JPWO2008093530A1 - Colorectal cancer stage detection method - Google Patents

Abstract

A method for detecting COX-2 for diagnosis of colorectal cancer stage and a kit for diagnosis of colorectal cancer stage containing COX-2. Steps: a) collecting and freeing stool, b) homogenizing frozen stool in the presence of an RNase inhibitor to prepare a suspension, c) extracting the obtained RNA A step of extracting RNA from the sample, d) reverse transcription of the extracted RNA to obtain cDNA, e) amplifying the obtained cDNA, f) step of detecting the amplified cDNA, g) detected A method for detecting COX-2 for diagnosing the stage of colorectal cancer, comprising the step of quantifying the amount of cDNA obtained and h) determining which stage of the colorectal cancer the obtained cDNA quantity belongs to.

Description

  The present invention relates to a method for detecting COX-2 for diagnosing the stage of colorectal cancer and a kit for diagnosing the stage of colorectal cancer containing COX-2.

  Deaths from colorectal cancer are increasing. The death from colorectal cancer is the fourth most common cancer in men and the second most common cancer in women (1999 cancer death statistics). The number of cancer cases in 2015 is estimated to be the first for both men and women, and comprehensive measures against colorectal cancer, including secondary prevention, are required. It is one of the effective methods.

  For mass screening of cancer, it is important to be a simple and non-invasive detection method. The only non-invasive method that can be used at present is a stool test for examining the presence or absence of occult blood, that is, a stool occult blood test, which is widely used as a standard method for mass screening of colorectal cancer.

  The present inventor has already developed a method for detecting tumor markers useful for diagnosis of colorectal cancer from feces by homogenizing an optionally frozen biological sample in the presence of an RNase inhibitor. (WO 2004 / 083856A1).

  The inventor further employs one or more tumor markers selected from the group consisting of COX-2, SNAIL and MMP-7 (excluding only COX-2 or MMP-7). A method has also been developed that improves the sensitivity, specificity and clinical utility of the above method.

  However, the information provided by the above method is limited to qualitative information such as the presence or absence of colorectal cancer, and it has been demanded to provide more qualitative information such as cancer stage.

  Accordingly, an object of the present invention is to provide a COX-2 detection method for diagnosing the stage of colorectal cancer and a kit for diagnosing the stage of colorectal cancer comprising the tumor marker.

More specifically, the present invention relates to the following.
1. The following process:
a) collecting and freezing feces;
b) homogenizing frozen stool in the presence of an RNase inhibitor to prepare a suspension;
c) extracting RNA from the sample for extracting the obtained RNA;
d) reverse transcription of the extracted RNA to obtain cDNA;
e) amplifying the obtained cDNA;
f) detecting the amplified cDNA;
g) quantifying the amount of cDNA detected; and h) diagnosing the stage of colorectal cancer and colorectal adenoma, including the step of determining to which stage of the colorectal cancer the obtained cDNA quantity belongs. COX-2 detection method.
2. The following means:
a) means for collecting and freezing feces;
b) means for homogenizing frozen stool in the presence of an RNase inhibitor to prepare a suspension;
c) means for extracting RNA from the sample for extracting the obtained RNA;
d) means to reverse-transcribe the extracted RNA to obtain cDNA;
e) means for amplifying the resulting cDNA;
f) means for detecting the amplified cDNA;
g) means for quantifying the amount of cDNA detected, and h) means for diagnosing the stage of colorectal cancer and colorectal adenoma, including means for determining to which stage of the colorectal cancer the obtained cDNA quantity belongs. COX-2 detection kit.

The result of COX-2 is shown. There is a statistically significant difference in the expression level of COX-2 between early cancer and advanced cancer (P = 0.034), and high in advanced cancer. If the expression level is (++) or higher, the probability of advanced cancer is 89.7%. The result of SNAIL is shown. There is no statistically significant difference in the expression level of SNAIL between early and advanced cancer, but it tends to be higher in advanced cancer (P = 0.060). If the expression level is (++) or higher, the probability of advanced cancer is 91.7%. The result of MMP-7 is shown. There is no statistically significant difference in the expression level of MMP-7 between early cancer and advanced cancer, but it tends to be higher in advanced cancer (P = 0.056). If the expression level is (++) or higher, the probability of advanced cancer is 85.7%. The result of COX-2 is shown. There is no statistically significant difference in the expression level of COX-2 between the left cancer and the right cancer (P = 0.21). If the expression level is (++) or higher, the probability of being left cancer is 77.1%. The left cancer is a cancer present in the descending colon, sigmoid colon, and rectum, and the right cancer is a cancer present in the cecum, ascending colon, and transverse colon. The result of SNAIL is shown. There is a statistically significant difference in the expression level of SNAIL between left and right cancers (P = 0.177), and it is high in left cancers. If the expression level is (++) or higher, the probability of left cancer is 83.3%. The definitions of left side cancer and right side cancer are the same as in FIG. The result of MMP-7 is shown. There is a statistically significant difference in the expression level of MMP-7 between left cancer and right cancer (P = 0.033), and it is high in left cancer. If the expression level is (++) or higher, the probability of left cancer is 76.2%. The definitions of left side cancer and right side cancer are the same as in FIG.

  Examples of the RNase inhibitor of the present invention include guanidine thiocyanate, Isogene, Ultraspec II (registered trademark), and Ultraspec II.

  As the freezing method, any conventional technique can be used, and liquid nitrogen is preferably used. The freezing temperature is a storage temperature of −1 to −196 ° C., preferably −20 to −196 ° C., preferably −75 to −196 ° C., more preferably −110 to −196 ° C., most preferably − 196 ° C.

  The frozen sample may be stored frozen. The storage temperature is −75 to −196 ° C., preferably −110 to −196 ° C., more preferably −196 ° C. The storage period is 1 day to 10 years, preferably 1 day to 3 years, and more preferably 1 day to 1 year.

  The tumor marker used in the present invention is COX-2.

  COX-2 is an inducing enzyme that converts arachidonic acid to prostaglandin H2. When the expression of COX-2 increases, apoptosis is inhibited, and angiogenic factors and substrate degrading enzymes required during invasion are induced. The SNAIL is a negative transcriptional regulator of E-cadherin, which is an intercellular adhesion factor. When SNAIL expression increases, E-cadherin expression decreases, intercellular adhesion decreases, and deep invasion and metastasis are affected. It is known. MMP-7 is a kind of matrix metalloprotease and is considered necessary for cancer invasion and metastasis.

  The above steps d) to f) are called RT-PCR methods, and are performed, for example, according to the description of Takeo Sekiya et al., PCR method forefront, 1997, Kyoritsu Shuppan, pages 187 to 196. Can do.

  For extracting RNA from the suspension, a conventionally known method can be used. For example, a commercially available kit such as RNeasy Mini Kit (QIAGEN) or RNA Extraction Kit (Pharmacia Biotech) can be used.

In the present invention, reverse transcription refers to conversion of RNA into complementary DNA (cDNA) using reverse transcriptase. The reverse transcription reaction is usually performed using a solution containing a buffer, salts such as MgCl ₂ and KCl, dithiothreitol (DTT), primers, deoxyribonucleotides, an RNase inhibitor and a reverse transcriptase. The above salts can be used by changing to other salts as appropriate. In addition, proteins such as gelatin and albumin, surfactants and the like can also be added.

PCR is usually used for amplification of cDNA performed following reverse transcription. A PCR reaction solution usually contains a buffer, salts such as MgCl ₂ and KCl, primers, deoxyribonucleotides, and a heat-resistant polymerase. The above salts can be used by changing to other salts as appropriate. Further, proteins such as gelatin and albumin, dimethyl sulfoxide, surfactants and the like can be added.

  For amplification of cDNA, the LAMP method (Japanese Patent No. 3313358) or the ICAN method (Japanese Patent Laid-Open No. 2001-136965) can also be used.

  In the present invention, a primer refers to an oligonucleotide that serves as a starting point for synthesis during cDNA synthesis or nucleic acid amplification. The primer is preferably single-stranded, but double-stranded can also be used. When the primer is double-stranded, it is desirable to make it single-stranded prior to the amplification reaction. The primer can be synthesized according to a known method, or can be isolated from an organism.

  The reverse transcriptase used for the reverse transcription reaction means an enzyme that can reverse transcribe RNA into cDNA. As reverse transcriptases, there are reverse transcriptases derived from retroviruses such as RAV (Rous associated virus) and AMV (Avian myeloblastosis virus), and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus). However, it is not limited to these.

  A thermostable polymerase used for PCR includes, but is not limited to, Taq polymerase.

  As a method for detecting the amplified DNA, electrophoresis using an agarose gel can be used, but the method is not limited thereto.

  The amplified cDNA is electrophoresed with at least two known concentrations of size markers, labeled with ethidium bromide, and visualized. Any conventionally known label can be used as a label for visualization. The signal intensity of the size marker at a known concentration is compared with the signal intensity of the amplification product, and the signal intensity of the amplification product is classified into five levels 0-4 (0: negative, 1: weak positive, 2: positive, 3: strong positive, 4: super strong positive) and semi-quantified. For example, 0: negative means no signal at all, 1: weak positive corresponds to a signal at a concentration of 40 bp of 200 bp of a 100 bp DNA ladder size marker, 2: positive means 500 bp of a 100 bp DNA ladder size marker Corresponding to a signal of 125 ng concentration, 3: Strong positive clearly exceeds the signal of 125 ng concentration, 4: Super strong positive corresponds to a particularly strong signal among 3: Strong positive. Electrophoresis can be performed using agarose gel electrophoresis, but is not limited thereto, and can be quantitatively performed using any conventionally known nucleic acid electrophoresis method such as Real-time PCR.

  Early colon cancer and advanced cancer are defined by the depth of wall penetration. Early cancer refers to cancer in which the advanced part of the cancer is confined to the mucosa or submucosa of the large intestine wall and does not exceed this, and advanced cancer refers to cancer that extends beyond the submucosa and reaches deeper than the intrinsic muscle layer. These are defined in the 7th edition of the rules for handling colorectal cancer (Edited by Colorectal Cancer Study Group, Kanehara Publishing, 2006). In addition, some adenomas are difficult to differentiate from early stage mucosal cancer, especially adenomas larger than 10 mm can develop into submucosal cancer in the future as well as mucosal cancer. It is assumed to be similar to cancer as a sex tumor.

  The kit of the present invention may also contain instructions describing the method of the present invention. Also included in the kit are reverse transcriptase, RNase inhibitor, DNA synthase, various deoxynucleotide triphosphates (dNTPs), specific primers for each tumor marker, positive control for each tumor marker, RNase / DNase Examples thereof include distilled water not contained, a fluorescence / visual detection reagent, and the like.

  The following examples illustrate the invention but do not limit the invention.

  The subjects were patients who were admitted to Hamamatsu Medical University's First Internal Medicine for the purpose of examination and treatment, and the presence of colon cancer was confirmed by total colonoscopy. Informed consent has been obtained from all patients.

  Feces were collected from 0.5 to 1 g each in 5 ml tubes as soon as possible after collection, frozen with liquid nitrogen, and stored at -80 ° C. The tissue was stored at −80 ° C. after freezing the biopsy material of the cancerous part and normal part with liquid nitrogen at the time of endoscopy received before treatment. Thereafter, homogenization was performed using a homogenizer, a guanidine salt, and phenol, and total RNA was extracted with chloroform and ethanol.

  1 μg of the obtained RNA was reverse-transcribed using River Script II (registered trademark) (reaction volume 20 μl, Wako Pure Chemical Industries) to obtain cDNA, and a portion thereof was nested PCR using Gene Taq (Wako Pure Chemical Industries). Amplified by The obtained PCR amplification product (10 μl of the second round PCR product) was electrophoresed using 4% agarose gel and stained with ethidium bromide. In addition, a 100 bp DNA ladder size marker (200 bp, 40 ng, 500 bp, 125 ng) was used as a standard for semi-quantification.

In addition, the primer used was a random primer in reverse transcription, and in PCR, those designed independently for COX-2, SNAIL, and MMP-7 were used. For the three molecules, the first round was performed in 20 cycles, and in the second round, COX-2 was performed in 20 cycles, and both SNAIL and MMP-7 were performed in 35 cycles.
The primers used are shown below.
<COX-2>
Forward 1: 5'-CTGAAACCCACTCCAAACACAG-3 '
Forward 2: 5'-GCACTACATACTTACCCACTTCAA-3 '
Reverse: 5'-ATAGGAGAGGTTAGAGAAGGCT-3 '
<SNAIL>
Forward 1: 5'-AGATGAGGACAGTGGGAAAGGCT-3
Forward 2: 5'-CCTTCGTCCTTCTCCTCTACTTCA-3 '
Reverse 1: 5'-AGGTATGGAGAGGAAGAGGGAG-3 '
Reverse 2: 5'-GCACTGGTACTTCTTGACATCTGAG-3 '
<MMP-7>
Forward 1: 5-ATGAGTGAGCTACAGTGGGAA-3 '
Forward 2: 5'-TTAAACTCCCGCGTCATAGAA-3 '
Reverse 1: 5-AAATGCAGGGGGATCTCTTTG-3 '
Reverse 2: 5-TCGATCCACTGTAATATGCGG-3 '

Results The expression levels of COX-2, SNAIL, and MMP-7 by stool RT-PCR in 70 colon cancer patients (14 early cancer patients, 56 advanced cancer patients) were compared between early cancer and advanced cancer.

  The signal was semi-quantified from 0 to 4 by comparison with the DNA size marker, and COX-2 showed 0: 4 cases, 1: 7 cases, 2: 2 cases, 3: 1 cases, and 0 for advanced cancers. : 6 cases, 1:24 cases, 2:10 cases, 3:14 cases, and 4: 2 cases. In SNAIL, there were 0: 9 cases, 1: 3 cases, 2: 2 cases in early cancer, and 0:25 cases, 1: 9 cases, 2: 3 cases, 3:11 cases, 4: 8 cases in advanced cancers. . In MMP-7, 0: 8 cases, 1: 0 cases, 2: 2 cases, 3: 4 cases in early stage cancers, 0:16 cases, 1: 3 cases, 2: 6 cases, 3:29 cases in advanced cancers. Example: 4: 1. Here, when comparison was made between early cancer and advanced cancer, the median level of COX-2 expression was 1 vs. 1 (P = 0.034 in Mann-Whitney U test), 0 vs. SNAIL. 1 (P = 0.060), and 0 vs. MMP-7. 3 (P = 0.056) (FIGS. 1 to 3).

  As a result, a statistically significant difference (P <0.05) was observed for COX-2, and a significant correlation was observed between the increase in the expression level of COX-2 and the stage.

  In other words, COX-2 is a very useful test method that is excellent in screening for relatively early colon tumors that can be completely cured by endoscope or surgery, and has high specificity.

  The method of the present invention can determine the stage of cancer and can replace the conventional fecal occult blood method as a non-invasive screening method for new colorectal cancer with high clinical usefulness. It is useful.

Claims (2)

The following process:
a) collecting and freezing feces;
b) homogenizing frozen stool in the presence of an RNase inhibitor to prepare a suspension;
c) extracting RNA from the sample for extracting the obtained RNA;
d) reverse transcription of the extracted RNA to obtain cDNA;
e) amplifying the obtained cDNA;
f) detecting the amplified cDNA;
g) quantifying the amount of cDNA detected; and h) diagnosing the stage of colorectal cancer and colorectal adenoma, including the step of determining to which stage of the colorectal cancer the obtained cDNA quantity belongs. COX-2 detection method. The following means:
a) means for collecting and freezing feces;
b) means for homogenizing frozen stool in the presence of an RNase inhibitor to prepare a suspension;
c) means for extracting RNA from the sample for extracting the obtained RNA;
d) means to reverse-transcribe the extracted RNA to obtain cDNA;
e) means for amplifying the resulting cDNA;
f) means for detecting the amplified cDNA;
g) means for quantifying the amount of cDNA detected, and h) means for diagnosing the stage of colorectal cancer and colorectal adenoma, including means for determining to which stage of the colorectal cancer the obtained cDNA quantity belongs. COX-2 detection kit.

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