KR101401749B1 - Diagnostic Methods and Kits for Colorectal Cancer - Google Patents

Abstract

The present invention relates to a novel cancer diagnosis method and a diagnostic kit, and more particularly, to (1) observing a recurrently altered genomic region (RAR) on a chromosome; And / or (2) measuring a change in gene expression in the RAR region; 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. The diagnostic method of the present invention allows statistically significant cancer-suppressing genes to be selected on the RAR region and precisely observes and measures changes in expression thereof, thereby enabling early diagnosis as well as prognosis of various cancers and tumors including colorectal cancer .

(RAR), colorectal cancer (CRC) prognosis, tumor suppressor genes,

Description

{Diagnostic Methods and Kits for Colorectal Cancer}

Fig. 1 shows the result of analyzing the genome of a colon cancer patient.

A: genomic profiles of patients with colorectal cancer; B: Frequency of acquisition and disappearance of chromosome images

FIG. 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;

Fig. 3 shows the result of analyzing the correlation between the reproduced genome 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

FIG. 4 shows the results of analysis of the expression profile of the cancer suppressor gene of the present invention.

A: ratio of tumor / normal intensity; B: Capran-Meyer Survival Curve; C: Missense mutation.

The present invention relates to a novel diagnostic method and diagnostic kit for colon cancer, and more particularly, to (1) a genomic change region (RAR) to be reproduced on a chromosome; And / or (2) measuring a change in gene expression in the RAR region; 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 account for one million patients worldwide (9.4% of the global total) in 2002. Colorectal cancer is the third most common cause of cancer among men in terms of incidence. It is estimated that prevalence is second only to breast cancer worldwide and mortality rate is approximately half of the incidence (approximately 529,000 deaths in 2002), 2.8 million patients diagnosed with colon cancer within five years and surviving . There is at least a 25-fold variability in the incidence of colorectal cancer worldwide. These colon cancer incidence is highest in developed countries, while it tends to be low in Africa and Asia. In Korea, colorectal cancer ranked 4th among cancer deaths in 2004, which is higher than the global average in both men and women, by age. These geographical differences are likely due to genetic background as well as environmental factors, since colon cancer is a multifactoral disease.

It is well known that various mutations accumulate in the pathogenesis of colorectal cancer. Genetic instability in colorectal cancer has been divided into two main types: microsatellite instability (MIN) and chromosomal instability (CIN). Approximately 13% of colorectal cancers are characterized by a lack of mismatch repair resulting in microsatisfactory instability, whereas in the remaining 87%, chromosomal instability is thought to result in genetic material acquisition and loss. Thus, studies of chromosomal instability can help identify potential oncogenes and / or tumor suppressor genes and further elucidate the pathogenesis of colorectal cancer.

In order to study such chromosomal instability, conventional comparative genomic hybridization (hereinafter abbreviated as "CGH") has been used to examine multiple chromosomal imbalances in samples from a single experiment. However, the conventional CGH method lacks the resolution to accurately identify microscopic changes other than microscopic changes. Experimental evidence gathered suggests that changes in the amount of genomes alter the degree of expression of genes associated with cancer, leading to tumorigenesis, so a more detailed analysis with higher resolution is needed. In recent years, the combination of the existing CGH method and the microarray technology has made it possible to analyze the genome-wide DNA replication number with high accuracy. The array CGH is seen as a useful tool for detecting genomic abnormalities in the presence of cancer genes or tumor suppressor genes. In addition, the array CGH can be used to molecularly classify tumors or to identify target genes for treatment or prophylaxis, since several genomes or more have been presented as prognostic markers in tumors.

The present inventors performed genome-wide array CGH using genomic DNAs extracted from finely cut tissues of 59 colon cancer patients to investigate genome changes and their clinicopathological significance in colorectal cancer. As a result, various changes in genome replication numbers associated with colorectal cancer were observed with a new recurrently altered region (hereinafter abbreviated as "RAR ") and genetic changes found by array CGH and clinical pathology We can investigate the relationship between the variables.

As a result of continuing efforts to develop a new method for diagnosing colorectal cancer, the inventors of the present invention confirmed the 27 regions of change (RAR) reproduced in colorectal cancer and measured the expression level of the genes related to the colon cancer obtained therefrom, (RAR-L1) and RAR-L20, and CAMTA1 gene which acts as a tumor suppressor from the RAR-L1, and using the same to diagnose and diagnose colon cancer, the present invention has been successfully completed Respectively.

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

In order to achieve the above object,

(1) observing the genomic change region (RAR) reproduced on the chromosome; And / or

(2) measuring the expression of a specific gene on the RAR; (CRC) is a diagnostic method that determines the prognosis of colorectal cancer (CRC).

In addition,

(1) a tool for observing a genomic change region (RAR) reproduced on a chromosome; And / or (2) a tool for measuring a change in the expression of a specific gene located on the RAR. The present invention also provides a diagnostic kit for determining the prognosis of a colorectal cancer (CRC).

The present invention also provides a cancer suppressor gene which can be used for the diagnosis of colon cancer.

Hereinafter, the present invention will be described in detail.

The present invention

(1) observing the genomic change region (RAR) reproduced on the chromosome; And / or

(2) measuring the expression of a specific gene on the RAR; (CRC) is a diagnostic method that determines the prognosis of colorectal cancer (CRC).

The genome change region (RAR) reproduced in the above (1) is preferably at least one 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, more preferably a cancer suppressor gene CAMTA1 located on the RAR.

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

In addition,

(1) a tool for observing a genomic change region (RAR) reproduced on a chromosome; And

(2) a tool for measuring a change in expression of a specific gene located on the RAR; The present invention provides a diagnostic kit for determining the prognosis of colorectal cancer (CRC).

In the above (1), the genome change region (RAR) to be reproduced is preferably at least one selected from RAR-L1 (deletion of chromosome 1p36) and RAR-L20 (deletion of chromosome 21q22).

It is preferable to measure the decrease in expression of the cancer suppressor gene CAMTA1 located on the RAR in (2) above.

The present invention also provides a cancer suppressor gene which can be used for diagnosis of various cancers including colon 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 illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Characterization of genomic changes in colorectal cancer

(1) Collection of colon cancer patients and cell samples

In the present invention, samples of 59 CRC patients who underwent surgery at Dankook University Hospital (Cheonan, Korea) during 1995 and 1997 were collected and used under approval of researchers at Kangnam St. Mary's Hospital. Tumors and nearby normal tissues from each patient were surgically removed and frozen and stored in the freezer. Each tissue was prepared on gelatin-coated slides using cryotom. After H & E staining, the tissue was screened and manually dissected under microscope in areas where the tumor cells were large (over 60%) and normal cells. Microdissected tissues were immersed in a cell lysis buffer (TE buffer solution containing 1% protease K) and reacted at 50 ° C for 12 hours to extract genomic DNA. DNA from normal tissue was used as a control for array CGH. The separated DNA was purified pure using a DNA purification kit (Solgent, Daejeon, Korea) and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Delaware, USA). Histopathological findings of the tumors were made by a pathologist trained according to the American Society of Clinical Oncology classification for cancer guidelines.

(2) array comparison genome Hybridization  And data processing

A human clone array with 1 Mb resolution was used in the entire genome of the Sanger Research Institute microarray researchers. DNA labeling, pre-hybridization, hybridization and post-hybridization 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 conventional methods. The array was scanned using a GenePix 4100A scanner (Exxon Instruments, USA), and the image was processed using GenePix Pro 6.0. The array CGH data was normalized (print-tip loss normalized) and rearranged using the array CGH analysis interface ArrayCyGHt on the web. Longer inserted clones were mapped according to genomic location on Ensembl and UCSC genome browsers. Overall, 2,981 BAC clones were first processed from 3,014 clones. Information about the entire clone set is available from the Ensembl human genome browser.

(3) Analysis of data on chromosome changes

To establish the cut-off values for chromosomal changes in each clone, four normal hybridization were performed in separate series. Based on the hybridization results of the control group, the cut-off value for the number of replications was set to be about three times the standard error in each individual's data. Changes in the number of copies in the region were defined as changes in the number of DNA copies following two or more BAC clones and were not targeted to the entire chromosome. A high degree of amplification in each clone was defined as a logarithmic value of greater than 1.0 for the logarithmic intensity and the opposite for homologous deletion. The range of change in the number of replications was determined to be about half of that of neighboring clones. The RAR was defined as the number of replicates on the area of at least 10 tumor samples.

(4) genome change investigation

Clinical pathological data of all 59 colorectal cancer patients are shown in Table 1 below. 39 male patients and 20 female patients were selected and the average age of the patients was 58.7 years (23 to 81 years). Of the 59 patients, 41 patients (69.5%) were rectosigmoid cancer and 36 patients (61.0%) were classified as early stage tumors. During the experiment, 23 patients died.

All of the genomic changes found in the 59 colorectal cancer patients are shown in FIG. The frequencies of chromosomal changes were all randomly distributed, but there were some hot regions concentrated throughout the entire genome. Data on the signal intensity ratio (log 2 units) of array CGH in 59 patients can be downloaded from the website.

On average, the changed clones for each patient were measured to be 764.8 (58 to 1,540) out of a total of 2,981 clones. The number of altered clones was significantly higher in men, in worsening symptoms, and in rectal cancer. The most frequent changes among the whole chromosomes were 13q (31/59, 52.5%), 20q (30/59, 50.8%), 20p (21/59, 35.6%), 7p / 59, 49.2%) and loss of 18q (29/59, 49.2%), 18p (27/59, 45.8%) and 17p (26/59, 44.1%

Example  2. Number of copies  Confirmation of Change

In order to investigate the change in the number of copies, the present invention was carried out by using MLPA-Aneuploidy test kit P095 (MRC, Netherlands), using multiplex-ligation- dependent probe amplification analysis ) Was performed as follows. 250 ng of the genomic DNA was denatured at 98 DEG C for 10 minutes and 3 mu l of the probe mixture solution containing the buffer solution was added. The reaction mixture was then heated at 95 ° C for 1 minute and allowed to react at 60 ° C for 16 hours. The ligation reaction was carried out at 54 캜 for 15 minutes using heat-stable Ligationase 65. 10 μl of the reaction solution was mixed with a PCR reaction mixture containing 40 μl of the universal primer. One primer was left alone, and only the remaining primers were labeled with FAM [N- (3-fluorenyl) maleimide]. The thermal reaction was repeated 35 times at 95 ° C for 1 minute followed by 95 ° C for 30 seconds, at 60 ° C for 30 seconds and at 72 ° C for 60 seconds. Amplified fragments were analyzed using an ABI PRISM 3730 XL DNA analyzer (Applied Biosystems, USA) and using ROX-500 as size marker. The peak region of the PCR product was estimated by Genotyper software and the data were analyzed using a simplified method from Coffalyser macro.

In order to investigate the number of clones by array CGH, MLPA analysis was performed on 13 patients with early colorectal cancer who showed more than the number of clones. The number of clones identified by the array CGH was roughly in agreement with the MLPA results. FIG. 2 illustrates the results of MLPA confirmation, in which the twelve numbered peaks show more than the number of copies of chromosome 13, 18, 21 and X, respectively.

Example  3. Identification of the area of change being reproduced

Along with the entire chromosomal change, the number of replications on many areas was observed. Of these recurrently altered regions, at least 10 patients were identified as being RARs that were reproducible in the chromosomes. Specifically, seven RAR acquisitions (RAR-G) and 20 RAR deletions (RAR- L) was observed. Table 2 lists the location, size, and cancer-associated genes on the map of 27 RARs. Five of these RARs were found in over 40% of patients: RAR-G4 (28/59, 47.5%), RAR-L2 (27/59, 45.8%), RAR- RAR-L14 (28/59, 47.5%) and RAR-L17 (28/59, 47.5%).

The RARs include several cancer-associated genes. For example, BLC11A , PLD1 , ECT2 , AGR2 , TWIST1, and BIRC4 , As well as existing cancer genes such as MYC and REL are included in the RAR-Gs. 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 Replica  change

Table 3 summarizes the high degree of amplification and homozygous deletion along with the cancer-associated genes located in RARs.

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

Example  5. Correlation between genomic changes

The association analysis between RARs was performed by examining the significance of these mutual incidence rates. Pairs between all possible RARs on different chromosome regions have been considered. Five pairs of RARs were found to be significantly related to each other as a result of optimization with multiple testing. Specifically, RAR-L5 on chromosome 4p15 is correlated 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 . Furthermore, RAR-L6 on chromosome 4p12 was correlated with RAR-L2 on chromosome 1p33.

We also investigated whether the significantly correlated RARs share functionally related genes using the gene database (Gene Ontology; GO). We also screened genes that have the same functional label but are located separately on the two associated RARs. In addition, three RAR pairs were identified to share functionally related genes along the twelve indications (see Table 4).

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

Four types of clinical variables (age, stage, sex, and tumor location) were analyzed to investigate the correlation with previously identified genomic changes. Acquisition of chromosomes 8q, 19p and X and loss of 14q, 15q, Xq and Y are associated with gender, as are RAR-G7, RAR-L11, RAR-L12, RAR-L13, RAR-L16, RAR-L17 and RAR- . RAR-G3, RAR-L1, RAR-L2, RAR-L5, RAR-L6 and RAR-L20 and the acquisition of chromosome 1p and 4p were associated with cancer progression. Acquisition of chromosomes 13q, 20p and 20q and loss of 18p and 18q were associated with rectal cancer sites, with RAR-G7, RAR-L4, RAR-L9, RAR-L11 and RAR-L12.

Example  7. Analysis of survival rate according to genome change

Survival analysis was performed to assess the value of the prognostic value of clinicopathologic variables and RARs. Cancer progression stages, RAR-L1, RAR-L4 and RAR-L20 were significantly associated with low survival rates (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 were analyzed using clinical variables such as age, sex, and tumor progression with significant genomic changes investigated in the above analysis, Confirming that it could be an independent predictor (see Table 5). Figure 3 shows two RARs significantly associated with patient survival.

Example  8. Associated with survival rate From RARs  Expression of cancer genes

(1) Real-time quantitative polymerase chain reaction analysis

One strand of cDNA was synthesized from total RNA from 44 pairs of cancer / normal tissues and three cell lines (RKO, HT29 and HCT116) using M-MLV reverse transcriptase (Invitrogen, Canada). To analyze the expression profiles of the CAMTA1 gene of the present invention, real-time quantitative PCR was performed using the Mx3000P qPCR system and MxPro version 3.0 software (Stratagene, USA). 20 [mu] l of the real time PCR reaction mixture was constructed to contain 10 ng cDNA, 1X SYBR Green Tbr polymerase combination (FINNZYMES, Finland), 05X ROX and 20 pmole primers. The GAPDH gene was used as an internal control in each experiment. The heat reaction was repeated 40 times at 95 ° C for 10 minutes and then at 94 ° C for 10 seconds, followed by 30 seconds at 54 ° C and 30 seconds at 72 ° C. To confirm amplification of a particular site, melting point analysis was performed at 55 ° C to 95 ° C at 0.5 ° C / sec. The comparative quantification was performed by the △ ΔCT method and the expression of CAMTA1 gene was decreased by 40% in the eye tissue. All experiments were repeated twice and the mean value of the intensity within the standard error was measured for each patient. The primers of the CAMTA1 gene used here were prepared so as to include the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

(2) CAMTA1  Mutation analysis of genes

The somatic mutation of the CAMTA1 gene was screened by PCR-direct sequencing. The primer set for amplification of a particular exon was constructed in the same manner as the conventional method with a slight modification. Detailed information on the primer sequences is shown in Table 6 below. All amplification procedures were performed using Phusion High-Fidelity DNA polymerase (FINNZYMES, Finland), and the PCR products were purified using a MEGA-spin gel extraction kit (iNtRON, Korea).

As a result, among the coding regions of the RAR-11 and RAR-20 genes, CAMTA1 The gene has been shown to be a tumor suppressor gene in neurogenic tumors. Therefore, the expression profiles of this gene in three colorectal cancer cell lines and 44 pairs of early colorectal cancer were examined by real-time quantitative PCR. The ratio between gene expression levels (cancer vs. normal) was measured. All three cell lines and 26 of 44 colon cancer showed low expression of the CAMTA1 gene compared to normal tissues (see FIG. 4). Specifically, CAMTA1 The low expression of the gene was significantly associated with a lower survival rate compared with the normal CAMTA1 gene. After optimization by age, sex, and stage by Cox regression analysis, low expression of CAMTA1 gene was found to be significantly associated with low survival rate as an independent predictor (see Table 7).

In addition, low expression of CAMTA1 gene was more frequently observed in RAR-L1-positive colon cancer (55.9%, 19/34) than in the absence of RAR-L1 (70%, 7/10) L1 in colorectal cancers, but not significantly. In addition, somatic mutations (26 colon cancer) and degree of methylation (38 colon cancer) were searched to investigate the mechanism of low expression of CAMTA1 gene. One mismatch mutation was found in early colorectal cancer (CCRC71), which resulted in low expression of the CAMTA1 gene without RAR-L1 (see FIG. 4). However, hypermethylation was not observed in the promoter region of the CAMTA1 gene.

As described above, the present invention relates to (1) observing a reproduced genome change region (RAR) on a chromosome; And / or (2) measuring a change in gene expression in the RAR region; A diagnostic method and a diagnostic kit for determining the prognosis of colorectal cancer (CRC), and a novel tumor suppressor gene that can be used for diagnosis of colorectal cancer. The diagnostic method of the present invention selects a statistically significant cancer suppressor gene in the RAR region and accurately observes and measures the change in its expression. Therefore, not only the prognosis of various cancers and tumors including colorectal cancer but also the early diagnosis, Help.

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Claims (8)

A method for providing information necessary for colon cancer diagnosis or prognosis determination, (1) detecting a Recurrently altered genomic region (RAR) -L1 defined in the following table from a sample of a patient; And (2) measuring the degree of expression of the cancer suppressor gene CAMTA1 belonging to the detected RAR-L1. RAR Clone size
(Mb) Saito band ^a
(Cytoband) Cancer-related genes L1 RP3-438L4-RP11-338N10 1.90 1p36.31-p36.23 CAMTA1 ^{a The} cyto-band follows the UCSC genome browser (2004 (NCBI35 / hg17) version). delete delete delete A DNA chip for colon cancer diagnosis prepared by immobilizing a nucleotide encoding a gene CAMTA1 belonging to Recurrently altered genomic region (RAR) -L1 defined in the following table, a cDNA thereof, or a nucleotide having a complementary sequence thereto. RAR Clone size
(Mb) Saito band ^a
(Cytoband) Cancer-related genes L1 RP3-438L4-RP11-338N10 1.90 1p36.31-p36.23 CAMTA1 ^{a The} cyto-band follows the UCSC genome browser (2004 (NCBI35 / hg17) version). A colon cancer diagnostic kit comprising the DNA chip of claim 5. delete delete

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