WO2010067722A1

WO2010067722A1 - Method for evaluating degree of malignancy of breast cancer, and kit for the evaluation

Info

Publication number: WO2010067722A1
Application number: PCT/JP2009/070078
Authority: WO
Inventors: 内野雅浩; 小島弘子; 和田健太; 宇津木孝彦
Original assignee: 株式会社バイオマトリックス研究所
Priority date: 2008-12-08
Filing date: 2009-11-20
Publication date: 2010-06-17
Also published as: JP2010130993A

Abstract

Disclosed are: a method for evaluating the degree of malignancy of breast cancer, which is simple and rapid and has high reliability; and an array and a kit for use in the evaluation method. Specifically disclosed are: a method for evaluating the degree of malignancy of breast cancer based on the expression patterns of at least three genes selected from a group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1 and SOCS2 in a test sample, wherein the expression patterns are determined based on the expression levels of the at least three genes; an array for the above-mentioned evaluation, which comprises a support and nucleic acids corresponding to the at least three genes and/or fragments of the nucleic acids immobilized on a predetermined area on the support; a kit for the above-mentioned evaluation, which can detect mRNA molecules expressed from the at least three genes and/or fragments of the mRNA molecules; and a kit for the above-mentioned evaluation, which comprises antibodies directed against polypeptides encoded by the at least three genes and/or fragments of the polypeptides and/or fragments of the antibodies.

Description

Breast cancer malignancy evaluation method and evaluation kit

The present invention relates to a method for evaluating the grade of malignancy of breast cancer, and an array and a kit used for the evaluation method.
Specifically, the present invention relates to a method for evaluating the possibility of metastasis and / or recurrence of breast cancer, and an array and kit used for the evaluation method.

Breast cancer is the most common female cancer in the UK, USA and Japan. Currently, the number of breast cancer patients in Japan exceeds 40,000, and the number is still increasing. In recent years, advancements in medical devices using mammography or other imaging techniques have enabled early diagnosis of breast cancer, and the cure rate of breast cancer is expected to increase gradually.
However, despite early surgical diagnosis and radiation therapy in early stages of breast cancer, many patients have metastasis recurrence due to early diagnosis. In fact, it is disclosed in Non-Patent Document 1 that approximately 40% of breast cancer patients die, despite the 5-year survival rate after resection surgery of the first breast cancer being 90% or more. That is, early diagnosis of breast cancer does not necessarily contribute to reducing the risk of metastasis or recurrence.
There are also many breast cancer patients who do not experience metastasis or recurrence, but in order to reduce the risk of metastasis recurrence, many patients are forced to administer therapeutic agents that cause side effects and are receiving unnecessary treatment.
Conversely, conservative treatment strategies may be presented for patients who are actually at high risk of metastatic recurrence. Therefore, it is necessary to assess the possibility of breast cancer metastasis recurrence early, appropriately and accurately for each breast cancer patient, and to determine an optimal prognostic treatment policy.
The risk of metastasis recurrence is added as a predictor of distant metastasis, such as tumor diameter, which is already known as a prognostic marker for breast cancer, and the presence or absence of axillary lymph node metastasis The degree of undifferentiation of cancer cells (the more undifferentiated, the higher the grade).
However, in Non-Patent Document 1, about 1/3 of patients without axillary lymph node metastasis have recurrent metastasis, and about 1/3 of patients with positive axillary lymph node metastasis have relapsed even after 10 years of local treatment. Is not allowed. Further, it is known that a low estrogen receptor expression level correlates with a high risk of metastasis, but Non-Patent Document 1 discloses that estrogen receptor-positive breast cancer tends to cause bone metastasis. Yes. In other words, at present, the risk of metastasis recurrence for each breast cancer patient cannot be sufficiently predicted. Non-patent document 1 discloses that these prognostic markers can be used to determine whether the prognosis is good or not in about 30% of patients, and about 30% of the remaining 70% have recurrence of metastasis. Therefore, a new prognostic marker that can determine the level of recurrence risk appropriately and accurately is needed.
While various biomarkers are listed as candidates for new breast cancer prognostic markers, ERBB2 (epidermal growth factor receptor 2 or HER2 / neu) has attracted attention as a prognostic prognostic marker. For example, the value as a prognostic marker for predicting the recurrence-free survival rate and overall survival rate of those who are positive for lymph node metastasis is considered to be low to moderate.
Research on methods for detecting cancer cells circulating in the bloodstream is also underway. In order to form metastases, cancer cells need to infiltrate the surrounding tissues, ride through the body circulation through the blood, stay in the capillary bed of the distant organ, and then infiltrate and proliferate there. Therefore, if a method for detecting this disseminated cancer cell before distant metastasis occurs is expected to be useful for predicting the prognosis of the patient. However, due to technical problems such as non-specific expression of cytokeratin 19 which is a mammary epithelial marker and the fact that less than 0.1% of tumor cells entering the systemic circulation can form metastatic foci, There is no consensus on the significance of tumor cells as a prognostic marker. Furthermore, a method has been proposed in which the activity values of uPA (urokinase-type plasminogen activator) and PAI1 (plasminogen activator inhibitor 1) in proteins extracted from breast cancer primary lesions are measured by ELISA. Not widely used in clinical practice.
Most studies that looked for new prognostic markers to predict the risk of metastatic recurrence in breast cancer patients have examined the correlation between just one to three markers and patient outcomes, but the heterogeneity of the disease In view of this, it is considered necessary to analyze various markers at the same time in order to predict the recurrence of breast cancer metastasis. DNA microarray technology has made this possible, and gene expression analysis can be performed across the entire chromosome. Based on the analysis of gene expression patterns using DNA microarray technology and unsupervised analysis (unsupervised analysis that does not specify objective variables and automatically generates factors), breast cancer has five subtypes (basal-like). Group, ERBB2 + group, normal tissue-like group, luminal A group, luminal B group) (Non-patent Documents 2-4). Importantly, these five subtypes represent patient fractions that are clinically distinct. For example, patients in the basal-like group and the ERBB2 + group have a short survival time, while the estrogen receptor positive luminal A group has the best prognosis among all patient groups. By classifying breast cancer patients into subtypes that each have a characteristic clinical picture, it is expected that subtype-specific treatment targets can be found and effective treatment can be performed.
Another approach to determine gene expression patterns that can predict the clinical picture of breast cancer is the supervised stratification method (supervised analysis specifying objective variables) that limits the target patient. These patient stratification reports identified 70 gene expression profiles that could predict recurrence of distant metastases in breast cancer patients under 55 years of age who did not have lymph node metastasis (Non-Patent Documents 5 and 6). This retrospective study was a particularly useful report because it had no adjunct therapy affecting patient outcomes and had an observation period of more than 10 years. With this gene set, the characteristics of primary breast cancer were grouped into either a poor prognosis group in which recurrence of metastasis is expected or a good prognosis group in which the possibility of metastasis recurrence is low. In addition, it was shown that retrospective analysis of the expression status of 76 genes by a microarray different from the above-mentioned test can predict the outcome of lymph node-negative breast cancer patients in all age groups (non- Patent Document 7). The expression profiles of these genes are considered useful for individualized treatment of individual breast cancer patients and are expected to reduce the number of patients receiving unnecessary adjuvant treatment.
However, only three genes overlap between these two gene sets disclosed in Non-Patent Documents 5 and 6 and Non-Patent Document 7. In practice, it is not easy to compare these gene expression profiles. This is because these microarray analyzes use different array chips and different mathematical analysis methods. It has long been known that different gene arrays can be identified using different microarray chips, even though they represent the same biological phenomenon. Therefore, it is necessary to verify the difference in gene expression level at the mRNA level by reverse transcription polymerase chain reaction (RT-PCR), or at the protein level by immunoassay. Analyzing genetic markers is not clinically practical. Moreover, it cannot be said that each marker of the prognostic marker group determined by the supervised stratification technique necessarily reflects the above-mentioned breast cancer tissue subtype. This is because the selection of candidate genes is strongly influenced by the composition and stratification of the patient group subjected to analysis.
Patent Document 1 discloses a molecular signature divided into three elements for breast cancer prognosis: breast cancer patient survival possibility, breast cancer recurrence possibility, and metastatic characteristics.
Patent Document 2 discloses a prognostic marker group for cancer.

Special table 2008-508895 gazette JP 2006-526998 A

According to the technique disclosed in Patent Document 1, it is possible to select a molecular signature that correlates with statistical data of the survival rate, recurrence rate, and metastasis rate of the breast cancer patient group, In an individual patient, these three elements are not easily splittable. Therefore, it is doubtful whether the molecular signature group disclosed in Patent Document 1 will lead to individualized treatment of breast cancer patients, which may lead to confusion when determining the optimal prognosis treatment policy.
Moreover, although patent documents 1 and 2 disclose various cancer prognostic marker groups including breast cancer, these cancer prognostic marker groups include individual genes included in the marker group, or The function of the signaling pathway to which the gene belongs is not fully taken into account, and genes whose expression varies indirectly or secondaryly to the risk of recurrence of metastasis are also included.
As described above, DNA microarray analysis is useful in searching for new prognostic markers for predicting the risk of recurrence of metastasis in breast cancer patients, but as mentioned above, the composition or stratification of sample groups used for selection of candidate genes Because of this problem and the practical problem of the number of selected genetic markers, or the problem of the significance of the marker group, there is currently no gene set that can sufficiently and significantly predict the risk of recurrence of metastasis in each breast cancer patient.
Therefore, the problem to be solved by the present invention is to overcome the above-mentioned problems, evaluate the malignancy of breast cancer to individual breast cancer patients early, appropriately and accurately, and determine the optimal prognosis treatment policy. An object of the present invention is to provide an evaluation method that enables determination, and an array and a kit used therefor.

As a result of intensive studies to solve the above problems, the present inventors have measured the expression levels of at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2. The present inventors have found that malignancy of breast cancer can be evaluated.
That is, the present invention provides the following method for evaluating the malignancy of breast cancer, and an array and kit used for the method.
[1] A method for evaluating the malignancy of breast cancer,
(1) measuring the expression levels of at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 in a test sample;
(2) evaluating the malignancy of breast cancer based on the expression pattern of the at least three genes.
[2] The method according to [1], wherein (1) is a step of measuring the expression level of mRNA and / or polypeptide.
[3] The method according to [2], wherein the expression level of the mRNA is measured using an array in which nucleic acids and / or nucleic acid fragments corresponding to the mRNA are immobilized.
[4] The method according to [2], wherein the expression level of the mRNA is measured using a primer and / or probe for detecting a nucleic acid and / or a nucleic acid fragment corresponding to the mRNA.
[5] The method according to [2], wherein the expression level of the polypeptide is measured using an antibody and / or antibody fragment against the polypeptide and / or polypeptide fragment.
[6] The method according to any one of [1] to [5], wherein (1) is a step of measuring an expression level of the gene in the test sample and an expression level of the gene in a control sample. Method.
[7] The method according to [6], wherein the expression pattern comprises an expression ratio of the expression level of the gene.
[8] The method according to [6], wherein the expression pattern includes a relative expression level obtained from the expression level of the gene in the test sample with respect to the expression level of the gene in the control sample.
[9] In (2), the gene expression pattern in the test sample is selected from the group consisting of healthy individuals, non-lesional sites, low metastatic cancer cell lines, and tissues having low metastatic cancer. The breast cancer malignancy is assessed to be low enough to approximate the gene expression pattern in the at least one control sample and / or the gene expression pattern in the test sample is high metastatic cancer cell lines and high [6] to [8] are steps for evaluating that the malignancy of breast cancer is higher as it approximates the expression pattern of the gene in at least one control sample selected from the group consisting of tissues derived from metastatic cancer. The method of any one of these.
[10] In the above (2), the gene expression pattern in the test sample is selected from the group consisting of healthy individuals, non-lesional sites, low metastatic cancer cell lines, and tissues having low metastatic cancer. Compared to the expression pattern of the gene in at least one control sample
(A) When an expression pattern of a gene in which the expression level of at least one gene selected from the group consisting of CD44 and SOCS2 is increased, and / or (B) from the group consisting of BMP7, CD24, CXCL12, and PIK3RI When the expression level of at least one selected gene shows a reduced gene expression pattern,
The method according to any one of [6] to [8], which is a step of evaluating that the malignancy of breast cancer is high.
[11] The method of [9] or [10], wherein the low metastatic cancer cell line is MCF7 cells.
[12] The method according to [9], wherein the highly metastatic cancer cell line is MDA-MB-231 cells.
[13] An array for assessing the malignancy of breast cancer,
An array in which nucleic acids and / or nucleic acid fragments corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 are immobilized on a defined region on a support.
[14] An array for assessing the malignancy of breast cancer,
An array in which only nucleic acids and / or nucleic acid fragments corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 are immobilized on a defined region on the support .
[15] A kit for evaluating the malignancy of breast cancer,
Primers and / or probes for detecting nucleic acids and / or nucleic acid fragments corresponding to mRNAs and / or mRNA fragments of three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 Contains a kit.
[16] A kit for evaluating the malignancy of breast cancer,
A kit comprising an antibody and / or an antibody fragment against a polypeptide and / or polypeptide fragment encoded by three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2.

According to the present invention, it is possible to evaluate the malignancy of breast cancer at an early stage, appropriately and accurately, and to determine an optimal prognosis treatment policy for individual breast cancer patients. Useful and can also reduce the number of patients receiving unnecessary adjunct therapy.

1 shows a diagram (photograph) of fluorescent immunostaining in Example 1. FIG.
FIG. 2 shows the expression level of mRNA measured by quantitative RT-PCR.
FIG. 3 shows clustering based on DNA microarray analysis.
FIG. 4 shows clustering (6 genes) based on DNA microarray analysis.
FIG. 5 shows clustering (3 genes) based on DNA microarray analysis.
FIG. 6 shows the expression level of mRNA measured by quantitative RT-PCR.
FIG. 7 shows a diagram of protein expression analysis by flow cytometry.

Hereinafter, the best mode for carrying out the present invention will be described in detail. The present invention is not limited to the best mode for carrying out the following, and can be implemented with various modifications within the scope of the gist thereof.
The method for evaluating the malignancy of breast cancer of the present invention comprises:
(1) measuring the expression levels of at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 in a test sample;
(2) evaluating the malignancy of breast cancer based on the expression patterns of the at least three genes.
In the present invention, the grade of malignancy of breast cancer means the bad nature of breast cancer, that is, the degree of ease of proliferation, metastasis, and recurrence.
In the present invention, breast cancer metastasis means that breast cancer cells reach lymph nodes (lymph node metastasis) and other organs (distant metastasis) and proliferate there.
In the present invention, recurrence of breast cancer means that the breast cancer becomes active again several months to several years after the treatment. Depending on the site of recurrence, local recurrence in the preserved breast and chest wall, regional recurrence in the axillary lymph nodes, and distant (systemic) recurrence (ie, metastasis) in organs and tissues away from the breast, such as lung, bone, liver, and brain Divided into
Further, in the present invention, by evaluating the malignancy of breast cancer, the possibility of metastasis recurrence of breast cancer can be evaluated early and appropriately and accurately, and the optimal prognosis treatment policy can be determined. .
In the method of evaluating the malignancy of breast cancer, simple selection of candidate genes to be evaluated is simple breast cancer metastasis that takes into account the tissue type and eliminates noise due to individual differences such as age and the presence or absence of adjuvant therapy A model is needed.
In the present invention, MCF7-14 cells disclosed in International Publication No. 2008/93886 pamphlet and MDA-MB-231 cells which are widely used are used as a breast cancer metastasis model, thereby considering the tissue type. Above, it can be set as the method of evaluating the malignancy of a breast cancer which excluded the noise by individual differences, such as age and the presence or absence of adjuvant therapy.
MCF7-14 cells are selected from the luminal type and low invasive / metastatic MCF7 breast cancer cell line, and confirmed to be highly invasive in vitro and highly metastatic in vivo. (International Publication No. 2008/93886 pamphlet).
MDA-MB-231 cells are known as basal / mesenchymal (mesenchymal) type and high malignant cells.
The evaluation method of the present invention compares gene expression profiles in MCF7 cells, MCF7-14 cells, and MDA-MB-231 cells as useful breast cancer metastasis models for evaluating the malignancy of breast cancer for selection of candidate genes. As a result, the malignancy of breast cancer, such as the possibility of recurrence of breast cancer metastasis, can be evaluated early and appropriately for each individual breast cancer patient.
In the present invention, as a result of comparing gene expression profiles in MCF7 breast cancer cells, MCF7-14 breast cancer cells and MDA-MB-231 breast cancer cells, a probe set whose expression level varies depending on the possibility of metastasis and / or recurrence, , A 163 probe set (corresponding to the 144 gene) was found in which the expression level was more than doubled in both MCF7-14 and MDA-MB-231 cells compared to MCF7 cells ( Table 1 and Table 2). In Tables 1 and 2, for EST (expressed sequence tag), GenBank Accession No. And Refseq ID was described for other cases.

These 144 genes include genes that have a direct causal relationship with the malignancy of breast cancer, and genes that change expression indirectly or indirectly, so the functional relationship of the genes in the selection of genetic markers. It is also necessary to consider the role in metastasis recurrence mechanisms. In addition, considering that it is not clinically practical to analyze dozens or more gene markers, pathway analysis and network analysis are performed on these 144 genes, and functionally significant in the mechanism of metastasis recurrence Candidate genes were narrowed down. As a result, genes encoding BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2, which are involved in pathways involved in foreign body metabolism such as cell migration / migration and drug excretion, were selected as markers for prognosis of breast cancer.
The gene set used in the present invention is selected from the group consisting of genes encoding BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2, and is composed of at least three genes.
In the present invention, by evaluating at least three types selected from BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 which are functionally significant genes in the mechanism of metastasis recurrence, The malignancy of breast cancer can be evaluated appropriately and accurately at an early stage.
BMP7 is Bone morphogenetic protein 7 gene (osteogenic protein 1, bone morphogenetic protein 7, NCBI Reference Sequences [RefSeq] ID: NM 001719).
CD24 is a glycosyl-phosphatidylinositol-linked sialoglycoprotein gene (NCBI Reference Sequences [RefSeq] ID: NM 012330), and the induction of CD24 expression in MDA-MB-231 cells suppresses the following cell migration promoting effect by CXCL12. (See Schabath H, Runz S, Joumaa S, Altevogt P. CD24 effects CXCR4 function in pre-Blymphosites and breast carcinoma cells. J Cell Sci.
CD44 is a hyaluronic acid receptor gene (NCBI Reference Sequences [RefSeq] ID: NM 000610), and CD44 (standard form) expression induction has been reported to enhance migration and invasion of MCF7 breast cancer cells ( Hill A, McFarlane S, Mulligan K, et al. Cortactin underpins CD44-promoted inversion and adhesion of breast cancer cells to bone mar.
CXCL12 is a chemokine [C-X-C motif] ligand 12 gene (stromal cell-derived factor 1 [SDF1], NCBI Reference Sequences [RefSeq] ID: NM 199168), and CCXCL12 cell induction PIK3R1 is known to promote phosphoinositide-3-kinase gene (regularity subunit 1 [alpha], phosphatidylinositol 3-phosphate enzyme subunit, NCBI Reference Sequences [RefSeq] ID 150 N overexpression of dominant negative PIK3R1, or PI3 kina It has also been reported that the addition of an inhibitor of zeotide suppresses the motility of MDA-MB-231 cells (Slivea D, Rizzo MT, England D. Phosphatidylinositol 3-kinase and NF-κB regulative motility in MD -231 human breast cancer cells by the section of urokinase-type plasminogen activator. J Biol Chem 2002; 277: 3150-7.).
SOCS2 is a suppressor of cytokine signaling 2 gene (CIS2, Cish2, SSI2, STATI2, RefSeq ID: NM 003877).
High expression of SOCS2 or low expression of BMP7 in primary breast cancer lesions is associated with the formation of bone metastases in breast cancer (Buijs JT, Henriquez NV, van Overveloped PG, et al. Bone morphogenetic protein 7 in the tent enveloment of the brain. methestes from breast cancer. Cancer Res 2007; 67: 8742-51. and Minn AJ, Kang Y, Serganova I, et al .. Distinct organic-specific biostatic. Invest 2005; 115: 44-55.).
Examples of the test sample used in the present invention include biological samples such as mammary gland, milk, sentinel lymph node and blood collected from an individual.
As a control sample used in the present invention, tissues derived from healthy individuals, biological samples such as milk, blood, non-lesional tissues, or mammary glands, milk, blood collected from breast cancer patients whose malignancy was confirmed by a prognostic survey , Biological samples such as metastatic diseased tissue, and breast cancer cell lines.
Preferably, the mammary gland having a cancer or a breast cancer cell line having a different degree of metastasis may be used. In view of data reproducibility and availability, the MCF7 breast cancer cell line and the MDA-MB- It is preferred to use the 231 breast cancer cell line.
The expression level of the gene is measured from the amount of mRNA and / or fragment thereof transcribed from the gene or the amount of protein and / or fragment thereof translated from the mRNA. In the present invention, the expression level of the gene in the test sample and the control sample can be measured by any method / means known to those skilled in the art.
Examples of the method for measuring the mRNA expression level include measuring the mRNA expression level using an array on which nucleic acids and / or nucleic acid fragments corresponding to mRNA are immobilized. Specifically, a DNA microarray ( DNA chip), oligonucleotide microarray, Northern blotting (Northern hybridization), in situ hybridization, RNase protection assay, reverse transcription polymerase chain reaction, etc. Can be used.
Examples of the nucleic acid and / or nucleic acid fragment corresponding to mRNA include the nucleic acid and / or nucleic acid fragment used in the method as described above, and nucleic acid and / or nucleic acid fragment for detecting mRNA, cDNA synthesized from mRNA , Nucleic acids and / or nucleic acid fragments for detecting cRNA.
Examples of the method for measuring the protein expression level include a method for measuring the expression level of a polypeptide using an antibody and / or antibody fragment against the polypeptide and / or the polypeptide fragment, and specifically, Western blotting. , ELISA assay, flow cytometry, protein array, immunohistochemical staining, immunochromatography, yeast Two-Hybrid and the like.
In the present invention, antibody fragments include, for example, Fab portions that bind to an antigen that is a polypeptide of an antibody.
In measuring the gene expression level, it is desirable to obtain a plurality of data for each gene of the same sample in consideration of the reproducibility of the data. In that case, the average value of these data can be used as the expression level of each gene. In addition, reproducibility can be verified from the variance or standard deviation of these data.
As a step of evaluating the malignancy of breast cancer based on the expression patterns of at least three kinds of genes, the numerical values of the measured gene expression levels are patterned and compared between the test sample and the control sample.
As a gene pattern, for example, the expression level of a housekeeping gene can be set as 1 as an internal control, and the relative expression level with respect to the internal control can be calculated for a control sample and a test sample to evaluate the malignancy of breast cancer. As the internal control, in addition to a housekeeping gene such as GAPDH, a gene whose expression level is considered not to change between samples can be used. Further, the relative expression level of the expression level in the test sample relative to the expression level in at least one control sample may be calculated. Specifically, the test sample when the expression level in the control sample is 1. The expression level in can be calculated as a relative expression level and evaluated as a gene expression pattern in a test sample. If the level of expression level in the control sample is known in advance, the result may be a high or low value for each gene depending on the relative expression level of the test sample with respect to the control sample.
In addition, for example, the expression pattern can be calculated as an expression ratio obtained by dividing the expression level in the test sample and the expression level in the control sample by the average value or the median value of the expression levels, respectively.
In the present invention, at least three genes are selected from the six genes, and the expression patterns of at least three genes are compared. Alternatively, the expression level of each of the genes is compared to whether the expression level of SOCS2 or CD44 is high or the expression level of BMP7, CD24, CXCL12 or PIK3R1 is low compared to the control sample.
Various methods can be employed for the analysis and display of gene expression patterns. The most common method is to arrange the expression ratio of the test sample to the control sample in a graphical dendogram in which the column indicates the test sample and the control sample, and the row indicates each gene. The expression ratio of each gene can be visualized by color. Such data display includes commercially available computer software, such as Silicon Genetics, Inc. “GeneSpring” can be used. In addition, display using a histogram and statistical analysis such as analysis of variance (ANOVA) or multiple comparison can be performed using general spreadsheet software.
By assessing the malignancy of breast cancer based on the expression patterns of at least three genes, the malignancy of breast cancer can be evaluated appropriately and accurately at an early stage for each individual breast cancer patient.
As the evaluation step, the gene expression pattern in the test sample is at least one selected from the group consisting of a healthy individual, a non-lesion site, a low metastatic cancer cell line, and a tissue having a low metastatic cancer. Evaluate that the malignancy of breast cancer is low enough to approximate the expression pattern of the gene in the control sample of the species, and the expression pattern of the gene in the test sample is derived from a tissue with a high metastatic cancer cell line and a high metastatic cancer A method of evaluating that the malignancy of breast cancer is higher as the expression pattern of the gene is approximated to at least one control sample selected from the group consisting of
In the step of evaluating, the gene expression pattern in the test sample is selected from the group consisting of a healthy individual, a non-lesion site, a low metastatic cancer cell line, and a tissue having a low metastatic cancer. Compared to the expression pattern of the gene in at least one control sample,
(A) When the expression level of at least one gene selected from the group consisting of CD44 and SOCS2 increases,
(B) A method for evaluating that the malignancy of breast cancer is high when the expression level of at least one gene selected from the group consisting of BMP7, CD24, CXCL12, and PIK3RI decreases.
In the present invention, examples of the control sample derived from a healthy individual include mammary gland tissue, lymph node tissue, milk, and blood derived from an individual who has not developed cancer.
In the present invention, the non-lesion site-derived control sample includes, for example, mammary gland tissues other than cancer lesions among mammary gland tissues having cancer lesions of individuals who have developed cancer.
In the present invention, the low metastatic cancer cell line includes, for example, the MCF7 human breast cancer cell line.
In the present invention, a control sample derived from a tissue having low metastatic cancer includes, for example, a mammary gland tissue having a cancer lesion collected from an individual whose metastasis was not observed by a prognostic survey.
In the present invention, examples of the highly metastatic cancer cell line include an MDA-MB-231 human breast cancer cell line.
In the present invention, the control sample derived from a tissue having highly metastatic cancer includes, for example, a cancer lesion collected from an individual in which metastasis is observed by a prognostic survey or a tissue having a metastatic cancer lesion.
The most common method for analyzing the degree of approximation of the gene expression pattern is cluster analysis, and analysis software such as “GeneSpring” can also be used. For example, gene expression patterns in test samples, MCF7 cells, and MDA-MB-231 cells are compared by cluster analysis, and if they are classified into the same cluster as MCF7 cells, the malignancy is evaluated to be relatively low. On the contrary, if it is classified into the same cluster as MDA-MB-231 cells, it can be judged that the malignancy is relatively high. The degree of approximation can be expressed visually by creating a tree diagram of the sample by hierarchical cluster analysis. Specifically, a hierarchical cluster analysis is performed, and a graphical dendrogram in which a column indicates a test sample and a control sample, and a row indicates each gene may be output. The closer to the expression pattern of the highly metastatic breast cancer cell line, the higher the malignancy can be evaluated.
In the present invention, by evaluating the expression patterns of at least three genes, the malignancy of breast cancer can be evaluated early and appropriately and accurately for individual breast cancer patients. By evaluating the expression patterns of four types, more preferably at least five types, and more preferably all six types, the malignancy of breast cancer can be evaluated with higher accuracy.
The array or kit of the present invention is used in a method for measuring the expression level of the gene in a test sample, that is, the amount of mRNA transcribed from the gene or the expression level of a protein encoded by the gene.
The array of the present invention is an array for evaluating the malignancy of breast cancer,
An array in which nucleic acids and / or nucleic acid fragments corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 are immobilized on a defined region on a support It is.
The array of the present invention is an array for evaluating the malignancy of breast cancer,
Only nucleic acids corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 and / or nucleic acid fragments thereof are immobilized in a defined region on the support. An array.
The kit of the present invention is a kit for evaluating the malignancy of breast cancer as a kit for measuring the expression level of mRNA,
A kit comprising primers and / or probes for detecting mRNA of at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 and / or mRNA fragments thereof.
Moreover, the kit of the present invention is a kit for evaluating the malignancy of breast cancer as a kit for measuring the expression level of a polypeptide,
A kit comprising a polypeptide encoded by at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 and / or an antibody and / or antibody fragment against the polypeptide fragment is there.
For example, the nucleic acid corresponding to at least three genes or the nucleic acid fragments thereof contained in the array or kit of the present invention, primers for detecting mRNA of at least three genes and / or mRNA fragments thereof, and The base sequences of the probes need only correspond to the nucleic acid sequences of the six genes in the present invention.
Examples of the nucleic acid and / or nucleic acid fragment, and the primer and / or probe include polynucleotides and / or oligonucleotides corresponding to the base sequences of at least a part of the six genes in the present invention, and the length is several It can be 10 base pairs.
Since the sequence information of the six genes in the present invention can be easily obtained from, for example, a database such as Genbank, nucleic acids and / or nucleic acid fragments corresponding to at least three genes based on the information used in the array or kit The primers and / or probes for detecting the mRNA and / or the mRNA fragment thereof can be appropriately selected, designed and adjusted by those skilled in the art. They can also be used as probes in Northern blotting, primers in PCR, and the like. In addition, an antibody against at least a part of a polypeptide encoded by the gene and / or an antibody fragment thereof contained in the kit of the present invention can be prepared by a person skilled in the art by any method, such as Western blotting or ELISA assay. It can be used not only for immunological assay methods, but also for immunostaining methods. Furthermore, if necessary, the polynucleotide / oligonucleotide, antibody or antibody fragment thereof may be labeled with an appropriate labeling substance such as a radioactive substance, a fluorescent substance, or a dye.
When a partial base sequence of the gene in the present invention is used as a primer, the sequence is not particularly limited, but preferably the sequence shown in Table 3 described later is used.
The above array or kit includes other elements or components known to those skilled in the art depending on the configuration and purpose of use, such as various reagents, enzymes, buffers, reaction plates (containers), and the like. It is done. Various reagents may include a reagent derived from a control sample for use as a control.

EXAMPLES Hereinafter, although an Example demonstrates this invention, the technical scope of this invention is not limited and interpreted by description of a following example at all. Also, unless otherwise noted, the standard methods known to those skilled in the art were used.
(Human breast cancer cell line)
The human breast cancer cell lines are luminal type and low malignancy MCF7 breast cancer cell lines, MCF7-14 breast cancer cell lines that are highly invasive and metastatic selected from MCF7 cells, and basal / mesenchymal types, And a high-grade MDA-MB-231 breast cancer cell line.
MCF7 cells and MDA-MB-231 cells were purchased from Tohoku University Institute of Aging Medicine (TKG 0479) and American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.
As the MCF7-14 cells, cells (accession number: FERM BP-10944) prepared according to the method described in International Publication No. 2008/093886 were used.
(Fluorescent immunostaining)
The above cells were fixed with a 4% paraformaldehyde (PFA) / phosphate buffer solution (PBS) solution and non-specific reaction was blocked with skim milk, and then a mouse anti-E-cadherin monoclonal antibody (clone, 67A4; Santa Cruz; Biotechnology, Inc .; dilution factor, 1: 100 dilution) and mouse anti-vimentin monoclonal antibody (V9, DAKO, 1: 300) solution were added and incubated for 1 hour at room temperature. After washing with PBS containing 0.1% Triton-X, incubation with Alexa Fluor 488-labeled goat anti-mouse IgG antibody solution was followed by counterstaining of cell nuclei with 4'6-diamidino-2-phenylindole (DAPI). The observation of the fluorescence image was performed using ZEISS AxioVision software.
(Total RNA extraction)
The above human breast cancer cell line was synchronized with the S phase of the cell cycle by the Thymidine-Hydroxyurea method, and then total RNA was extracted using Qiagen's RNeasy Mini kit.
(DNA microarray analysis)
Double-stranded cDNA was synthesized from the extracted total RNA by One-Cycle cDNA Synthesis Kit (Affymetrix) and purified by GeneChip Sample Cleanup Module (Affymetrix). Biotin-labeled cRNA was synthesized by GeneChip IVT Labeling Kit and purified by GeneChip Sample Cleanup Module. 15 mg of labeled cRNA was hybridized to the Human Genome U133 Plus 2.0 Array (Affymetrix) and the array image was scanned with GeneChip operating software (Affymetrix).
(DNA microarray data analysis)
The DNA microarray data was analyzed using GeneSpring GX 7.3.1 software and MultiExperiment Viewer (MeV) 4.2 software (part of the TM4 Software Suite, open source). After normalizing the microarray data on each chip to the 50th percentile, the data value of each probe was normalized with the median of the data in all samples.
The average linkage hierarchical clustering analysis was performed on genes of which detection flag was “present” in two or more of the three cells and the signal intensity was 50 or more in all cells.
(Reverse transcription polymerase chain reaction)
From 2 μg of the extracted total RNA, using SuperScript III reverse transcriptase (Invitrogen), reverse transcription (RT) reaction was performed at 50 ° C. for 1 hour to synthesize cDNA, and the reaction was stopped by heating at 85 ° C. for 5 minutes. It was. 2.5 μL of Power SYBRGreen PCR Master Mix (Applied Biosystems) is added to 7.5 μL of the reaction solution containing 0.2 μL of cDNA and 0.5 μM of the primer, and polymerase chain reaction (PCR) is performed. The fluorescence intensity of the amplification product at the end was measured. Table 3 shows the base sequences of the primers used in this experiment. PCR and fluorescence intensity measurements were controlled by 7900HT Fast Real-Time PCR System (95 ° C., 10 min pre-incubation; 95 ° C., 15 sec denaturation, 60 ° C., 1 min annealing / elongation 40 cycles). Triplicate experiments for each primer were performed twice on all cells.

(Quantitative RT-PCR data analysis)
Quantitative RT-PCR data for each gene was normalized to the mRNA expression level of GAPDH as an internal control and used as a relative value to the expression level in MCF7 cells. Statistical analysis was performed by ANOVA and Tukey-Kramer multiple comparison, and P <0.05 was considered significant.
(Flow cytometry)
Cloned MCF-7 and MCF-7-14 cells are stained with Physoerythrin (PE) labeled anti-CD44 monoclonal antibody (G44-26, BD Biosciences) or PE labeled anti-CD24 monoclonal antibody (ML5, BD Biosciences), The expression levels of CD44 and CD24 proteins were measured using FACSCalibur (BD Biosciences).
(Expression comparison of epithelial and mesenchymal markers)
As a result of the fluorescence immunostaining, E-cadherin expression was observed on the intercellular adhesion surface in MCF7 cells and MCF7-14 cells, but no vimentin expression was observed (FIG. 1). On the other hand, in MDA-MB-231 cells, expression of E-cadherin was not observed, but expression of vimentin was observed in the cytoplasm (FIG. 1). FIG. 2 shows the mRNA expression levels of epithelial and mesenchymal markers measured by quantitative RT-PCR as relative mRNA expression levels normalized by the expression level of GAPDH mRNA. Although no significant difference was observed between MCF7 cells and MCF7-14 cells, the expression level of E-cadherin was significantly higher in MDA-MB-231 cells than in MCF7 cells and MCF7-14 cells. Low (P <0.01), the expression level of the mesenchymal marker was significantly higher (P <0.01, FIG. 2). It was found that the expression level of epithelial / mesenchymal marker in MCF7-14 cells showed the expression pattern of epithelial type, similar to MCF7 cells.
(Cluster analysis of DNA microarray data)
Informal gene expression profiles of 13,092 genes in MCF7 cells, MCF7-14 cells and MDA-MB-231 cells are shown in a graphical dendrogram by relative comparison (FIG. 3). The gene expression profile of MCF7-14 cells was found to be more similar to MCF7 cells than MDA-MB-231 cells.
(BMP7, CD24, CD44, CXCL12, PIK3R1, SOCS2 gene expression comparison)
When BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 expression data were extracted from gene expression profiles of MCF7 cells, MCF7-14 cells, and MDA-MB-231 cells, and compared by cluster analysis, MCF7-14 cells were The cells were classified into the same cluster as MDA-MB-231 cells but not MCF7 cells (FIGS. 4 and 5).
The mRNA expression level of the gene measured by quantitative RT-PCR is shown in FIG. 6 as the relative mRNA expression level for MCF7 cells. MCF7-14 and MDA-MB-231 cells did not show significant differences for CXCL12 compared to MCF7 cells, but the expression levels of CD44 and SOCS2 were significantly higher (P <0.01). The expression levels of BMP7, CD24 and PIK3R1 were significantly lower (P <0.01, FIG. 6).
Also in the expression analysis at the protein level by flow cytometry, the cloned MCF7-14 cells had higher expression levels of CD44 and lower expression levels of CD24 than the cloned MCF7 cells (FIG. 7). .
As described above, MCF7-14 cells exhibit an epithelial type expression pattern with a relatively good prognosis at the expression level of the epithelial / mesenchymal marker, and the results of gene expression profiling with all the informative genes are also shown in FIG. It is very similar to MCF7 cells.
However, by comparing the expression level of BMP7, CD24, CD44, CXCL12, PIK3R1, SOCS2 gene mRNA, or the protein encoded by them, MCF7-14 cells are clearly distinguished from MCF7 cells and have high malignancy. It was found to show an expression pattern similar to that of MDA-MB-231 cells. This indicates that the expression level of the gene truly reflects the risk of recurrence of metastasis. In fact, in vitro wound healing assay and mouse transplantation model, high invasiveness and high metastasis of MCF7-14 cells have been confirmed. In this example, BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 gene expression It was shown that the risk of metastatic recurrence can be evaluated by measuring the amount.
That is, when MCF7 cells are used as a negative control for breast cancer malignancy, MDA-MB-231 cells are used as a positive control for breast cancer malignancy, and MCF7 cells are evaluated for malignancy of breast cancer. The degree was judged to be high.
From the comparison of the expression of epithelial and mesenchymal markers, it is determined that the MCF7-14 cell is a cell with low malignancy of breast cancer, like the MCF7 cell, but according to the method for evaluating malignancy of breast cancer of the present invention, Similar to MDA-MB-231 cells, it was determined that the cells had high malignancy of breast cancer. This result was consistent with the results in the nude mouse transplantation model of MCF7 cells and MCF7-14 cells disclosed in WO2008 / 93886.
In this example, the expression level of the 6 gene genes was measured by measuring the amount of mRNA transcribed from the gene using a DNA microarray and quantitative RT-PCR. Any other method that can be used to assess expression levels can be used. Further, by measuring the expression level of the protein encoded by the gene, for example, it can be performed by an immunological measurement method such as an ELISA assay or an immunostaining method. Since both methods are used for clinical examinations of other diseases, the versatility is high, and the time from obtaining a sample to obtaining a result is within several days, and clinical application can be sufficiently expected. In addition, the present invention is a method for evaluating the malignancy of breast cancer based on the expression levels of at most six genes, which is more practical than other molecular marker groups and has a high-throughput prognosis of breast cancer. I can expect.
As described in the embodiment, the present invention can perform data analysis using general spreadsheet software or analysis software such as GeneSpring. In particular, the results obtained by the cluster analysis are easy to visually understand and are less likely to cause confusion in the prognosis of individual patients, and therefore are particularly useful when dealing with data from multiple samples simultaneously. In addition, these expression level data are accumulated in a database and are reused for cluster analysis, so that it is expected that the accuracy and reliability of prognosis can be further improved, and are considered to be clinically useful data. .

To date, treatment methods have been determined according to the histopathologically diagnosed tissue type, but because the accuracy of prognosis prediction is low, it may be necessary to perform treatment that may be unnecessary It is in. The present invention is considered to be a useful tool for early, appropriate and accurate assessment of breast cancer malignancy and determination of an optimal prognostic treatment policy for individual breast cancer patients.

Claims

A method for assessing the malignancy of breast cancer,
(1) measuring the expression levels of at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 in a test sample;
(2) evaluating the malignancy of breast cancer based on the expression pattern of the at least three genes.
The method according to claim 1, wherein (1) is a step of measuring the expression level of mRNA and / or polypeptide.
The method according to claim 2, wherein the expression level of the mRNA is measured using an array corresponding to the mRNA and on which nucleic acids and / or nucleic acid fragments are immobilized.
The method according to claim 2, wherein the expression level of the mRNA is measured using a primer and / or probe for detecting a nucleic acid and / or a nucleic acid fragment corresponding to the mRNA.
The method according to claim 2, wherein the expression level of the polypeptide is measured using an antibody and / or antibody fragment against the polypeptide and / or polypeptide fragment.
6. The method according to claim 1, wherein (1) is a step of measuring the expression level of the gene in the test sample and the expression level of the gene in a control sample.
The method according to claim 6, wherein the expression pattern comprises an expression ratio of the expression level of the gene.
The method according to claim 6, wherein the expression pattern comprises a relative expression level obtained from the expression level of the gene in the test sample with respect to the expression level of the gene in the control sample.
(2) is at least selected from the group consisting of a healthy individual, a non-lesion site, a low metastatic cancer cell line, and a tissue having a low metastatic cancer, wherein the gene expression pattern in the test sample is It is evaluated that the malignancy of breast cancer is low enough to approximate the expression pattern of the gene in one control sample, and / or the expression pattern of the gene in the test sample is a highly metastatic cancer cell line and a highly metastatic cancer. 9. The step of evaluating that the malignancy of breast cancer is higher as it approximates the expression pattern of the gene in at least one control sample selected from the group consisting of tissue-derived tissue. The method described in 1.
(2) is at least selected from the group consisting of a healthy individual, a non-lesion site, a low metastatic cancer cell line, and a tissue having a low metastatic cancer, wherein the gene expression pattern in the test sample is Compared to the gene expression pattern in one control sample,
(A) When an expression pattern of a gene in which the expression level of at least one gene selected from the group consisting of CD44 and SOCS2 is increased, and / or (B) from the group consisting of BMP7, CD24, CXCL12, and PIK3RI When the expression level of at least one selected gene shows a reduced gene expression pattern,
The method according to any one of claims 6 to 8, which is a step of evaluating that the malignancy of breast cancer is high.
The method according to claim 9 or 10, wherein the low metastatic cancer cell line is MCF7 cells.
The method according to claim 9, wherein the highly metastatic cancer cell line is MDA-MB-231 cells.
An array for assessing the grade of breast cancer,
An array in which nucleic acids and / or nucleic acid fragments corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 are immobilized on a defined region on a support.
An array for assessing the grade of breast cancer,
An array in which only nucleic acids and / or nucleic acid fragments corresponding to at least three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 are immobilized on a defined region on the support .
A kit for evaluating the malignancy of breast cancer,
Primers and / or probes for detecting nucleic acids and / or nucleic acid fragments corresponding to mRNAs and / or mRNA fragments of three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2 Contains a kit.
A kit for evaluating the malignancy of breast cancer,
A kit comprising an antibody and / or an antibody fragment against a polypeptide and / or polypeptide fragment encoded by three genes selected from the group consisting of BMP7, CD24, CD44, CXCL12, PIK3R1, and SOCS2.