KR20080003472A - Diagnostic methods and kits for colorectal cancer - Google Patents

Abstract

Diagnostic methods and kits for colorectal cancer are provided to select tumor suppressor genes in recurrently altered genomic region(RAR) and determine their expression changes, so that they are useful for predicting prognosis of various cancers including colorectal cancer and diagnosing cancers at an early stage. A diagnostic method for colorectal cancer comprises the steps of: (1) observing the recurrently altered genomic region(RAR) selected from RAR-L1(deletion of chromosome 1p36) and RAR-L20(deletion of chromosome 21q22) on chromosome; and (2) measuring the expression changes of a specific gene such as tumor suppressor gene CAMTA1(calmodulin binding transcription activator 1) on the observed RAR, wherein the expression reduction of CAMTA1 gene on the RAR indicates negative prognosis of colorectal cancer.

Description

Diagnostic Methods and Kits for Colorectal Cancer

1 is a result of analyzing the genome of colorectal cancer patients.

A: genomic profiles of colorectal cancer patients; B: Frequency of acquisition and loss on chromosome

2 shows the result of analyzing the copy number profile using the array CGH.

A: normal tissue; B: CCRC80 tumor tissue; C: tumor tissue versus normal tissue;

3 shows the result of analyzing the correlation between the reproduced genomic change region (RAR) and the survival rate.

A: step; B: RAR-L1 on chromosome 1q36; C: RAR-L4 on chromosome 1p31; D: RAR-L20 on chromosome 21q22

4 is a result of analyzing the expression profile of the cancer suppressor gene of the present invention.

A: ratio of tumor / normal intensity; B: capran-meyer survival curves; C: missense mutation.

The present invention relates to a new method for diagnosing colorectal cancer and a diagnostic kit, more specifically: (1) observing a reproducing genomic change region (RAR) on a chromosome; And / or (2) measuring changes in gene expression of said RAR region; The process relates to diagnostic methods and diagnostic kits for determining the prognosis of colorectal cancer (CRC) and new tumor suppressor genes that can be used to diagnose colorectal cancer.

Colorectal cancer (CRC) is estimated to have occurred in 2002, approximately 1 million patients worldwide (9.4% of the world). Colorectal cancer ranks fourth in males and third in females. It is also estimated that prevalence is the second highest in the world after breast cancer, with mortality rates approximately half of the incidence (about 529,000 deaths in 2002), with 2.8 million patients diagnosed and living within five years of colon cancer. . At least 25 times the diversity of colorectal cancers worldwide. The incidence of colorectal cancer is highest in developed countries, while it is low in Africa and Asia. In South Korea, colorectal cancer ranked fourth among the causes of cancer deaths in 2004, which is higher than the world average for colorectal cancer by age in both men and women. This geographical difference is probably due to genetic background as well as environmental factors because colorectal cancer is a multifactoral disease.

It is well known that multiple mutations accumulate during the development of colorectal cancer. Genetic instability in colorectal cancer has been divided into two main forms: microsatellite instability (MIN) and chromosomal instability (CIN). In approximately 13% of colorectal cancers, the lack of mismatch repair causes microsatellite instability, while in the remaining 87%, chromosomal instability is believed to result in the acquisition and loss of genetic material. Thus, research on chromosomal instability can help identify potential oncogenes and / or tumor suppressor genes and further elucidate the course of colorectal cancer.

To study this chromosome instability, conventional comparative genomic hybridization (abbreviated as "CGH") has been used to investigate multiple chromosomal imbalances in samples from a single experiment. However, the conventional CGH method lacked the resolution to accurately identify the microscopic changes in addition to the microscopic changes. Because the collected experimental evidence suggests that changes in the amount of the genome change the expression of genes associated with cancer, causing tumorization, more detailed analysis with high resolution is needed. Recently, the combination of the existing CGH method and microarray technology enables the analysis of DNA genome-wide copy number with high accuracy. Array CGH is in the spotlight as a useful tool for detecting genomic abnormalities, whether cancer genes or tumor suppressor genes are present. In addition, array CGH can be used to molecularly classify tumors or to identify target genes for treatment or prevention since several genomic abnormalities have been shown as prognostic markers in tumors.

We performed genome-scale array CGH using genomic DNAs extracted from finely cut tissues of 59 colorectal cancer patients to investigate genomic changes and their clinicopathological significance in colorectal cancer. As a result, various changes in the number of genome copies associated with colorectal cancer were observed with a new, recurrently altered region (abbreviated as "RAR"), and genetic changes and clinical pathology found by array CGH. The association between enemy variables could be investigated.

As a result, the present inventors continued to develop a new method for diagnosing colorectal cancer. As a result, the present inventors identified 27 change regions (RARs) that are reproduced in colorectal cancer and measured the expression level of genes associated with colorectal cancer. The present invention has been successfully completed by providing two genes, RAR-L1 and RAR-L20, and a CAMTA1 gene, which acts as a tumor suppressor, from the RAR-L1. It was.

It is an object of the present invention to provide a diagnostic method and diagnostic kit for determining a new colorectal cancer prognosis and cancer suppressor genes used therein.

In order to achieve the above object, the present invention

(1) observe reproducing genomic change regions (RARs) on chromosomes; And / or

(2) measuring the change in expression of a particular gene on said RAR; Process provides a diagnostic method for determining the prognosis of colorectal cancer (CRC).

In addition, the present invention

(1) a tool for observing reproducing genomic change regions (RARs) on chromosomes; And / or (2) a tool for measuring a change in expression of a specific gene located on the RAR. It provides a diagnostic kit for determining the prognosis of colorectal cancer (CRC).

The present invention also provides a cancer suppressor gene that can be used for diagnosing colorectal cancer.

Hereinafter, the present invention will be described in detail.

The present invention

(1) observe reproducing genomic change regions (RARs) on chromosomes; And / or

(2) measuring the change in expression of a particular gene on said RAR; Process provides a diagnostic method for determining the prognosis of colorectal cancer (CRC).

The genomic change region (RAR) reproduced in step (1) is preferably one or more selected from RAR-L1 (loss of chromosome 1p36) and RAR-L20 (loss of chromosome 21q22).

In addition, in the step (2), the specific gene is preferably a cancer suppressor gene located on the RAR, and more preferably the cancer suppressor gene CAMTA1 located on the RAR.

The cancer suppressor gene CAMTA1 shows a negative prognosis of various cancers including colorectal cancer when gene expression is decreased, and a positive prognosis of various cancers including colorectal cancer when gene expression is increased.

In addition, the present invention

(1) a tool for observing reproducing genomic change regions (RARs) on chromosomes; And

(2) a tool for measuring a change in expression of a particular gene located on said RAR; A diagnostic kit is provided for determining the prognosis of colorectal cancer (CRC), and the like.

The genomic change region (RAR) to be reproduced in (1) is preferably one or more selected from RAR-L1 (loss of chromosome 1p36) and RAR-L20 (loss of chromosome 21q22).

It is preferable that it is a tool which measures the expression reduction of the cancer suppressor gene CAMTA1 located on said RAR in said (2).

The present invention also provides a cancer suppressor gene that can be used for the diagnosis of various cancers, including colorectal cancer.

The cancer suppressor gene preferably includes the CAMTA1 gene.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1. Characterization of Genomic Changes in Colorectal Cancer

(1) Collecting colorectal cancer patients and cell samples

In the present invention, samples of 59 colon cancer (CRC) patients who underwent surgery at Dankook University Hospital (Cheonan, Korea) during 1995 and 1997 were collected and used with the approval of researchers at Gangnam St. Mary's Hospital. Tumors and nearby normal tissues from each patient were surgically extracted and stored frozen in the freezer. Each tissue was prepared on gelatin coated slides using cryotom. After tissue H & E staining, the areas with high tumor cells (more than 60%) and normal cell areas were selected under a microscope and dissected by hand. The microdissected tissues were soaked in cytolysis buffer (TE buffer containing 1% protease K) and reacted at 50 ° C. for 12 hours to extract genomic DNA. DNA obtained from normal tissue was used as a control for the array CGH. The isolated DNA was purely purified using a DNA purification kit (Solgent, Daejeon, Korea) and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Delaware, USA). Histopathological findings of the tumor were made by pathologists trained according to the American Society of Standards TNM Classification of Cancer Guidelines.

(2) array comparison genome Hybridization  And data processing

We used a human clone array with 1 Mb resolution across the entire genome created by Sanger Labs microarray researchers. DNA labeling, charge hybridization, hybridization and posthybridization were performed as follows. Genomic DNA from cancer tissue was labeled with Cy3-dCTP and DNA from tumor tissue was labeled with Cy5-dCTP. Open-well hybridization was performed according to existing methods. Arrays were scanned using a GenePix 4100A scanner (Exon Instruments, USA) and images were processed using GenePix Pro 6.0. Array CGH data was normalized and rearranged using the array CGH analysis interface ArrayCyGHt on the web. Long insertion clones were mapped according to genomic location on the Ensembl and UCSC genomic browsers. In total, 2,981 BAC clones were processed from the first 3,014 clones. Information on the full set of clones is available through the Ensembl Human Genome Browser.

(3) data analysis for chromosomal changes

To set the cut-off values for chromosomal changes in each clone, four normal hybridizations were performed in separate series. Based on the hybridization results of the control group, the cut-off value for the copy number abnormality was set to be about 3 times the standard error in the data of each individual. The copy number change on the region is defined as the DNA copy number change following two or more BAC clones and not the entire chromosome. A high degree of amplification in each clone was defined by the intensity of log 2 above 1.0 and vice versa in homology loss. The extent of copy number variation was set at about half between neighboring clones. RAR was defined as the copy number change on a region appearing in at least 10 tumor samples.

(4) genome change investigation

The clinicopathological data of all 59 colorectal cancer patients are shown in Table 1 below. Thirty-nine male and twenty female patients were selected and the average age of the patients at surgery was 58.7 years (23 to 81 years). Of the 59 patients, 41 patients (69.5%) were rectal cancer (rectosigmoid cancer) and 36 patients (61.0%) were classified as early stage tumors. 23 patients died during the trial.

All of the genomic changes found in the 59 colorectal cancer patients are shown in FIG. 1. Investigations of the frequency of chromosomal changes revealed a random distribution of all of them, but several hot regions were concentrated throughout the entire genome. Data on the signal intensity ratio (in log 2 units) of the array CGH in 59 patients can be downloaded from the website.

The clones changed for each patient averaged 764.8 (58-1,540) out of a total of 2,981 clones. The number of clones changed was significantly higher in men, in groups with worse symptoms, and in rectal cancer. The most frequent changes among the chromosomes were 13q (31/59, 52.5%), 20q (30/59, 50.8%), 20p (21/59, 35.6%), 7p (20/59, 33.9%) and 8q (29 / 59, 49.2%) acquisition and loss of 18q (29/59, 49.2%), 18p (27/59, 45.8%) and 17p (26/59, 44.1%).

Example  2. Clones  Confirmation of change

The present invention uses multiplex ligation-dependent probe amplification analysis (hereinafter abbreviated as "MLPA") to examine copy number changes using MLPA-Aneuploidy test kit P095 (MRC, Netherlands ) Was performed as follows. The 250 ng of genomic DNA was denatured at 98 ° C. for 10 minutes and 3 μl of the probe mixture containing the buffer solution was added. The reaction mixture was then heated at 95 ° C. for 1 minute and reacted at 60 ° C. for 16 hours. The ligation reaction was reacted at 54 ° C. for 15 minutes using a thermally stable Lagease 65. 10 μl of reaction solution was mixed with a PCR reaction mixture containing 40 μl of universal primer. One primer was left intact and only the remaining primers were labeled with FAM [N- (3-fluantyl) maleimide]. The thermal reaction was repeated 35 minutes for 1 minute at 95 ° C. followed by 30 seconds at 95 ° C., followed by 30 seconds at 60 ° C. and 60 seconds at 72 ° C. Amplified fragments were analyzed using an ABI PRISM 3730 XL DNA analyzer (Applied Biosystem, USA) and ROX-500 as size marker. Peak regions of PCR products were estimated with Genotyper software and data were analyzed using a simplified assay obtained from Coffalyser macro.

In fact, to examine the number of copies by array CGH, MLPA analysis was performed on 13 early colon cancer patients with abnormal numbers. The copy number abnormalities identified by array CGH were approximately consistent with MLPA results. FIG. 2 illustrates the results of MLPA identification, with twelve numbered peaks showing the abnormal number of copies of 13, 18, 21 and X chromosomes, respectively.

Example  3. Identify the area of change to be reproduced

In addition to total chromosomal changes, copy number changes on many regions were observed. Of these recurrently altered regions, the reproducible change regions of the chromosome in at least 10 patients were named RAR, specifically 7 RAR acquisitions (RAR-G) and 20 RAR loss (RAR- L) was observed. Table 2 below lists the location, size and cancer related genes on the map of 27 RARs. Five of these RARs were observed in over 40% of patients: RAR-G4 (28/59, 47.5%), RAR-L2 (27/59, 45.8%), RAR-L5 (25/59, 42.2%), RAR-L14 (28/59, 47.5%) and RAR-L17 (28/59, 47.5%).

The RARs include several cancer related genes. For example, BLC11A , PLD1 , ECT2 , AGR2 , TWIST1, and BIRC4 Cancer genes such as oncogenes as well as existing oncogenes such as MYC and REL are included in the RAR-Gs. In addition, many cancer suppressor genes such as CAMTA1 , FAF1 , CTH , PTGER3 , TEC , CLDN22 , ING2 , IRF2 , ACSL5 , ANXA2 , RORA and SCO1 are located in the RAR-Ls.

Example  4. high To clone  change

A high degree of amplification and homology deletion along with cancer related genes located in RARs are summarized in Table 3.

Eleven amplified genomic segments and two homologous deletions were identified in at least one patient. The highest copy number change was observed in one patient, but amplification on 17q12, 20q11 and 20q13 was observed in two or more patients. In the amplification region, known cancer genes such as EGFR , CCND2 , ERBB2 and MYBL2 were located and also included in the high copy number variation region associated with several cancers (see Table 3).

Example  5. Examining correlations between genomic changes

Association analysis between RARs was performed by examining the significance of these interoccurrence rates. Pairs between all possible RARs on different chromosomal regions were considered. Five pairs between RARs were found to be significantly related to each other as a result of optimizing multiple testing. Specifically, RAR-L5 on chromosome 4p15 correlates with RAR-L2 on chromosome 1p33 and RAR-G7 on chromosome Xq24, and RAR-L17 on chromosome 17p13 correlates with RAR-L5 on chromosome 4p15 and RAR-L14 on chromosome 14q32. Appeared to be. RAR-L6 on chromosome 4p12 also correlated with RAR-L2 on chromosome 1p33.

In addition, it was investigated using Gene Ontology (GO) whether significantly correlated RARs share functionally related genes. Genes with the same functional markers but also located separately on two related RARs were selected. In addition, three RAR pairs have been identified that share functionally related genes along the twelve markers (see Table 4).

Example  6. Differentiated distribution of genetic changes according to clinicopathological variables

Four types of clinical variables (age, stage, sex, tumor location) were analyzed to examine their correlation with pre-identified genomic changes. Acquisition of RAR-G7, RAR-L11, RAR-L12, RAR-L13, RAR-L16, RAR-L17 and RAR-L18, chromosomes 8q, 19p and X and loss of 14q, 15q, Xq and Y correlated with sex It was. The acquisition of RAR-G3, RAR-L1, RAR-L2, RAR-L5, RAR-L6 and RAR-L20 and chromosomes 1p and 4p has been investigated to be associated with cancer progression stage. Acquisition of RAR-G7, RAR-L4, RAR-L9, RAR-L11 and RAR-L12, chromosomes 13q, 20p and 20q and loss of 18p and 18q were associated with rectal cancer sites.

Example  7. Survival analysis according to genome change

Survival analysis was performed to evaluate the clinical pathology variables and the prognostic value of RARs. Cancer progression stages, RAR-L1, RAR-L4 and RAR-L20, were significantly associated with low survival (see FIG. 3). In particular, the presence of RAR-L1 was observed to have the highest statistical significance.

Two RARs (RAR-L1 and RAR-L20), age and stage, showed significant prognosis for colorectal cancer using clinical variables such as age, sex and stage of tumor progression, with significant genomic changes investigated in this analysis. It confirmed that it could be an independent predictor (see Table 5). 3 shows two RARs significantly associated with patient survival.

Example  8. Associated with survival rate In RARs  Expression investigation of cancer gene

(1) Real time quantitative polymerase chain reaction analysis

One strand of cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen, Canada) from total RNA obtained from 44 pairs of cancer / normal tissues and three cell lines (RKO, HT29 and HCT116). In order to analyze the expression profile of the CAMTA1 gene of the present invention, real time quantitative PCR was performed using the Mx3000P qPCR system and the MxPro version 3.0 software (Stratagene, USA). 20 μl of real time PCR reaction mixture was configured to contain 10 ng cDNA, 1 × SYBR Green Tbr polymerase combination (FINNZYMES, Finland), 05 × ROX and 20 pmole primers. The GAPDH gene was used as an internal control for each experiment. The thermal reaction was repeated 40 times for 10 minutes at 95 ° C. followed by 10 seconds at 94 ° C., followed by 30 seconds at 54 ° C. and 30 seconds at 72 ° C. To confirm amplification of specific sites, melting point analysis was performed at 55 ° C to 95 ° C at 0.5 ° C / sec. Comparative quantitation was performed by the ΔΔCT method and was considered low when the expression of the CAMTA1 gene in ocular tissues was reduced by 40%. All experiments were repeated twice and the mean value of intensity within standard error was measured for each patient. The primers of the CAMTA1 gene used at this time were produced to include the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

(2) CAMTA1  Mutation Analysis of Genes

Somatic mutations of the CAMTA1 gene were examined according to PCR-direct sequencing. Primer sets for the amplification of specific exons were constructed in the same manner as the conventional method with slight modifications. Detailed information about the primer sequence is shown in Table 6 below. All amplifications were carried out using Phusion High-Fidelity DNA polymerase (FINNZYMES, Finland) and PCR products were purely separated using MEGA-spin gel extraction kit (iNtRON, Korea).

As a result, among the coding regions of the RAR-1 and RAR-20 genes, CAMTA1 The gene has been suggested as a tumor suppressor gene in neuronal tumors. Therefore, the expression profiles of these genes in three colorectal cancer cell lines and 44 pairs of early colorectal cancers were examined by real-time quantitative PCR. The ratio between gene expression levels (cancer to normal) was measured. All three cell lines and 26 of 44 colorectal cancers showed low expression of the CAMTA1 gene compared to normal tissue (see FIG. 4). Especially CAMTA1 Low expression of the gene was significantly associated with low survival compared to the normal CAMTA1 gene. Cox regression analysis showed that after optimization by age, sex and stage, low expression of the CAMTA1 gene was more significantly associated with lower survival as an independent predictor (see Table 7).

In addition, low expression of the CAMTA1 gene was observed more frequently in colorectal cancer with RAR-L1 (55.9%, 19/34) than in the absence of RAR-L1 (70%, 7/10), and the expression level was also RAR- Although not significantly lower in colorectal cancer with L1, it was confirmed. In addition, to investigate the mechanism of low expression of the CAMTA1 gene, somatic mutations (26 colorectal cancers) and methylation degrees (38 colorectal cancers) were searched. One missense mutation was found in early colorectal cancer (CCRC71), indicating that this resulted in low expression of the CAMTA1 gene without RAR-L1 (see Figure 4). However, no hypermethylation was observed in the promoter region of the CAMTA1 gene.

As described above, the present invention provides a method for the detection of (1) reproducing genomic change regions (RARs) on chromosomes; And / or (2) measuring changes in gene expression of said RAR region; The present invention relates to diagnostic methods and diagnostic kits for determining the prognosis of colorectal cancer (CRC) and new tumor suppressor genes that can be used for the diagnosis of colorectal cancer. The diagnostic method of the present invention selects a statistically significant cancer suppressor gene on the RAR region and accurately observes and measures its expression change, so that not only the prognosis of various cancers and tumors including colorectal cancer but also early diagnosis and the like can be effectively made. Help.

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Claims (8)

(1) observe reproducing genomic change regions (RARs) on chromosomes; And / or (2) measuring the change in expression of a particular gene on said RAR; A diagnostic method that determines the prognosis of colorectal cancer (CRC) by the process. The method of claim 1, The genome change region (RAR) reproduced in step (1) is at least one selected from RAR-L1 (loss of chromosome 1p36) and RAR-L20 (loss of chromosome 21q22). The method of claim 1, In (2) the specific gene diagnostic method, characterized in that the cancer suppressor gene located on the RAR. The method according to claim 1 or 3, A diagnostic method of determining the negative prognosis of colorectal cancer by measuring the decrease in expression of the cancer suppressor gene CAMTA1 located on the RAR in the step (2). (1) a tool for observing reproducing genomic change regions (RARs) on chromosomes; And / or (2) a tool for measuring a change in expression of a particular gene located on said RAR. The method of claim 5, And wherein said regenerated genomic change region (RAR) is at least one selected from RAR-L1 (loss of chromosome 1p36) and RAR-L20 (loss of chromosome 21q22). The method of claim 5, The diagnostic method, characterized in that (2) is a tool for measuring the decrease in expression of the cancer suppressor gene CAMTA1 located on the RAR. Cancer suppressor gene CAMTA1 that can be used to diagnose colorectal cancer.

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