (NMIBC) relates to a prognostic diagnostic marker.

Bladder cancer is the most frequent cancer in the urinary system. It is reported that 16.5 cases per 100,000 population occur in the West and 4.5 cases occur in Korea. Although the incidence rate is lower than in the West, the incidence rate is increasing year by year, and it is known as the most frequent cancer among the urinary system cancers in Korea (Lee C, et al., 1992).

Bladder cancer is classified into non-muscle invasive bladder cancer and invasive bladder cancer according to the degree of invasion. Noninvasive bladder cancer is a lesion in which the cancer is confined to the mucous membrane without invasion of the muscle layer, and can be treated relatively simply by transurethral resection of bladder tumor or by injecting an anticancer agent or BCG into the bladder. Progression becomes a problem. Invasive bladder cancer, on the other hand, refers to a condition in which the cancer has infiltrated into the muscle layer. For the treatment of the invasive bladder cancer, in addition to radical bladder extraction, complex urinary diversion may be performed, and the patient may have fatal results. Therefore, prediction and early detection and prevention of recurrence and progression after primary treatment are very important.

Recently, methods for diagnosing cancer by measuring DNA methylation have been proposed. DNA methylation mainly occurs in the cytosine of CpG island of the promoter region of a specific gene, whereby the binding of transcription factors interferes. As the expression of certain genes is blocked, detection of the methylation of the promoter CpG island of tumor suppressor genes is very helpful for cancer research, which is called methylation specific PCR (hereinafter referred to as "MSP") or Attempts have been made to use such methods as automatic base analysis to diagnose and screen cancer.

Promoter methylation of tumor-related genes is an important indicator of cancer and can be used in many ways, including the diagnosis and early diagnosis of cancer, the prediction of carcinogenic risk, the prognosis of cancer, the follow-up of treatment, and the prediction of response to anticancer therapy. Attempts have recently been made to investigate the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, and urine and 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).

Accordingly, the present inventors have hypermethylated HOXA9, ISL1 and ALDH1A3 genes that are specifically methylated in bladder cancer cells through microarray analysis from non-muscle Invasive Bladder Cancer (NMIBC) tissue. The present invention was completed by confirming that is down-regulated.

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

An object of the present invention is to provide a composition for markers for the diagnosis of bladder cancer, containing one or more of the above genes.

Another object of the present invention to provide a variety of uses for the diagnosis of bladder cancer and bladder cancer prognosis of the genes.

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

In order to solve the above problems, the present invention provides a biomarker of one or more genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3 and a composition for markers for bladder cancer diagnosis containing them. In particular, it is effective for the diagnosis of non-muscle invasive bladder cancer (NMIBC) prognosis.

Since the genes are hypermethylated in bladder cancer and their expression is reduced, bladder cancer can be diagnosed. If possible, the genes are preferably used in a combination of two or more, and the more genes used in combination, the more preferable. Most preferably, they are used in combination of all three genes.

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

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

Determining the expression level of the gene includes measuring the level of methylation, mRNA or protein, most preferably measuring the methylation level of the gene.

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

At this time, the measurement of methylation level is known PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyro sequencing and Methods such as bisulfite sequencing can be used.

Agents for measuring the level of gene mRNA may comprise primers that specifically bind to one or more genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3, and agents for measuring the levels of the proteins may be HOXA9, ISL1 and It may comprise an antibody specific for one or more proteins selected from the group consisting of ALDH1A3.

In another aspect, the present invention provides a kit for diagnosing bladder cancer prognosis comprising the diagnostic composition. In this case, the kit may be an RT-PCR kit, a DNA chip kit or a protein chip kit.

In another aspect, the present invention provides a method for providing information for diagnosing the prognosis of non-muscular invasive bladder cancer, including:

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

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

In another aspect, the present invention utilizes the gene function of the biomarker, and provides a method for screening a material for treating bladder cancer, in particular, non-invasive (superficial) bladder cancer, comprising the following steps.

(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 for treating bladder cancer when methylation of one or more genes selected from the group consisting of HOXA9, ISL1 and ALDH1A3 without candidates is inhibited.

At this time, too, the genes are preferably used in combination of two or more.

As described above, the present invention provides a variety of uses based on the novel function of HOXA9, ISL1 and ALDH1A3 genes as a prognostic diagnostic factor for bladder cancer, most preferably Non-muscle Invasive Bladder Cancer (NMIBC). To provide.

As described above, the present invention relates to a novel function of a particular gene, and has the effect of providing a composition and method for diagnosing the prognosis of bladder cancer, especially non-muscle invasive bladder cancer (NMIBC). . The present invention may also provide a method for screening a therapeutic agent for bladder cancer by screening for substances that inhibit hypermethylation of the HOXA9, ISL1 and ALDH1A3 genes.

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

Figure 1 schematically shows the experimental design and strategy for the present embodiment.

2 and 3 show the results of gene methylation and expression patterns in microarray and PSQ values in NMIBC and NC.

4-10 are graphs showing Kaplan-Meier curves predicting the likelihood of relapse and progression according to methylation status in NMIBC patients [HOXA9 (FIG. 4), ISL1 (FIG. 5), ALDH1A3 (FIG. 6), And M score (FIG. 7); And ISL1 (FIG. 8), ALDH1A3 (FIG. 9), and M score (FIG. 10) for progression]

Definitions of terms used in the present invention are 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 the prognosis of bladder cancer. Especially, it is useful for the diagnosis of non-muscle invasive bladder cancer (NMIBC) prognosis.

"Diagnostic markers or diagnostic markers are substances that can differentiate bladder cancer cells from normal cells. Polypeptides or nucleic acids (e.g. mRNA, etc.) show an increased pattern in cells with bladder cancer compared to normal cells, Organic biomolecules such as lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.), etc. For the purposes of the present invention, bladder cancer prognostic markers are hypermethylated in bladder cancer tissue with HOXA9, ISL1 and ALDH1A3. Are genes with reduced expression.

"Prognosis" means the prediction of the course and outcome of a disease, and the present invention encompasses the prediction of the recurrence progression of bladder cancer. Therefore, the indicators that can accurately predict the recurrence and progression of bladder cancer is very important, and factors that can predict the response of treatment while complementing clinical indicators such as tissue differentiation and staging, the genes of the present invention HOXA9, ISL1 and Since ALDH1A3 functions as an indicator, it can be used as a prognostic factor for bladder cancer. That is, the expression characteristics of these genes can be used as a prognostic indicator (diagnosis marker) useful for predicting the degree of differentiation, stage, and progression of bladder cancer.

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

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. One or both strands of a double stranded nucleic acid molecule and any fragment or portion of an 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 in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

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 a cell. In that sense, gene expression includes not only transcriptional and translational processes, but also posttranscriptional and posttranslational processes that can affect the biological activity of a gene or gene product. The processes include, but are not limited to, RNA synthesis, processing and transport, as well as post-translational modifications of polypeptide synthesis, transport and polypeptide. In the present invention, aspects of gene expression include all cases of methylation of gene promoter, mRNA expression and protein expression.

A "coding region" or "coding sequence" refers to a nucleic acid sequence, complement thereof, or portion thereof that encodes a particular gene product or fragment thereof that requires expression, depending on the conventional base pair and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are linked together by a cell's biochemical machinery to provide mature mRNA. Antisense strands are complements of the nucleic acids, and coding sequences can be estimated from them. The coding sequence is placed in a relationship with transcriptional regulatory elements and translation initiation and termination codons such that transcripts of the appropriate length are produced and translated in the appropriate reading frame to produce the desired functional product.

An “primer” is an oligonucleotide sequence that hybridizes to complementary RNA or DNA target polynucleotides and functions as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example by the action of nucleotidyltransferases that occur in polymerase chain reactions. Means.

A “protein” is also to include fragments, analogs and derivatives of a protein that possess essentially the same biological activity or function as the reference protein.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates the detection of a reagent conjugated to, fused, conjugated or fused to a reagent, eg, a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

The expression “down-regulation” refers to a significant decrease in the expression level of a particular gene into mRNA or protein by intracellular transcription or translation compared to normal tissue cells. it means.

"Antibody" is used in its broadest sense and specifically refers to intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies and Antibody fragments that exhibit the desired biological activity.

"Epigenetics" refers to changes in gene expression that are inherited by offspring without alteration of DNA sequences. Mostly, researches on the concept that abnormal genetic information due to changes in four nucleotide sequences (loss, substitution, amplification, etc.) accumulate in tumorigen genes or tumor suppressor genes, and their function is amplified or lost, affecting cancer development. But this alone has not been enough to explain the development, growth, and metastasis of cancer. Recently, epidemiology that regulates the expression of genes without mutations is developing into a new field of cancer-related research. Epigenetic changes occur through processes such as DNA methylation, histone modification, and genomic imprinting.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include the treatments required for the disorders that have already occurred as well as the disorders to be prevented. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

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

Bladder cancer is the most frequent urinary tract tumor in Korea, and the mechanism and progression of bladder cancer are known to occur through various causes and stages. Recent studies on chromosomes and genetic abnormalities and the occurrence, recurrence and progression of bladder cancer There are many studies on prognostic factors that can predict.

Bladder cancer can be divided into superficial and invasive cancers at the time of initial diagnosis and appears superficial in about 75% of patients. Superficial tumors are more common, with about 30% of the recurrent superficial tumors showing higher malignancy or stage progression, and at 10%, the involvement into the muscle layer.1 For low malignant Ta lesions Relapses in 50-70%, progression to invasive bladder cancer in about 5%, high malignant T1 lesions relapse in more than 80% and progress to invasive bladder cancer in 50% of patients within 3 years. Factors recognized to affect the progression of these tumors include bladder cancer found in T1 stage and G3 malignancy (T1G3), multiple epithelial cancer, high recurrence rate, residual tumor after bladder BCG therapy, and expression of p53 gene. Reported.

In invasive bladder cancer, bladder extraction is known as a standard treatment. The prognosis after invasive bladder cancer is T stage, N stage, lymph node density, malignancy, tumor size, number, type, lymph node / vascular involvement, and urinary tract epithelium. It is known to be related to the state of. Eighty to ninety percent of invasive bladder cancers are primary invasive bladder cancers without a history of previous superficial bladder cancers, but about 15% of those have previously progressed from superficial bladder cancers to invasive bladder cancers. There were also cases of primary bladder cancer diagnosed as invasive bladder cancer at the first diagnosis and superficial bladder cancer at the first diagnosis, but recurred and advanced to invasive bladder cancer (advanced invasive cancer).

Therefore, such frequent recurrences and progression of stages are a frequent problem in bladder cancer, and it is necessary to find indicators or develop treatments that can effectively predict the progression of recurrence and invasiveness of bladder cancer.

As a result of studies on genetic abnormalities, there are reports 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 ; 17: 1025-9, Korean J Urol 2005; 46: 211-20, Cancer Genet Cytogenet 1999; 110: 87-93, Cancer Res 1998; 58: 3555-60), the HOXA9, ISL1 and ALDH1A3 genes and bladder cancer prognosis The relevance of is not known at all.

We have found that the HOXA9, ISL1 and ALDH1A3 genes are hypermethylated in Bladder Cancer cancer tissues. That is, the expression relationship between bladder cancer and specific genes was first identified.

Accordingly, the present invention relates to the use of one or more genes selected from HOXA9, ISL1 and ALDH1A3 as a diagnostic marker for bladder cancer prognosis, and to predict and diagnose the bladder cancer prognosis by confirming whether the gene is hypermethylated and thus reduced expression. Can be. In particular, it is very useful for prognostic diagnosis of non-muscle Invasive Bladder Cancer (NMIBC).

Prognosis of the bladder cancer includes recurrence and progression.

In this specification, recurrence is defined as the relapse of primary NMIBC at lower or equivalent pathological stage, and progression is defined as muscle infiltration (TNM stage T2 or higher) or metastatic disease.

[Use of bladder cancer prognosis diagnostic marker]

Accordingly, the present invention relates to the use of at least one gene selected from HOXA9, ISL1 and ALDH1A3 as a prognostic diagnostic marker of bladder cancer and bladder cancer in one aspect.

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements. Bladder cancer prognosis diagnostic marker of the present invention shows the same result in repeated experiments with genes whose expression changes by direct or indirect factors with the onset of bladder cancer, and the difference in expression level is very large when compared with the control group, resulting in incorrect results. Highly reliable markers with little probability. Therefore, the result of diagnosis based on the result obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

Certain genes of the present invention are characterized in that they are hypermethylated and downregulated in bladder cancer tissues.

Thus, bladder cancer can be predicted through methylation and / or downregulation of HOXA9, ISL1 and ALDH1A3 genes.

In particular, the present invention can be used individually as a diagnostic or predictive marker, or in combination with several marker genes in the form of a panel display, where several marker genes are reliable through an overall expression pattern or a list of methylated genes. And to improve the efficiency. The genes identified in the present invention can be used individually or as a set of genes in which the genes mentioned in this example are combined. Alternatively, genes can be ranked, weighted, and selected for the level of likelihood of developing cancer, depending on the number and importance of the genes methylated together.

Preferably it can be determined by a combination of two or more of the genes, most preferably a combination of all three genes.

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

In the same aspect, the present invention relates to a composition for diagnosing bladder cancer prognosis comprising an agent for measuring the expression level of one or more genes selected from HOXA9, ISL1 and ALDH1A3.

At this time, the 'gene expression level measurement' includes both measuring the level of methylation, mRNA or protein thereof. Most preferably the methylation level is measured.

1. Measurement of DNA Methylation Level

"DNA methylation" refers to the binding of methyl groups (CH3) to the 5 carbon sites of CpG by DNA methyl transferase (DNMT), which occurs in the promoter CpG islands, and this DNA methylation is responsible for cell cycle or apoptosis. Regulate, repair DNA, and are involved in cell adhesion and intercellular interaction.

CpG islands are sites of 0.2-3kb in length with a C + G content of at least 50% and a CpG ratio of at least 3.75%. There are about 45,000 CpG islands in the human genome, most of which are found at promoter sites that regulate gene expression. Indeed, the CpG islands are found in promoters of housekeeping genes, about 50% of human genes (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5: 309, 1995). . The CpG is a site where most epigenetic changes occur frequently in mammalian cells.

In particular, when the HOXA9, ISL1 and / or ALDH1A3 promoter CpG islands of the invention are methylated, such methylation inhibits expression and function of the gene in the same way as mutations in the coding sequence, thereby developing, recurring and progressing cancer. This will be facilitated.

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

(a) isolating sample DNA from clinical samples;

(b) amplifying the isolated DNA using 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 the presence or absence of the amplified product produced in step (b).

The measurement of the DNA methylation level can use various known methods. For example, the methylation measurement method may be PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing and vi It may be characterized in that it is selected from the group consisting of sulfite sequencing.

Methylation specific PCR method

When bisulfite is treated on genomic DNA, cytosine at the 5'-CpG'-3 site remains cytosine when methylated, and uracil when unmethylated. Therefore, PCR primers corresponding to the sites where the 5'-CpG-3 'nucleotide sequence exists were prepared for the converted nucleotide sequence after bisulfite treatment. In this case, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated were prepared. When pyro DNA is converted to bisulfite, and PCR is performed using the two kinds of primers, PCR products are produced by using the primers corresponding to the methylated sequences when methylated. In the PCR product is produced by using a primer corresponding to unmethylation. Methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real time methylation specific PCR

Real-time methylation-specific PCR converts the methylation-specific PCR method into a real-time measurement method. After treating bisulfite on genomic DNA, a PCR primer corresponding to methylation is designed and real-time PCR is performed using these primers. It is. At this time, there are two methods of detection using a TaqMan probe complementary to the amplified base sequence, and two methods of detection using Sybergreen. Thus, real-time methylation specific PCR can selectively quantitate only methylated DNA. In this case, a standard curve may be prepared using an in vitro methylated DNA sample, and the standardization may be quantitatively analyzed by amplifying a gene without a 5'-CpG-3 'sequence in a nucleotide sequence into a negative control group.

Pyro Sequencing

The pyro sequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. As in bisulfite sequencing, genomic DNA was converted by bisulfite treatment, and PCR primers corresponding to sites without the 5'-CpG-3 'sequence were prepared. The genomic DNA was treated with bisulfite, amplified with the PCR primers, and then subjected to real-time sequencing using the sequencing primers. Quantitative analysis of the amount of cytosine and thymine at the 5'-CpG-3 'site can indicate the degree of methylation as the methylation index.

PCR or quantitative PCR and DNA chips using methylated DNA specific binding proteins

In the PCR or DNA chip method using methylated DNA-specific binding proteins, when a protein that specifically binds to methylated DNA is mixed with DNA, only methylated DNA can be selectively separated because the protein specifically binds to methylated DNA. . After genomic DNA was mixed with methylated DNA specific binding proteins, only methylated DNA was selectively isolated. After amplifying these separated DNA using a PCR primer corresponding to the promoter site, it can be determined whether or not methylation by agarose electrophoresis.

In addition, methylation can also be determined by quantitative PCR.Methylated DNA separated by methylated DNA-specific binding proteins can be labeled with a fluorescent dye and hybridized to DNA chips having complementary probes to measure methylation. Can be.

Detection of Differential Methylation-Bisulfite Sequencing Method

Other methods of detecting nucleic acids containing methylated CpG include contacting a sample containing nucleic acid with an agent that modifies unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using CpG-specific oligonucleotide primers. It includes. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and unmethylated nucleic acid. The amplification step is optional and desirable but not necessary. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and unmethylated DNA. Such methods are disclosed in US Pat. No. 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

That is, in one specific aspect of the present invention, by using the above-described method and the like to measure the DNA methylation level of the HOXA9, ISL1 and ALDH1A3 gene can be used to diagnose the prognosis of bladder cancer when hypermethylated.

2. mRNA expression level measurement

"Measurement of mRNA expression level" is to measure the amount of mRNA in the process of confirming the presence and expression of mRNA of the marker genes in a biological sample to diagnose bladder cancer.

Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and RNase protection assay (RPA). , Northern blotting, DNA chips, etc., but are not limited to these.

At this time, the primers used may initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers 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 can also be modified with a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

Thus, in one specific aspect of the present invention, it is possible to provide a bladder cancer diagnostic marker composition comprising a primer sequence specific for one or more genes selected from HOXA9, ISL1 and ALDH1A3. At this time, the down-regulation by measuring the mRNA expression level of the genes may provide a use for diagnosing the prognosis of the bladder cancer.

3. Determination of protein expression level

In addition, protein expression level measurement is a process of confirming the presence and 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 specifically binds to the protein of the gene. You can check the amount of protein using.

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

Analytical methods for this purpose include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited to these. .

Thus, as another aspect, the present invention can provide a bladder cancer diagnostic marker composition comprising an antibody specific for at least one protein selected from HOXA9, ISL1 and ALDH1A3. At this time, when down-regulated by measuring the protein expression level of the genes can provide a use for diagnosing the prognosis of bladder cancer.

[Sample]

The following protocol related to the detection of specific markers in a sample is shown for illustration.

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

Tissue samples can be fixed (ie preserved) by conventional methods. Those skilled in the art will appreciate that the fixative may be selected depending on the purpose for which the sample is histologically stained or otherwise analyzed. One skilled in the art will also understand that the length of fixation is determined by the size of the tissue sample and the fixative used.

The method further includes a protocol for examining methylation levels, mRNA or protein expression in tissue or cell samples. As described above, methods for evaluating methylation, mRNA, protein in cells are well known in the art.

The method may also include a protocol for investigating or detecting methylation and mRNA in tissue or cell samples by microarray technology.

Microarray methods can simultaneously study the RNA expression of thousands or even tens of thousands of genes in a tumor, making it possible to more effectively gain comprehensive insight into the molecular basis of human disease. It can also be used to assess gene expression patterns, clinical outcomes and responses to chemotherapy in tumor classification.

In this way, the prognostic diagnosis of bladder cancer is possible by measuring the expression of one or more genes selected from HOXA9, ISL1 and ALDH1A3 by the above protocol using a sample. As mentioned above, the gene is preferably measured in many different combinations.

Important probes can be labeled for detection and can be labeled, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Proper labeling of such probes is a technique well known in the art and can be carried out by conventional methods.

In a similar aspect, the present invention relates to a kit for diagnosing 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 one or more genes selected from HOXA9, ISL1 and ALDH1A3. This can be done in two ways: genetic analysis and immunoassay.

To this end, the kit may comprise, for example, a primer or probe that specifically binds one or more gene sequences selected from HOXA9, ISL1 and ALDH1A3; Or an antibody that specifically binds one or more proteins selected from HOXA9, ISL1, and ALDH1A3.

That is, if 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 contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus). thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. In addition, when the kit for diagnosing human bladder cancer of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate of a label.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

As such, 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 kits of the invention comprise a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing a primer for amplifying CpG containing nucleic acid and a truncated or It may include one or more containers including a third container containing means for detecting the presence of the uncleaved nucleic acid.

[Bladder Cancer Diagnosis Method]

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

Since the 'gene expression level measurement' includes all of the levels of methylation, mRNA or protein thereof, methylation and / or downregulation of the HOXA9, ISL1 and ALDH1A3 genes may be used to predict information on the prognosis of bladder cancer. Can be.

Preferably, the combination of HOXA9 and ISL1 can be used to obtain information about the case of progression during the prognosis of bladder cancer and the case of recurrence with the combination of HOXA9, ISL1 and ALDH1A3.

In one embodiment, the method of the present invention

Measuring the expression level of at least one gene selected from HOXA9, ISL1 and ALDH1A3 from a 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 expression of the gene in the normal control sample. In particular, it is characterized by measuring the methylation level of the genes. Other details are as described above.

That is, the expression of the HOXA9, ISL1 and ALDH1A3 gene is characterized in that it is an indicator for bladder cancer.

In other words, the method for diagnosing bladder cancer prognosis relates to investigating the expression of specific markers according to non-muscle Invasive Bladder Cancer (NMIBC), in particular the method disclosed herein It may provide a convenient, efficient and cost effective means for obtaining useful data and information when evaluating appropriate or effective therapies.

[Screening method]

Meanwhile, in another aspect, the present invention relates to a method for screening a substance for treating bladder cancer, which inhibits methylation of HOXA9, ISL1, and ALDH1A3, based on the above-described fact.

The method is an embodiment,

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

(b) selecting a candidate substance for the treatment of bladder cancer when methylation of the genes is inhibited, compared to when one or more genes selected from HOXA9, ISL1, and ALDH1A3 are expressed without the candidate substance. have.

 The present invention extends to a genetic approach to up-regulation or down-regulation of expression of genes involved in methylation of HOXA9, ISL1 and ALDH1A3.

As such, the present invention shows increased methylation and decreased expression profile of at least one gene selected from HOXA9, ISL1 and ALDH1A3 in patients with bladder cancer, preferably Non-muscle Invasive Bladder Cancer (NMIBC). These results suggest that the genes characterized by the present invention can be used as target genes for developing or screening bladder cancer diagnosis and treatment, utilizing the use of these genes as diagnostic markers for bladder cancer prognosis.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be 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: six 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.

Patients were followed-up for less than six months or patients with disease relapse within six months were excluded from the study to rule out the possibility of confounding variables that could have an incomplete resection or overly impact analysis. Samples of normal bladder urinary 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) was reviewed in our previous study Mol Cancer. 2010; 9: 3 and J Clin Oncol. 2010; 28: 2660-7.

Experimental design

This experimental design and strategy is schematically shown in FIG.

All tumors were macro-dissected within 15 minutes of surgical resection. Each NMIBC sample was identified by pathological analysis of the tissue sample portion (ie, sections from TUR samples were snap-frozen in liquid nitrogen and stored at -80 ° C). Samples used in the experiment were provided by Chungbuk National University Hospital. The collection and analysis of all samples was approved by the Institutional Review Board of Chungbuk National University Hospital and agreed with each patient.

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

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

DNA methylation profile

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

β values represent quantitative measurements of DNA methylation levels of specific CpG islands from 0 (completely unmethylated) to 1 (complete methylation).

PSQ Analysis

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

 PSQ primers were designed to include CpG sites analyzed on Illumina Infinium array. The primer sequences and amplification conditions are listed in Table 1 below.

Primers were designed using NCBI Reference Sequences build version 36.1. PCR reactions included 0.01 μM primers, Bioneer Taq (Bioneer, Daejeon, Korea), and 20 ng bisulfite-treated DNA. Thermal cycling parameters are as follows: denaturation at 94 ° C. for 5 minutes; 45 cycles at 94 ° C. for 30 seconds, 52 ° C. for 30 seconds, and 72 ° C. for 30 seconds; And final elongation at 72 ° C. for 5 minutes.

Table 1

bp, base pairs; TSS, transcription start sit

Statistical analysis

DNA methylation and gene expression profile data were normalized using displacement normalization in the R language environment (version 2.10.0, available at http://www.r-project.org/). Specific analytical methods are described in Mol Cancer. 2010; 9: 3; J Clin Oncol. 2010; 28: 2660-7; And Clin Cancer Res. 2011; 17: 4523-30, and the like.

Using 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) Difference in DNA methylation level between NMIBC and NC (Δβ value)> 0.4; (2) mean β value for NC <0.15; And (3)> 3-fold difference in gene expression levels between NMIBC and NC.

To assess 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 populations were used. Differences in continuous variables between groups were analyzed using ANOVA trend analyses using two sample t-tests or polynomial controls. Pearson's correlation coefficient was used to evaluate the relationships between groups with continuous variables. Receiver operating characteristic (ROC) curves were used to determine the optimal cut-off point that represents the highest combined sensitivity and specificity for prediction (relapse or progression). The patients were then divided into subgroups (hypermethylated or hypermethylated).

To cover 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 regression coefficient derived from Cox regression analysis used to assess the predictable value of each gene. . For multivariate Cox proportional hazards regression models, the prognostic values of methylation sites are individually assessed and well-known clinicopathology (gender, age, tumor size, tumor count, urinary tract). Intra-bladder treatment, extent and stage).

Example 1 Characteristics of NC and NMIBC Patients

Baseline characteristics of NC and NMIBC patients are listed in Table 2 below.

TABLE 2

As such, on average, relapse-free survival and progression-free survival were 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, among NMIBC patients.

Example 2 Identification of Differently Methylated and Expressed Genes in NMIBC and NC

Genome-wide methylation and expression profiles from 18 NMIBC patients were compared with those of six NCs. A complete set of human bladder tissue derived microarray data was used online (http://www.ncbi.nlm.nih.gov/geo/) under the data series accession number GSE37817.

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

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

Genes as methylation markers for NMIBC were evaluated using an independent set of Infinium microarray methylation data from Western populations (Clin Cancer Res. 2011; 17: 5582-92). Δβ values are almost identical to those in other experiments. This is shown in Table 3 below.

TABLE 3

Example 3 PSQ Analysis

To assess DNA methylation levels of candidate genes using another method, PSQ analysis was performed using bisulfite-modified genomic DNA obtained from 187 human bladder samples (NC = 6, NMIBC = 181).

Four out of six candidate genes [HOXA9 (Homeobox A9), ISL1 (ISL LIM homeobox 1), ALDH1A3 (Aldehyde dehydrogenase 1 family, member A3), EOMES (Eomesodermin) PSQ analysis were technically possible and analyzed by PSQ It was. To test the reliability of the bisulfite PSQ technique, 24 bladder samples obtained from Infinium array and PSQ were compared.

As a result, as shown in Table 4, the Pearson's correlation coefficient showed an acceptable range of 0.715 to 0.940.

Table 4

P-values are calculated using Pearson's correlation coefficient.

Example 4 Methylation and Gene Expression Patterns in NMIBC and NC

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

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

.

Example 5 Association Between Methylation Levels and Clinical Pathology Variables

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

As a result, as shown in Table 5 below, in general, increased methylation values for HOXA9, ISL1, ALDH1A3, and EOMES were deeply related to the increase in tumor number, size, extent, and stage.

Table 5

^a P-value is calculated using the Students t-test

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

Example 6 Methylation Status as a Prognostic Predictor

To determine whether the selected methylation markers correlated with prognosis (relapse or progression), methylation values of each gene were divided into two by the optimal cut-off point based on the ROC curve analysis.

The results are shown in Table 6 below.

Table 6

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

To integrate the methylation sites of the prognostic justification methylates, M scores for relapse (HOXA9, ISL1 and, ALDH1A3) and progression (ISL1 and ALDH1A3) were calculated, respectively. The patients were then classified into two groups accordingly. According to the multivariate Cox regression analysis, the M score classifier was an independent predictor of relapse (Hazard ratio [HR], 1.93; P = 0.033) and progression (HR, 10.86; P = 0.004, Table 7).

TABLE 7

^a HR (hazard ratios) predict disease outcome according to methylation status (low methylation vs. high methylation).

^b HR includes clinical pathologic factors [gender, age, tumor size (<3 cm vs. ≥ 3 cm), single vs. multiple, urinary bladder therapy (no vs. yes), tumor extent. (G1 vs. G2 vs. G3), and step T (Ta vs. T1)]. In multivariate analysis, HR is determined by each gene methylation status and clinicopathological factors.

And similarly, as shown in Figures 4 to 10, Kaplan-Meier evaluation confirmed a significant difference in time versus relapse or progression with methylation status (log-rank test, P <0.05).

So far I looked at the center of the preferred embodiment for the present invention. Those of ordinary skill in the art to which the present invention pertains may include the present invention

It will be appreciated that the present invention may be embodied in a modified form without departing from the quality thereof. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (14)

A composition for bladder cancer diagnostic markers, which contains at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3. The method of claim 1, Said genes are hypermethylated in bladder cancer, the composition for markers, characterized in that the low expression. The method of claim 1, The bladder cancer is a composition for markers, characterized in that non-muscle Invasive Bladder Cancer (NMIBC). The method of claim 1, Bladder cancer diagnosis is a composition for markers, characterized in that it comprises the progression and recurrence of bladder cancer. The method of claim 4, wherein The composition for markers characterized in that it is contained in the combination of HOXA9 and ISL1 in the case of progression, and in the combination of HOXA9, ISL1 and ALDH1A3 in case of relapse. Comprising an agent for measuring the expression level of at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3, bladder cancer diagnostic composition. The method of claim 6, The gene is bladder cancer diagnostic composition, characterized in that measured in combination of two or more. The method of claim 6, The expression level of the gene is bladder cancer diagnostic composition, characterized in that for measuring the level of methylation, mRNA or protein. The method of claim 6, The bladder cancer is a composition for diagnosing bladder cancer, characterized in that the non-muscle Invasive Bladder Cancer (NMIBC). The method of claim 6, Bladder cancer progressive composition in the case of bladder cancer progression, the expression level is measured by the combination of HOXA9, ISL1 and ALDH1A3 in case of relapse. Bladder cancer diagnostic kit comprising the composition according to claim 6. Determining the methylation level of at least one gene selected from the group consisting of HOXA9, ISL1 and ALDH1A3 from biological samples 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 the normal control sample comprising the steps of providing information for diagnosing bladder cancer. The method of claim 12, The bladder cancer is characterized in that the non-muscle Invasive Bladder Cancer (NMIBC). The method of claim 12, The sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.

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