JP7024957B2 - Methods for predicting the presence or absence of metastatic metastasis of colorectal cancer and kits used for it - Google Patents

Description

The present invention relates to a gene associated with metastasis of colorectal cancer and a method for predicting metastasis using the same. More specifically, the gene group useful for predicting metastatic metastasis of colorectal cancer obtained by statistically analyzing the expression information of the gene group specifically expressed in the colorectal cancer tissue of a colorectal cancer stage 2 patient, and Regarding its use.

Colorectal cancer has the highest prevalence among Japanese people, and is ranked second in males and first in females in statistics by major site. As a carcinogenic process of this colorectal cancer, a multi-step process in which multiple oncogenes are involved with the development and progression of cancer is known. Specifically, a colon polyp due to an abnormality of a tumor suppressor gene such as APC. When abnormalities such as activation due to mutation of the oncogene ras and inactivation due to mutation of the tumor suppressor gene p53 are added, the colon polyp becomes enlarged and cancerous. In addition, abnormalities in other oncogenes can cause the cancer to invade or metastasize. Therefore, even if colorectal cancer is diagnosed, the details vary from benign ones, which have no problem if the tumor tissue is removed, to highly malignant ones, which are small but easily metastasize. Among them, since there are many highly malignant tumors that are easily metastasized even if the tumor tissue is small, liver metastasis and lung metastasis after excision have become serious problems.

In addition, not only for colorectal cancer but also for cancer in general, it is important to improve the accuracy of cancer diagnosis by adding not only surgical findings and pathological examination of tissues but also molecular biological judgment. The social significance of accurately determining the prognosis of the patient and improving the QOL of the patient after surgery is extremely great.

In addition, information on predicting colorectal cancer prognosis and metastasis potential may suggest antibody drug susceptibility in drug selection for colorectal cancer metastasis, and it is not possible to analyze the expression profile of individual colorectal cancer tissues. , It may be very important information for personalized medicine in the future.

Therefore, it is desired to improve the accuracy of prognosis prediction after excision of colorectal cancer tumor tissue and to establish rational follow-up based on it.

The present invention has been made in view of such a situation, and provides a method for predicting the presence or absence of metastatic metastasis in a colorectal cancer patient from the gene expression level in a tumor tissue obtained from the colorectal cancer patient. With the goal.

It is also an object of the present invention to provide a kit used in the above method.

The present inventors have attempted to develop a prognostic prediction method based on molecular biology by advancing the deformation analysis of the control system network by RNA for nearly 1000 cases of gastrointestinal cancer for more than 10 years, and are related to metachronous metastasis. Succeeded in identifying candidate genes to be used. In addition, the present inventors can narrow down candidate genes as a result of performing retrospective analysis and cluster analysis, especially in colorectal cancer stage 2 patients, and by performing cluster analysis with prospective samples, they are different. We were able to confirm the predictability of temporal metastasis.

As a result of intensive studies to solve the above problems, the present inventors can predict metastasis of colorectal cancer stage 2 patients by measuring the expression levels of five genes. I found.

Specifically, according to the first main aspect of the present invention, there is a method for predicting the presence or absence of metastatic metastasis in a colorectal cancer stage 2 patient, which is GNL3L, SLC35A2 in the colorectal cancer tissue obtained from the patient. , UBE2I, IL2RB, and TYMP, and a step of measuring the expression level of at least one gene, and a step of calculating a score for predicting the presence or absence of metastasis from the measured expression level. However, a method is provided to show that metastasis does not occur in the patient when the calculated score is smaller than the reference value.

With such a configuration, it is possible to find out the possibility of cancer metastasis in the future only by acquiring the tumor tissue of a colorectal cancer stage 2 patient.

Further, according to one embodiment of the present invention, in such a method, the score is calculated by normalizing the expression level of the at least one gene.

Further, according to another embodiment of the present invention, in such a method, the reference value is the measurement in a colorectal cancer tissue obtained from a colorectal cancer stage 2 patient whose presence or absence of metastatic metastasis is found. It is determined based on the expression level of the same gene as the gene used in the step.

Further, according to another embodiment of the present invention, in such a method, the measuring step measures the expression level of two genes selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP. In this case, the two genes are preferably selected from the group consisting of IL2RB and GNL3L, IL2RB and SLC35A2, IL2RB and UBE2I, TYMP and GNL3L, TYMP and SLC35A2, and TYMP and UBE2I. More preferably, the measuring step measures the expression levels of all genes of GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP.

Furthermore, according to another embodiment of the present invention, in such a method, the metastatic metastasis is a metastasis within 3 years after the tissue acquisition.

Also, according to the second main aspect of the present invention, differences in colorectal cancer stage 2 patients having a probe that specifically binds to at least one gene selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP. A kit is provided to predict the presence or absence of temporal metastasis.

The features and remarkable actions / effects of the present invention other than those described above will be clarified to those skilled in the art by referring to the sections and drawings of the following embodiments of the present invention.

FIG. 1 is a table showing the results of the expression level T-test of the candidate gene for predicting metastatic metastasis in one embodiment of the present invention. FIG. 2 is a photograph and a table showing the results of expression analysis obtained using five metastatic metastasis predicting genes in one embodiment of the present invention. FIG. 3 is a graph showing the relationship between the threshold value of the score value obtained by using the five metastatic metastasis prediction genes and the prediction probability in one embodiment of the present invention. FIG. 4 is a table showing the results of predicting metastasis obtained by using two genes for predicting metastasis in one embodiment of the present invention. FIG. 5 is a table showing the results of predicting metastasis obtained by using one gene for predicting metastasis in one embodiment of the present invention.

Hereinafter, embodiments and examples according to the present invention will be described with reference to the drawings.
In one embodiment of the present invention, the method of predicting the presence or absence of metastatic metastasis in a colorectal cancer stage 2 patient is selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP in the colorectal cancer tissue obtained from the patient. At least one gene is used. Here, GNL3L, SLC35A2, and UBE2I are genes that are overexpressed in colorectal cancer patients with metastatic metastasis as compared with patients without metastatic metastasis, and IL2RB and TYMP are It is a gene whose expression is reduced in colorectal cancer patients who develop metastasis as compared with patients who do not develop metastasis.

In addition, the method for obtaining colorectal cancer tissue from the patient may be a surgical operation performed to remove the colorectal cancer, or a biopsy performed to diagnose the cancer or examine the degree of enlargement of the lesion. Any method of obtaining colorectal cancer tissue from a patient can be adopted.

In one embodiment of the present invention, the score for predicting the presence or absence of metastatic metastasis measures the expression level of at least one gene selected from the above gene cluster in colorectal cancer tissue obtained from a patient. , It is calculated by normalizing the expression level by the number of cycles and the like. For example, such normalization is calculated by the following formula.
Score value = Σ (ΔΔCt (gene whose expression level increases in the group with metastasis))-Σ (ΔΔCt (gene whose expression level decreases in the group with metastasis)))
Therefore, for example, when the method according to the present invention is carried out using only the GNL3L gene, (score value) = (ΔΔCt (GNL3L)). As the control gene used for calculating ΔCt, any control gene known in the present technology such as a housekeeping gene represented by ACTB, GAPDH, GUSB, HPRT, TBP and the like can be used.

In one embodiment of the present invention, the score value calculated as described above is compared with the reference value, and if the score value is smaller than the reference value, metastasis does not occur and the score value is the reference. If it is larger than the value, it can be shown that metastasis occurs. This reference value may be any score value, for example, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV). Any situation that functions as a genetic marker for predicting the presence or absence of metastatic metastasis, such as whether to predict the presence or absence of metastatic metastasis. The standard value according to the above can be adopted. Therefore, for example, if it is desired to predict only the presence of metastasis, it is possible to use a score value such that PPV is high as a reference value. Further, when predicting the presence or absence of metastasis, a score value in which PPV and NPV are kept high can be adopted as a reference value with a value at which the sensitivity and specificity do not decrease too much.

Further, in one embodiment of the present invention, since this reference value can be determined in consideration of sensitivity, specificity, PPV, and NPV as described above, the presence or absence of metastatic metastasis is already known for the colon. Using colorectal cancer tissue obtained from a cancer stage 2 patient, it can be determined based on the expression level of the same gene as the gene used to calculate the score value.

The gene expression level can be measured by using a known gene expression level measuring method such as microarray method, Northern blotting method, RT-PCR method, etc. using a probe or primer that specifically hybridizes with each gene. can.

Further, in one embodiment of the present invention, the “metastasis” refers to metastasis confirmed 1 year or 6 months after the discovery of the primary site of the tumor, preferably a colorectal cancer tissue. Refers to metastasis within 3 years of acquisition. In addition, the metastatic site includes metastasis to all organs such as liver metastasis, lung metastasis, and peritoneal metastasis.

In one embodiment according to the present invention, a kit for predicting the presence or absence of metastatic metastasis in a colorectal cancer stage 2 patient can be provided, and the kit is selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP. It also has a probe that specifically binds to at least one gene, and can also have reagents and the like well known in the art.

Such probes are known to specifically hybridize to a contiguous sequence of at least 15 bases to total bases, preferably 18 to total bases in the base sequence of each gene. It is designed as a polynucleotide having each of the above base lengths according to the method.

The probe does not have to be completely complementary as long as it specifically hybridizes to each gene. Such a polynucleotide is preferably 70% or more, preferably 80% or more, more in the base sequence, as compared with a polynucleotide consisting of at least 15 consecutive base sequences in the base sequence of each gene or a complementary polynucleotide thereof. It is preferably a polynucleotide having 90% or more, more preferably 95% or more, and particularly preferably 98% or more identity.

In addition, the probe may be labeled with a conventional radioactive substance, fluorescent substance, chemical luminescent substance, or enzyme so that the gene can be easily detected.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Experimental Methods and Materials The experimental methods and materials used in the present invention will be described below. Although the following experimental methods are used in one embodiment of the present application, similar results can be obtained by using other experimental methods.

Specimen As the target sample, a frozen sample of stage 2 colorectal cancer whose prognosis was clearly determined 3 to 5 years after the operation was used.

RNA extraction and real-time PCR
RNA was extracted from colorectal cancer tissue using TRIZOL. Concentration measurement and quality check were performed with a bioanalyzer, and real-time PCR was performed using TaqMann Probe (Life Technologies) using total RNA. The procedure is shown below.

The extracted RNA sample was adjusted to a concentration of 100 ng / μL with DW. Using the High Capacity cDNA Reverse Transcription Kit (Life Technologies), the cDNA was synthesized according to the manual. Using 20 μL of the prepared RNA solution, synthesis was performed with a total volume of 40 μL. The temperature conditions were 25 ° C. for 10 minutes, 37 ° C. for 120 minutes, 85 ° C. for 5 seconds, and 4 ° C. Hold. After completion of the reaction, 60 μL of DW was added to make a total volume of 100 μL.

100 μL of TaqMan Universal PCR Master Mix was added to the reverse transcriptase reaction solution adjusted to 100 μL and stirred to prepare 200 μL of the reaction solution. 100 μL of the reaction solution was dispensed and injected into a TaqMan Low Density Array plate (cDNA concentration was 1000 ng cDNA equivalent / Fill port). The measurement was performed using an Applied Biosystem 7900 HT Fast real-time PCR system (Life Technologies) at 50 ° C. for 2 minutes, 94.5 ° C. for 10 minutes, (97 ° C. for 30 seconds, 59.7 ° C. for 1 minute) / 40 cycles. .. The measurement was performed by double measurement using the same reaction solution.

After the reaction, the Ct value of each sample was obtained using RQ Manager 1.2 software.

Data processing The expression levels of 38 genes that are candidates for predicting metachronous metastasis were normalized for each of the five control genes (ACTB, GAPDH, GUSB, HPRT, TBP). Five data are obtained per marker gene. Based on clinical information, the group was classified into a group with metastasis and a group without metastasis, and a T-test was performed to examine whether there was a difference in the expression level of each.

The score value was calculated by the following formula.
Score value = Σ (ΔΔCt (gene whose expression level increases in the group with metastasis))-Σ (ΔΔCt (gene whose expression level decreases in the group without metastasis)))

Results Expression analysis of 38 genes and 5 control genes The expression levels of each gene were separately normalized with 5 control genes, and compared between the group with metastasis and the group without metastasis. As a result of the T-test, there was a significant difference (p <.01) for the combination of 5 genes (GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP) and 3 controls (FIG. 1). Of these, when one of the controls having the most significant difference was selected for the five genes, there were five combinations of GNL3L vs GAPDH, SLC35A2 vs GAPDH, UBE2I vs GAPDH, IL2RB vs GUSB, and TYMP vs GUSB. Cluster analysis was performed using these five expression information (Fig. 2). When the score values were classified by -1.9, PPV: 33.7%, NPV: 88.7%, Sensitivity: 49.3%, and Specialty: 80.4%. In other words, it was found that it is possible to roughly distinguish between metastasis and non-metastasis depending on the expression pattern.

Prediction of the presence or absence of metastasis by scoring All cases 398 (67 with metastasis, 331 without metastasis) were combined with a training set (n = 199; 34 with metastasis, 165 without metastasis) and a test set (n = 199; 33 with metastasis, metastasis). The analysis was performed separately from None 166). As a result, PPV: 50.0%, NPV: 78.8%, ACC: 73.9% in the training set, PPV: 48.5%, NPV: 81.9%, ACC: 76.4 in the test set. %Met.

FIG. 3 shows the relationship between the threshold value of the score value and the prediction probability. The correctness rate, PPV, and NPV change depending on which value the threshold value is set, but setting it between -2.5 and 0 is considered to be useful for clinical application.

Prediction of metastasis by 2 marker genes Using the same 2 sets (training set and test set) of classification samples, we examined whether 2 of the 5 genes could be used for discrimination. The combination is a combination of genes whose expression is increased and decreased in patients with metastasis, IL2RB and GNL3L, IL2RB and SLC35A2, IL2RB and UBE2I, TYMP and GNL3L, TYMP and SLC35A2, TYMP and UBE2I, It is a combination of six.

As a result, PPV: 29.1% to 36.4%, NPV: 86.7% to 88.3%, ACC: 71.4% to 78.4% in the training set, and PPV: 26 in the test set. It was .1% to 40.0%, NPV: 86.2% to 89.9%, and ACC: 72.4% to 80.9% (FIG. 4). From this result, it is possible to predict the presence or absence of metastatic metastasis in colorectal cancer stage 2 patients even when two genes selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP are used. I understand.

Prediction of metastasis by one marker gene Next, it was examined whether or not the presence or absence of metastasis can be confirmed for the GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP genes when only one gene is used.

As a result, PPV: 18.8% to 34.5%, NPV: 83.2% to 86.2%, ACC: 71.9% to 78.4% in the training set, and PPV: 24 in the test set. It was 3.3% to 37.0%, NPV: 85.1% to 87.1%, and ACC: 73.9% to 79.9% (FIG. 5). From this result, it is possible to predict the presence or absence of metastatic metastasis in colorectal cancer stage 2 patients even when only one gene selected from GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP is used. It turned out that there was.

In addition, the present invention is not limited to the above-described embodiment, and can be variously modified without changing the gist of the invention.

Claims (9)

A method for predicting the presence or absence of metastatic metastasis in patients with stage 2 colorectal cancer.
The step of measuring the expression level of the SLC35A2 gene in the colorectal cancer tissue obtained from the patient, and
It has a step of calculating a score for predicting the presence or absence of metastatic metastasis from the measured expression level, and when the calculated score is smaller than the reference value, metastatic metastasis occurs in the patient. Show that there is no , here,
The reference value is the group with metastasis of the patient with respect to the expression level of the same gene as the gene used in the measurement step in the colorectal cancer tissue obtained from the colorectal cancer stage 2 patient whose presence or absence of metastatic metastasis was found. And / or metastasis-free group, and the value determined based on the relationship between the value of the expression level of the gene in the patients in each group and the predicted probability including the positive predictive value and / or the negative predictive value. And a method selected between -2.5 and 0 . The method according to claim 1, wherein the score is calculated by normalizing the expression level of the SLC35A2 gene. In the method according to claim 1, the reference value is
Score value = Σ (ΔΔCt (gene whose expression level increases in the group with metastasis))-Σ (ΔΔCt (gene whose expression level decreases in the group without metastasis)))
A method , which is a value determined based on the relationship between the threshold value of the score value indicated by and the predicted probability including the positive predictive value and / or the negative predictive value . In the method of claim 1, the measuring step is a method of measuring the expression level of two genes selected from SLC35A2, GNL3L, UBE2I, IL2RB, and TYMP. The method of claim 4, wherein the two genes are IL2RB and SLC35A2, or TYMP and SLC35A2. In the method of claim 1, the measuring step is a method of measuring the expression levels of all genes of GNL3L, SLC35A2, UBE2I, IL2RB, and TYMP. The method according to claim 1, wherein the metastatic metastasis is a metastasis within 3 years after the tissue acquisition. A kit for predicting the presence or absence of metastatic metastasis in colorectal cancer stage 2 patients with a probe that specifically binds to the SLC35A2 gene. The method according to claim 3, wherein the reference value is a value determined from the positive predictive value and the score value at which the negative predictive value is kept high.

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