JP2001017200A - Diagnosis of lymph node metastasis of esophageal cancer by detection of chromosomal abnormality - Google Patents

Abstract

PROBLEM TO BE SOLVED: To diagnose cancer and treat cancer in an early stage and diagnose the lymph node metastasis of esophageal cancer by detecting the amplification of a specific site of the long arm of the human 8th chromosome or the long arm of the human 20th chromosome in the chromosome of a cancerous cell derived from a patient. SOLUTION: This method is to diagnose the lymph node metastasis of esophageal cancer by detecting the amplification of the long arm 23-long arm ter of the human 8th chromosome or the long arm of the human 20th chromosome in the chromosome of a cancerous cell derived from a patient. A DNA of the cancerous cell of the patient labeled with a first label and a DNA of a normal cell labeled with a second label are competitively hybridized with the chromosome of the normal cell to observe the chromosmal site where the DNA of the cancerous cell is hybridized with the first label and the chromosomal site where the DNA of the normal cell is hybridized with the second label. The chromosomal site where both are hybridized is observed with the mixture of the first label with the second label. Thereby, the labels of the respective sites are discriminated to detect the amplification. As a result, the lymph node metastasis of the esophageal cancer is diagnosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for diagnosing esophageal cancer. More specifically, the present invention provides a method for diagnosing lymph node metastasis of esophageal cancer by detecting amplification and deletion at a specific site of a chromosome derived from a patient.

[0002]

2. Description of the Related Art Cancer is a disease in which some cells in abnormally proliferate and invade surrounding normal tissues, thereby destroying normal functions. Since 1981, cancer has been the leading cause of death in Japan, and establishing an effective diagnostic and therapeutic method is urgently needed in society. To date, various gene-level studies have been performed on cancer. As a result, it was revealed that deletion or amplification of a part of the chromosome was related to the development of cancer. For example, it has been confirmed that the Rb gene on human chromosome 13 is always deleted in retinoblastoma, and that the p53 gene on the short arm of human chromosome 17 is deleted in many cancers. Has been issued. These genes are tumor suppressor genes, and their deletion is thought to cause cancer. It has also been confirmed that certain types of cancer amplify specific sites on the chromosome. These days, instead of just one gene in the genome,
It has also been proposed that cancer is caused by multi-step mutation caused by deletion or amplification of a plurality of genes.

As described above, active research activities are being conducted on the relationship between cancer development and chromosomal abnormalities, but there are still many points to be elucidated for the relationship between them, and the mechanism of cancer development has not been clarified. .

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to clarify specific structural changes of chromosomes in esophageal cancer with lymph node metastasis. Also,
An object of the present invention is to provide a method for diagnosing lymph node metastasis of esophageal cancer by detecting a specific structural change of chromosome in esophageal cancer. Another object of the present invention is to provide a method that can easily perform this diagnosis with a single operation.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors analyzed the DNA of esophageal cancer cells by using the CGH (comparative genomic hybridization) method. As a result, it was found that the chromosome of esophageal cancer cells with lymph node metastasis had DNA amplification at a specific site, and that it was possible to specifically correlate the structural change of the chromosome with cancer metastasis. became. The present inventors have developed a method for diagnosing lymph node metastasis of esophageal cancer by detecting cancer-specific amplification based on this fact, and have provided the present invention.

[0006] That is, according to the present invention, lymphoma of esophageal cancer comprising detecting amplification of the long arm 23-long ter of human chromosome 8 or the long arm of human chromosome 20 in the chromosome of a cancer cell derived from a patient. A method for diagnosing node metastasis is provided.

[0007] In one embodiment for carrying out the diagnostic method provided by the present invention, the chromosome of a normal cell is labeled with the DNA of a cancer cell derived from a patient labeled with the first label and the normal cell labeled with the second label. The DNA of the cell is competitively hybridized, the chromosome site where the DNA of the cancer cell hybridizes is observed by the first label, the chromosome site where the DNA of the normal cell hybridizes is observed by the second label, and both are observed. A diagnostic method is provided for detecting the amplification by allowing the hybridized chromosomal site to be observed in a mixture of the first label and the second label and identifying the label at each site.

[0008] In another embodiment for carrying out the diagnostic method provided by the present invention, the first chromosome is added to the chromosome of a normal cell.
The DNA of the cancer cell derived from the patient labeled so as to be detected as the second color and the DNA of the normal cell labeled so as to be detected as the second color are competitively hybridized, and the DNA of the cancer cell is hybridized. The soybean chromosome site is observed as the first color, the chromosome site where normal cell DNA hybridizes is observed as the second color, and the chromosome site where both hybridized is the first color and the second color. A diagnostic method for detecting the amplification is provided by identifying the color of each site by observing the third color as a mixture of the third color. In this aspect,
Amplification can be detected by measuring the intensity ratio of the first color and the second color at each site of the chromosome.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method for carrying out the present invention will be specifically described. The method for diagnosing lymph node metastasis of esophageal cancer according to the present invention is characterized by detecting an abnormality in a chromosomal site specific to esophageal cancer with lymph node metastasis.
Therefore, the diagnosis of the present invention may be performed using any means capable of confirming a change in a chromosomal site whose abnormality is to be detected. For example,
In the chromosome of a cancer cell derived from a patient, any method may be used as long as it can detect amplification of the long arm of human chromosome 8 (23-long arm ter) and the long arm of human chromosome 20 (chromosome 20).

As a method for detecting such a chromosomal abnormality, an in situ hybridization method can be used. The in situ hybridization method uses a labeled nucleic acid probe as a DNA-D
This is a method of elucidating a chromosome site where a nucleic acid probe has hybridized by NA hybridization or RNA-DNA hybridization, and then detecting a label. By using this method, a probe prepared by labeling genomic DNA prepared from normal cells can be hybridized to a chromosome prepared from a cancerous tissue of a patient. At this time, the chromosome site of the cancer cell to which the probe did not hybridize indicates the site where the chromosome of the cancer cell was amplified. Thus, when detecting chromosome amplification of a cancer cell, the in situ hybridization method can be effectively applied.

Further, the CGH method can be used.
In the CGH method, a DNA labeled with a first label and another DNA labeled with a second label are simultaneously hybridized on a chromosome, and each of the two labels is detected to detect a chromosome site where each DNA hybridizes. (Du Manoir, S et al., Hum. Genet., 90; 590-
610, 1993). In the diagnostic method of the present invention, a DNA probe prepared from DNA of a cancer cell derived from a patient labeled with the first label and a DNA probe prepared from DNA of a normal cell labeled with the second label are placed on the chromosome of the normal cell. At the same time. Among the chromosomes, the first label is observed at the site amplified in the cancer cell because the DNA of the cancer cell mainly hybridizes. In addition, the second label is observed at the site deleted in the cancer cell because only the DNA of the normal cell hybridizes. Furthermore, in a site which is the same in both normal cells and cancer cells without change, the first label and the second label are mixed and observed because they hybridize competitively. Thus, chromosome deletion and amplification due to cancer can be detected by a single operation. Hereinafter, the diagnostic method of the present invention using the CGH method, which is a preferred embodiment of the present invention, will be specifically described step by step.

[0012] DN of cancer cells used in the diagnostic method of the present invention
A is prepared by collecting cells from the cancerous tissue of a patient suspected of having esophageal cancer. The term “cancer cell” or “cancer tissue” of a patient referred to in the present specification is a concept that includes not only cells and tissues that have already become cancerous but also cells and tissues that may become cancerous in the future. The D of normal cells used in the diagnostic method of the present invention is
NA is the DNA of cells derived from healthy individuals without cancer. The types of tissues and cells to be obtained are not particularly limited.
Genomic DNA can be used as DNA for cancer cells and DNA for normal cells to be prepared without selecting DNA at a specific chromosomal site. As the genomic DNA, a full-length genome can be used, and a DNA prepared from a collected cell using an appropriate restriction enzyme according to a method known to those skilled in the art can be used. The DNA prepared from the genome is 10
Preferably, the size is in the range of ~ 1000 kb.
Each D used as a probe for hybridization
The length of NA is preferably from 200 to 1000 bp. However, DNA with a length outside this range can also be used.

Notwithstanding the above, DN of cancer cells to be prepared
If the DNAs of A and normal cells contain a portion corresponding to a chromosomal site whose amplification is to be detected, there is no problem in performing the diagnostic method of the present invention. In fact, D
Since selecting an NA is complicated, a chromosome derived from a normal cell to which the probe is hybridized (hereinafter referred to as target DN) is used.
A) is more practical. In order to detect the amplification of the long arm 23-long ter of human chromosome 8 or the long arm of human chromosome 20, those sites on the chromosome may be used as target DNA. When the target DNA is used for the two chromosomes, the influence of the background is reduced when measuring the label of the probe, compared with the case where the entire chromosome is used as the target DNA.

The target DNA may be a YAC vector or BA containing the long arm 23-long ter of human chromosome 8 and the long arm of human chromosome 20 instead of the chromosome of a normal cell.
There is also a method in which each probe is hybridized to a C vector or the like, or each probe is hybridized to the specific region cut out from the YAC vector or BAC vector to detect the degree of amplification or deletion. YA
As the C vector and the BAC vector, those in which human chromosomes are integrated are commercially available, and can be used. However, since the entire desired chromosome region is not always integrated in one YAC vector or the like, a plurality of YAC vectors or the like are selected and used so as to obtain the entire desired chromosome region.

The DNA of cancer cells and the DNA of normal cells are labeled with different labels (first label and second label) before hybridization. The type of label is not particularly limited as long as it can be detected separately after hybridization. Therefore, labels that can be detected spectroscopically, biochemically, chemically, and immunologically can be widely used. For example, fluorescent dyes, electron density reagents, enzymes, biotin, digoxigenin, and the like can be used. Preferred labels are labels that are visually recognizable for developing color. Above all, it is particularly preferable to select a combination of the markers so that the color of the first marker and the color of the second marker are mixed to obtain a third color that can be distinguished from the two colors. For example,
By selecting a sign that emits green as the first sign and selecting a sign that emits red as the second sign, the mixture can be made yellow when mixed. By selecting and hybridizing a label so that three colors that can be distinguished from each other are formed in this way, a site that is deficient in cancer, a site that is amplified by cancer, and a site that is not changed by cancer can be easily visually determined. Can be distinguished. As a green fluorescent dye that can be used in the present invention, FI
TC (fluorescein isothiocyanate) can be exemplified. Also, as a red fluorescent dye, TRIT
C (tetramethyl rhodamine isothiocyanate), rhodamine and Texas Red can be exemplified.

The conditions under which the DNA of cancer cells and the DNA of normal cells are hybridized to chromosomes are not particularly limited.
As a chromosome for hybridizing cancer cell DNA and normal cell DNA, a chromosome specimen prepared from cells such as lymphocytes can be used. However, it is also possible to carry out hybridization using unfixed chromosomes and then drop them on a slide glass or the like for fixation.

The method of analysis after hybridization is as follows:
It is determined appropriately according to the type of the label. When a fluorescent dye is used as a label, analysis can be performed while visually observing using a fluorescence microscope or the like having a high resolution. In addition, an image observed with a fluorescence microscope is read by a color image scanner, and the color at each point is separated into three colors to quantify which is more dyed at each point (that is, each point is dyed). Determining the ratio of labeled substances). Thus, by detecting deletion and amplification in the chromosome of a tissue derived from a patient using the present invention, it is possible to easily diagnose whether or not the tissue is esophageal cancer with lymph node metastasis. . This is expected to open the way for early diagnosis and treatment of cancer or more appropriate postoperative treatment.

[0018]

The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the present invention. <Example 1> (1) Preparation of chromosome specimen A chromosome specimen was prepared from cells having normal chromosomes (normal human blood cells) according to the following procedure. 10 ml of blood was collected from each person, and a culture solution (RPMI1640,
About 1.5% with 10% FCS and PHA (20 μl / ml)
Diluted 1-fold. This diluted blood is placed in a tube (Ficol Paque;
Pharmacia) and gently pour it for 2,000r.
Centrifuged at pm for 20 minutes. The operation of removing the leukocyte layer and centrifuging the culture solution at 1,200 rpm for 5 minutes was repeated twice. Further, after the above culture solution was added and sufficiently stirred, the culture was started by putting 15 ml each in two 10 cm dishes.

48 hours after the start of the culture, thymidine (manufactured by Sigma) was added to a final concentration of 300 μg / ml. After further culturing for 18 hours, a washing operation of centrifuging at 1,200 rpm for 5 minutes using the above culture solution was repeated three times. After further culturing for 6 hours, colcemide (manufactured by Gibco) was added to a final concentration of 0.05 μg / ml. One hour later, the above culture solution was added to the cells, and 1,2
Wash by centrifugation at 00 rpm for 5 minutes, 0.075MKC
1 to 3 ml and hypotonic treatment was performed at room temperature for 30 minutes.
Thereafter, Carnoy's fixative (methanol: acetic acid = 3: 1) was added in several portions until the color of the red blood cells disappeared, and the cells were fixed. The fixed cells were dropped on slides and stored at -80 ° C until use.

(2) Preparation of Genomic DNA Derived from Cancer Part Tissue Genomic DNA was prepared from cancer part tissues of esophageal cancer patients (27 cases) according to the following procedure. The breakdown of patients was 20 cases with lymph node metastasis and 7 cases with no metastasis.

A solid tumor tissue of 5 mm square or more was slightly moistened with PBS and cut into small pieces with ophthalmic scissors. The operation of sucking and discharging the cell suspension into a syringe (1 ml) was repeated several times, and then filtered through a nylon mesh. The cell suspension was placed in a centrifuge tube, 0.2% NaCl was added, and the mixture was allowed to stand for several minutes. After centrifugation at 2,000 rpm for 5 minutes, the supernatant was discarded and resuspended in a small amount of PBS. This cell suspension was placed in a microtube, centrifuged at 2,000 rpm for 5 minutes, and the supernatant was discarded. Hereinafter, DNA was prepared using the nucleic acid extractant Sepagene (registered trademark, manufactured by Sanko Junyaku Co., Ltd.) according to the instruction manual.

In addition, according to the following procedure, genomic DNA to be used as DNA of normal cells was prepared from lymphocytes of blood of a healthy person. Twenty ml of blood was collected from a healthy person, anticoagulated with heparin, and diluted 2-fold with PBS. This diluted blood was gently layered on a centrifuge tube containing 30 ml of Ficoll solution, and allowed to stand for 10 minutes.
Thereafter, when the mixture was centrifuged at 2,000 rpm for 20 minutes, a milky white lymphocyte layer was formed under the pale yellow layer. The milky layer was collected in another centrifuge tube containing 30 ml of Ficoll solution, and PBS was added. 5 at 2,000 rpm
After centrifugation for 1 minute, the supernatant was removed with an aspirator. 0.2
After adding 3 ml of NaCl to cause hemolysis,
After centrifugation at 0 rpm for 5 minutes, the supernatant was removed with an aspirator. Hereinafter, using a nucleic acid extractant Sepagene (registered trademark, manufactured by Sanko Junyaku Co., Ltd.), according to the instruction manual, DN
A was prepared.

(3) Preparation of labeled DNA The DNA of esophageal cancer tissue is labeled with FITC that emits green fluorescence, and the DNA of normal cells is TRIT that emits red fluorescence.
Labeled with C. DNA labeling is performed by mixing dTTP, dNTP mixture, labeled d-UTP, template DNA, nick translation buffer, nick translation enzyme, and water according to the instruction manual of the nick translation kit (Vysis). By incubating for an appropriate time.

(4) Hybridization (FISH
Method) (A) Single-strand DNA probe Human COT-1 DNA (20 μl), DNA of normal cells labeled with TRITC (10 μl), DNA of esophageal cancer tissue labeled with FITC (10 μl), sodium acetate ( 4 μl) and 100% ethanol (1 ml) were mixed and centrifuged at 14,000 rpm for 30 minutes at 4 ° C. The supernatant was discarded by decantation and dried sufficiently in the light. Then, add 10 Master Mix # 1
μl was added and thoroughly pipetted. Then 75
Single-stranded by treating at 5 ° C. for 5 minutes.

(B) Single-strand chromosome preparation The DNA of the chromosome preparation was subjected to 70% formamide / 2 × SS
It was made single-stranded by treating with C (pH 7.0) at 75 ° C. for 2.5 minutes. Then, the sample was treated with 70% ethanol, 85% ethanol, and 100% ethanol sequentially for 2 minutes to dehydrate, and the sample was air-dried. The position and number of bands appearing on the chromosome confirmed that the chromosome was a normal chromosome.

(C) Hybridization 9.5 μl of the single-stranded probe mix was placed on a slide glass of a chromosome specimen. A cover glass with a size of 18 × 18 was applied, and the surroundings were attached with a paper bond. Place on a hot plate for about 10 minutes,
Hybridization was performed for 2 to 3 days in a ₂ incubator. After hybridization, the operation of washing with 45% formamide / 2 × SSC (pH 7.0) at 45 ° C. for 12 minutes was repeated three times. Then 2x
The operation of washing with SSC at 45 ° C. for 10 minutes, the operation of washing with PN buffer at room temperature for 10 minutes was repeated twice, and the operation of washing with distilled water at room temperature for 5 minutes was repeated twice. Then, 0.1 to 0.2 mg / ml DAPI-a
8 μl of ntifade was mounted and sealed with an 18 × 18 cover glass.

(5) Observation with Fluorescence Microscope The slide glass was observed with a fluorescence microscope, and a fluorescence microscope photograph (color photograph) was taken. Observing each chromosome, the site where green color is observed, that is, DNA specific to esophageal cancer
And the site where red color was observed, that is, the site where DNA deletion specific to esophageal cancer was observed were determined.

In 20 esophageal cancer patients with lymph node metastasis, 14 human chromosome 8 long arm 23-long arm te
The amplification of r and the amplification of the long arm of human chromosome 20 in 8 cases were observed. In non-metastatic cases, human chromosome 8 long arm 23
-Amplification of long arm ter was observed in one case, and no amplification of long arm of human chromosome 20 was observed at all. from this result,
It has been found that esophageal cancer in which amplification of the long arm of human chromosome 8 23-long arm ter or amplification of the long arm of human chromosome 20 has been recognized has a high possibility of metastasis to lymph nodes.

[0029]

According to the above test, it was revealed that the chromosome of esophageal cancer cells metastasizing to the lymph node has DNA amplification at a specific site. By utilizing the present invention applying the relationship between such chromosomal structural changes and cancer development,
It is possible to accurately diagnose whether esophageal cancer may cause lymph node metastasis. This is expected to open the way for early diagnosis and treatment of cancer or more appropriate postoperative treatment.

Further, according to the present invention, since the prognosis of a patient can be objectively estimated, it is possible to select a treatment considered to be optimal for each patient. For patients determined to be at risk of metastasis, aggressive treatment using anticancer drugs or radiation can be administered, and for patients determined to be at risk of metastasis, There is no need for such treatment. This not only raises the quality of life of cancer patients, but also brings great benefits from the viewpoint of medical economics.

Continued on the front page (72) Inventor Toshihiko Kishimoto 1-chome, Taya-cho, Sakae-ku, Yokohama-shi, Kanagawa Prefecture Sumitomo Electric Industries, Ltd. Yokohama Works (72) Inventor Kosunori Sasaki 1-1-1, Minamikogushi, Ube City, Yamaguchi University Yamaguchi University 4B024 AA01 AA12 CA01 CA09 DA03 HA11 HA12 HA14 4B063 QA01 QA19 QQ03 QQ08 QQ42 QR56 QR59 QR66 QS24 QS34 QS36 QX01

Claims (4)

[Claims] 1. A method for diagnosing lymph node metastasis of esophageal cancer, comprising detecting amplification of the long arm of human chromosome 8 (23-ter) or the long arm of human chromosome 20 in the chromosome of a cancer cell derived from a patient. 2. A chromosome of a normal cell is competitively hybridized with a DNA of a cancer cell derived from a patient labeled with a first label and a DNA of a normal cell labeled with a second label,
The chromosomal site where the DNA of the cancer cell hybridized is the first
The chromosomal site where the normal cell DNA was hybridized was observed by the second label, and the chromosome site where both were hybridized was observed by mixing the first label and the second label. The diagnostic method according to claim 1, wherein the amplification is detected by identifying a label at the site. 3. The method according to claim 2, wherein the DNA of the cancer cell derived from the patient is labeled so that the DNA of the cancer cell derived from the patient labeled with the first label can be detected as a first color. The DNA of normal cells labeled with a label is
Of normal cells labeled so that they can be detected as the color of
A, the chromosomal site where the DNA of the cancer cell hybridized is observed as the first color, and the DN of the normal cell
The chromosome site where A hybridized is observed as a second color, and the chromosome site where both hybridized is observed as a third color in which the first color and the second color are mixed, The diagnostic method according to claim 2, wherein the amplification or the deletion is detected by distinguishing the color of each site. 4. The diagnostic method according to claim 3, wherein the amplification or the deletion is detected by measuring the intensity ratio of the first color and the second color at each site of the chromosome.