WO2015115545A1 - Method for evaluating of metastasis or risk of recurrence of breast cancer - Google Patents

Abstract

Provided is a technique for objectively, rapidly and highly accurately evaluating the presence or absence of the effect of a medicine, the risk of recurrence or the possibility of metastasis in a chemical therapy for breast cancer. A method for evaluating the metastasis of breast cancer or the risk of recurrence of breast cancer in a breast cancer patient, said method comprising a step of measuring the level of expression of at least one expression product of DNA containing a transcription initiation domain in a biological sample derived from a cancer tissue separated from the patient.

Description

Methods for assessing the risk of breast cancer metastasis or recurrence

(Related applications and incorporation by reference)
This application claims the priority of Japanese Patent Application No. 2014-017946 filed on January 31, 2014, the entire contents of which are hereby incorporated by reference.
Also, all documents cited herein are incorporated by reference in their entirety for all purposes. Citation of any document should not be construed as an admission that it is prior art with respect to the present invention.

The present invention relates to a method for evaluating the risk of breast cancer metastasis or recurrence at the molecular level.

Breast cancer is classified into four types, Luminal A, Luminal B, HER2-positive and Triple-negative (TN), based on biological characteristics. There is standard treatment as a suitable treatment method for each type, but it is said that the prognosis and the effect of chemotherapy differ depending on the type. For example, even within the group that selected the same treatment method at the same type, stage, early Some patients relapse and others do not recur.

Until now, it has not been possible to determine whether it will recur after comprehensive treatment has been performed for breast cancer. Moreover, the predictive factor regarding a certain specific drug has not been identified and has not yet been clinically applied. In other words, for example, in preoperative chemotherapy, the corresponding disease type and stage are factors of a large indication, but if the disease type and stage are the same, they are usually all administered with the same drug (regimen). However, the current situation is that accurate treatment suitable for individual breast cancer has not been made. Until now, although there are multiple chemotherapy options for TN type breast cancer, there was no basis for determining which treatment should be selected.

In addition, Mammaprint (Agendia-DNA chip research institute) has been put into practical use as a test for research (Patent Document 1), which roughly measures postoperative recurrence risk in patients with early breast cancer using a 70-gene signature. Therefore, there is a demand for more detailed diagnosis and prediction of recurrence according to the presence or absence of chemotherapy and the type of breast cancer.

On the other hand, in recent years, gene expression analysis (expression) analysis), in which genes expressed in cells in a certain state are comprehensively analyzed by comparing gene expression states, and their types and expression levels are compared between cells. Techniques for have been developed. For example, RNA-seq (Non-Patent Document 1) and CAGE (Cap Analysis Gene Expression: Non-Patent Document 2) that comprehensively analyze the expression state of the gene at the transcription start site as sequence information are known. Among these, the CAGE method has a feature that the activity of the transcription start site can be comprehensively quantified by selecting a long capped RNA such as mRNA and sequencing the 5 'end randomly and in large quantities.

However, the relationship between the expression level of the transcription initiation region in the human genome and a specific disease has never been reported so far.

Japanese Patent No. 4222835

The present invention relates to providing a method for objectively and rapidly discriminating the presence / absence of the drug effect of chemotherapy in breast cancer, the risk of recurrence, or the possibility of metastasis with high accuracy.

The present inventors extract RNA from breast cancer tissues obtained by surgery with or without recurrence, and those with or without lymph node metastasis, and use the CAGE analysis method for transcription initiation region (Transcript). As a result of comprehensive analysis of the expression state in the vicinity of Start Site (TSS) as sequence information, the expression level of DNA containing a specific transcription start region is significantly different between the two groups, and using this as an index, “with relapse” And “no recurrence”, “with lymph node metastasis” and “without lymph node metastasis”.

That is, the present invention relates to the following 1) to 4).
1) For a biological sample derived from a cancer tissue isolated from a breast cancer patient, the method includes measuring the expression level of one or more expression products of DNA including a transcription initiation region, A method to assess the risk of recurrence.
2) It contains an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes the translation product of DNA, and is used for testing for evaluating the risk of metastasis or recurrence of breast cancer used in the method of 1) kit.
3) Use of one or more expression products of DNA containing a transcription initiation region as a marker for evaluating the risk of breast cancer metastasis or recurrence.
4) Breast cancer patients whose disease type is TN type and stage 2a or 2b, or living tissue derived from cancer tissue isolated from breast cancer patients whose disease type is HER2-positive type and stage 2a or 2b A method for evaluating the risk of metastasis or recurrence of breast cancer in a patient, comprising measuring the expression level of HLA-DQA1 RNA or protein for a sample.

ADVANTAGE OF THE INVENTION According to this invention, the drug effect prediction of chemotherapy and the presence or absence of recurrence can now be determined quickly and accurately, and high-risk patients can be identified with high accuracy. For example, it is expected to establish a treatment strategy that is appropriate for individual breast cancer, such as aggressive chemotherapy for breast cancer that is effective for chemotherapy, and preoperative surgery and change of regimen for breast cancer that is not effective for chemotherapy. .
Of the above-mentioned disease types, the TN type has a lower response rate and higher recurrence rate than the other three types, but by using the method of the present invention, the TN type has a biological basis. This will open the way to the selection of better treatments and the development of new treatments.
Further, according to the present invention, the same or higher classification can be objectively performed by using the present invention without depending on subjectivity of experts such as pathologists and clinical technologists who have been trained. Therefore, it can be suitably used for a test (POCT: Point of Care Testing) performed by a medical worker alongside a patient from collection to analysis of a patient specimen.

Image of immunohistochemistry. (Left) Recurrence / metastasis, Brown: HLA-DQA1-expressing cells, magnification: 200 times, (Right) No recurrence / metastasis, magnification: 200 times

In the present invention, breast cancer means a carcinoma that occurs in breast tissue. Breast cancer is classified into four types, Luminal A, Luminal B, HER2-positive, and Triple-negative (TN), based on biological characteristics, and is divided into 0 to 4 stages depending on the stage. .
In the present invention, metastasis means that breast cancer grows in lymph nodes and other organs, and the evaluation of metastasis is the presence or absence of metastasis or the ability to metastasize (the ability of breast cancer to metastasize and proliferate). It means to evaluate or measure the presence or absence. Preferable examples include evaluating or measuring lymph node metastasis.

In the present invention, “risk of recurrence” means the likelihood of recurrence. “Relapse” refers to a case in which a malignant tumor reappears at the same site after the tumor has been removed from the living body for a certain period of time, and when tumor cells are separated from the primary lesion and carried to a distant tissue where they proliferate autonomously. Refers to cases. Whether or not recurrence occurs depends on the proliferative ability, viability, and migration ability of tumor cells.
The evaluation method of the present invention is particularly useful for evaluating the risk of recurrence in patients who have not received neoadjuvant chemotherapy and in patients who have been treated with TN type and who have not received neoadjuvant chemotherapy. It is suitable for evaluating lymph node metastasis of patients with stage 2a or 2b and patients with type HER2-positive type and stage 2a or 2b.
Here, the TN type disease type is an estrogen receptor negative, progesterone receptor negative and HER2 negative type in immunopathological examination, and the HER2-positive type disease type is HER2 in immunopathological examination. Is a type that is overexpressed.
Stage 2a refers to a tumor having a maximum tumor diameter of 2 cm or less and 1 to 3 regional lymph node metastases (including at least one tumor having a maximum diameter exceeding 2 mm), or a maximum tumor diameter of 2 cm. 2b or less and no regional lymph node metastasis. 2b means that the maximum diameter of the tumor exceeds 2 cm and is 5 cm or less and 1 to 3 regional lymph node metastases (maximum diameter is 0.2 mm or more) Or a tumor with a maximum diameter of more than 5 cm and no regional lymph node metastasis.

The biological sample used in the present invention is a breast cancer tissue separated from a breast cancer patient to be evaluated. The biological sample is appropriately prepared and processed for use in measurement. For example, when the sample is subjected to measurement at the nucleic acid level, an RNA extract is prepared, and when the sample is subjected to measurement at the protein level, a protein extract is prepared.
Any known method can be used as a method for extracting RNA from a biological sample. Specific examples include Ambion RiboPure kit manufactured by Life Technologies, miRNeasy manufactured by Qiagen, and RNeasy manufactured by Qiagen. Among these, the miRNeasy kit manufactured by Qiagen is preferably used.

In the present specification, the term “nucleic acid” or “polynucleotide” means DNA or RNA. In addition, “DNA” includes not only double-stranded DNA but also single-stranded DNAs comprising a sense strand and an antisense strand constituting the DNA. Accordingly, the DNA includes double-stranded genomic DNA, single-stranded cDNA, single-stranded DNA having a sequence complementary to the DNA, and the like. “RNA” includes any of total RNA, mRNA, rRNA, and synthetic RNA.

In the present invention, the transcription product of DNA consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 199 (human genomic DNA consisting of a transcription initiation region and 100 bases continuous downstream thereof) is 1) preoperative chemistry as shown in the Examples. Relapsed and non-recurrent specimens in breast cancer patients who received postoperative chemotherapy without therapy 2) Recurrence occurred in breast cancer patients who were TN type and did not receive preoperative chemotherapy Specimen with no recurrence 3) Breast cancer patient with stage TN type and stage 2a or 2b, specimen without lymph node metastasis and specimen with lymph node metastasis 4) Disease type HER2 -For patients with positive type and stage 2a or 2b, CAGE (Cap As a result of comprehensive analysis of the expression state of DNA of 100 bases or more downstream including the transcription initiation region on the genome using the Analysis Gene Expression) analysis method, the expression level (transcription activity) is significant between the two groups. The difference is recognized. Specifically, the RNA transcriptional activity of the specimens 1) to 4) is obtained from the clinical specimen-derived profile group obtained from “with recurrence” or “with metastasis”, “without recurrence” or “without metastasis”. The R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40) was used for differential analysis between the clinical specimen-derived profile groups, and a 5% FDR (false discovery rate) was set as a threshold. It has been extracted.
Therefore, a DNA consisting of a base at an arbitrary position (transcription start point) in the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 199 and one or more consecutive bases downstream thereof (hereinafter referred to as “transcription in SEQ ID NOs: 1 to 199”). The expression product (referred to as “the expression product of the present invention”) (referred to as “the DNA containing the starting point”) (or encoded thereby) can be a biomarker for assessing the risk of breast cancer metastasis or recurrence.

More details are as follows.
1) Expression product of DNA containing the transcription start site in SEQ ID NOs: 1-32 A biomarker for evaluating the risk of recurrence in breast cancer patients who have not undergone preoperative chemotherapy.
Among these, the DNA expression product containing the transcription start point in SEQ ID NOs: 1 to 5 is a marker whose expression level increases when the risk of recurrence is high, and the DNA expression product containing the transcription start point in SEQ ID NOs: 6 to 32 is It is a marker that decreases the expression level when the risk of recurrence is high.
2) A biomarker for evaluating the risk of recurrence in breast cancer patients whose expression product disease type of DNA containing the transcription start site in SEQ ID NOs: 33 to 107 is TN type and who have not undergone preoperative chemotherapy.
The DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
3) A biomarker for evaluating lymph node metastasis in a breast cancer patient whose expression product type of DNA containing the transcription start site in SEQ ID NOs: 108 to 177 is TN type and stage is 2a or 2b.
Among these, the DNA expression product containing the transcription start point in SEQ ID NOs: 108 to 164 is a marker whose expression level increases when the possibility of lymph node metastasis is high, and the DNA containing the transcription start point in SEQ ID NOs: 165 to 177 The expression product is a marker that decreases the expression level when the possibility of lymph node metastasis is high.
4) A biomarker for evaluating lymph node metastasis in breast cancer patients whose expression product type of DNA containing the transcription start site in SEQ ID NOs: 178 to 199 is HER2-positive type and stage is 2a or 2b.
Among these, the DNA expression product containing the transcription start point in SEQ ID NOs: 178 to 184 is a marker whose expression level increases when the possibility of lymph node metastasis is high, and the DNA containing the transcription start point in SEQ ID NOs: 185 to 199 The expression product is a marker that decreases the expression level when the possibility of lymph node metastasis is high.

In the present invention, the “transfer start region” refers to a region including a transfer start point. The transcription start point from a specific promoter is not limited to a single base, and may be a base present at a plurality of positions downstream of the promoter on the genome. A region including a plurality of these transfer start points is referred to as a transfer start region in this specification. More specifically, the transfer start area is an area between the transfer start point located on the most 5 ′ side and the transfer start point located on the most 3 ′ side among the plurality of transfer start points. In each of the base sequences represented by SEQ ID NOs: 1 to 199, the transcription initiation region has a 5 ′ end corresponding to a region defined at both ends by the base at position 1 (5 ′ end) and the 101st base from the 3 ′ end. This is a base region to be formed. In other words, each of the base sequences represented by SEQ ID NOs: 1 to 199 shows a transcription start region and 100 bases following the transcription start point located on the most 3 ′ side in the transcription start region. In the present specification, such a transcription initiation region is also referred to as “a transcription initiation region represented by SEQ ID NOs: 1 to 199”.
The positions of the transcription start regions shown in SEQ ID NOs: 1 to 199 on the genome, and related gene information, etc. are described in Tables 1-1 to 1-2, Tables 2-1 to 2-3, and Tables 3-1 to 3 below. -3 and Table 4.

In the present invention, the DNA for which the expression level of the expression product is measured is the base sequence at any position (transcription start point) in the transcription start region and the downstream 1 in the base sequence represented by SEQ ID NOs: 1 to 199. It is DNA consisting of a base sequence of bases or more.
Here, the number of bases in the downstream base sequence may be any number that can identify the expression product. Examples of the number of bases include 1 base or more, 5 bases or more, 10 bases or more, 15 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, 40 bases or more, 50 bases or more. Moreover, as the said base number, 10 bases or less, 15 bases or less, 20 bases or less, 25 bases or less, 30 bases or less, 40 bases or less, 50 bases or less, 100 bases or less are mentioned, for example.
The downstream base is not particularly necessary in the case of measurement by the CAGE method, but in the case of measurement by hybridization or PCR, any part up to about 100 bases downstream is targeted in order to ensure the accuracy. In addition, if the DNA has a length of at least 20 bases out of DNA consisting of the transcription start region and 100 bases downstream from it, there is a high probability that it can be identified even in an experimental system for the entire genome.
The DNA also includes DNA having a base sequence substantially identical to the base sequence of the DNA as long as the expression product can be a biomarker for evaluating the risk of breast cancer metastasis or recurrence. Here, the substantially identical base sequence is, for example, using the homology calculation algorithm NCBI BLAST and expecting value = 10; allowing a gap; filtering = ON; match score = 1; mismatch score = −3 When searching, it means that there is 90% or more, preferably 95% or more, even more preferably 98% or more identity with the base sequence shown in SEQ ID NOs: 1 to 199.

Such expression products of the present invention can be used to assess the risk of breast cancer metastasis or recurrence by appropriately combining one or more of the expression products and grasping the expression level.
Among these, when the threshold values shown in Tables 5 to 7 below are set, DNA that can be classified with specificity 100% and sensitivity 100%, that is, DNA that can be reliably discriminated only with one expression level, The following are mentioned.

1) An expression product of DNA containing the transcription start point in SEQ ID NO: 29 in the assessment of recurrence risk of breast cancer patients who have not undergone preoperative chemotherapy.
3) An expression product of DNA containing the transcription start point in SEQ ID NO: 150 or SEQ ID NO: 147 in the evaluation of lymph node metastasis of a breast cancer patient whose disease type is TN type and stage is 2a or 2b.
4) SEQ ID NO: 194, SEQ ID NO: 187, SEQ ID NO: 186, SEQ ID NO: 192, or SEQ ID NO: 193 in the evaluation of lymph node metastasis of a breast cancer patient whose disease type is HER2-positive type and stage is 2a or 2b An expression product of DNA containing the transcription start site in FIG.

In addition, a suitable combination in the case where at least two DNA expression products are used, that is, for all two combinations selected from the above transcription start regions, these expression levels are used as explanatory variables, and recurrence related to the transcription start region extraction sample. Alternatively, a logistic regression model that estimates the presence or absence of lymph node metastasis is constructed, and the DNA shown in Tables 8 to 11 below can be classified as 100% specificity and 100% sensitivity for both the transcription start region extraction sample and the verification sample. The expression products are listed respectively.
For the purpose of further improving the accuracy, these can be used in combination of two sets or three sets or more as appropriate. Further, in addition to the combination of the expression products of these two DNAs, it may be combined with the expression products of DNAs other than those shown as the combination among the DNAs containing the transcription start sites in SEQ ID NOs: 1 to 199, May be combined with DNA expression products consisting of any other base sequence within a range that can contribute to the evaluation of the present invention.

The expression product of the present invention includes a transcription product and a translation product expressed from the DNA. Specific examples of the transcription product include RNA generated by transcription from the DNA, preferably mRNA. Specific examples of the translation product include a protein encoded by the RNA. For example, among the DNA expression products containing the transcription start sites shown in Tables 1 to 4, the protein expressed from the DNA containing the transcription start sites in SEQ ID NO: 112 and SEQ ID NO: 196 is “HLA-DQA1” (Major Histocompatibility Complex, Class II, DQ Alpha 1; UniProtKB / Swiss-Prot: DQA1_HUMAN, P01909).
The target of the measurement or detection of the expression product is cDNA artificially synthesized from the RNA, DNA encoding the RNA, protein encoded by the RNA, molecule interacting with the protein, and interaction with the RNA. Also included are molecules that act or molecules that interact with the DNA. Here, as molecules that interact with RNA, DNA or protein, DNA, RNA, protein, polysaccharide, oligosaccharide, monosaccharide, lipid, fatty acid, and their phosphates, alkylated products, sugar adducts, and the like, and Any of the above-mentioned complexes can be mentioned.
The expression level comprehensively means the expression level and activity of the expression product.

When RNA, cDNA or DNA is targeted as a method for measuring the expression level, nucleic acid amplification represented by PCR method, real-time RT-PCR method, SmartAmp method, LAMP method, etc. using DNA hybridizing to these primers Method, hybridization method using nucleic acids that hybridize to these as probes (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, northern blot, hybridization, etc.), method for determining the base sequence, or these You can choose from a combination of methods.

Here, the probe or primer used for the measurement, that is, the primer for specifically recognizing and amplifying the expression product (transcription product) of the present invention or the nucleic acid derived therefrom, or the RNA or the nucleic acid derived therefrom is specific. This corresponds to the probes for detection in the above, and these can be designed based on the nucleotide sequences represented by SEQ ID NOs: 1 to 199. Here, “specifically recognize” means that, for example, in the Northern blot method, substantially only the expression product (transcription product) of the present invention or a nucleic acid derived therefrom can be detected. This means that the detection product or product can be determined to be the transcription product or a nucleic acid derived therefrom so that only the nucleic acid is produced.
Specifically, DNA comprising a base sequence represented by SEQ ID NOs: 1 to 199 or an oligonucleotide containing a certain number of nucleotides complementary to its complementary strand can be used. Here, the “complementary strand” refers to the other strand with respect to one strand of double-stranded DNA comprising A: T (U in the case of RNA) and G: C base pairs. In addition, “complementary” is not limited to the case where the certain number of consecutive nucleotide regions are completely complementary sequences, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more. What is necessary is just to have the identity on arrangement | sequence. The identity of the base sequence can be determined by the algorithm such as BLAST.
When such an oligonucleotide is used as a primer, it only needs to be capable of specific annealing and chain extension, and is usually 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and for example 100 bases or less. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are mentioned. Further, when used as a probe, it only needs to be capable of specific hybridization, and has at least part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence represented by SEQ ID NOs: 1 to 199, for example, A chain length of 10 bases or more, preferably 15 bases or more and, for example, 100 bases or less, preferably 50 bases or less, more preferably 25 bases or less is used.
Here, the “oligonucleotide” may be DNA or RNA, and may be synthesized or natural. Alternatively, the probe used for hybridization is usually labeled.

For example, when using the Northern blot hybridization method, the probe DNA is first labeled with a radioisotope, a fluorescent substance, etc., and then the obtained labeled DNA is transferred to a nylon membrane or the like according to a conventional method. Hybridize with RNA. Thereafter, a method of detecting and measuring a signal derived from the labeled product of the formed duplex of labeled DNA and RNA can be used.

When using the RT-PCR method, first, cDNA is prepared from RNA derived from a biological sample according to a conventional method so that the target expression product of the present invention (in this case, transcription product) can be amplified. A pair of prepared primers (a normal strand that binds to the cDNA (− strand) and a reverse strand that binds to the + strand) are hybridized therewith. Thereafter, PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected. For detection of the amplified double-stranded DNA, use a method of detecting the labeled double-stranded DNA produced by performing the above PCR using a primer previously labeled with RI, a fluorescent substance, etc. Can do.

When measuring the expression level of mRNA in a sample using a DNA microarray, at least one nucleic acid (cDNA or DNA) derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support. By using an array, labeled cDNA or cRNA prepared from mRNA is bound on the microarray, and the label on the microarray is detected, whereby the mRNA expression level can be measured.
The nucleic acid immobilized on the array may be any nucleic acid that hybridizes specifically (ie, substantially only to the target nucleic acid) under stringent conditions. For example, the expression product of the present invention (transcription) The product may be a nucleic acid having the entire sequence or a partial sequence. Here, the “partial sequence” includes a nucleic acid consisting of at least 15 to 25 bases.
Here, stringent conditions can usually include washing conditions of about “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent hybridization conditions include “0.5 × SSC, 0.1%. As a more stringent hybridization condition, a condition of “0.1 × SSC, 0.1% SDS, 65 ° C.” can be mentioned. Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001) and the like.

Examples of the method for determining the base sequence include the CAGE method, the TSS-seq method, the RNA-seq method, the DGE method, and the SAGE method, and the CAGE method is preferable.
When the expression level is measured using the CAGE method, it can be carried out in accordance with the method described in Examples below.

In addition, a protein (ie, translation product) encoded by DNA containing the transcription start site in SEQ ID NOs: 1 to 199, a molecule that interacts with the protein, a molecule that interacts with RNA, or a molecule that interacts with DNA is measured. In this case, protein chip analysis, immunoassay (for example, ELISA etc.), 1-hybrid method (PNAS 100, 12271-12276 (2003)) and 2-hybrid method (Biol. Reprod. 58, 302-311 (1998) )) Can be used and can be appropriately selected depending on the target.
For example, when a protein is used as a measurement target, an antibody against the expression product of the present invention (in this case, a translation product) is brought into contact with a biological sample, the polypeptide in the sample bound to the antibody is detected, and the level is detected. This is done by measuring. For example, according to Western blotting, after using the above-described antibody as a primary antibody, an antibody that binds to a primary antibody labeled with a radioisotope, a fluorescent substance, an enzyme, or the like as a secondary antibody is used. Labeling is performed, and signals derived from these labeling substances are measured with a radiation measuring instrument, a fluorescence detector or the like.

The antibody against the translation product may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a known method. Specifically, the polyclonal antibody is used to immunize non-human animals such as rabbits by using a protein expressed and purified in E. coli or the like according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method.
On the other hand, a monoclonal antibody is obtained by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from the prepared hybridoma cells.
When immunohistochemical analysis is performed, a biological sample isolated from a patient is fixed in formalin by a conventional method, embedded in paraffin, sliced into tissue pieces, and pasted on a slide glass. It is preferably used as a section sample. Further, a biological sample separated from a patient can be frozen immediately, sliced into tissue pieces, attached to a slide glass, and fixed in formalin, and used as a section sample. As the secondary antibody, an enzyme-labeled antibody such as alkaline phosphatase or peroxidase can be used, but highly sensitive detection is performed using a three-step method such as ABC method or LSAB method, or the DAVision EnVision detection system. Is preferred.

Thus, the expression level of the expression product of the present invention in a biological sample derived from a cancer tissue separated from a breast cancer patient is measured, and the risk of breast cancer metastasis or recurrence is evaluated based on the expression level. Specifically, the expression level of the detected expression product of the present invention is evaluated by comparing it with a control level.
Here, the “control level” refers to, for example, the expression level of the expression product in a cancer tissue isolated from a breast cancer patient without metastasis or recurrence or a normal tissue isolated from a breast cancer patient, or breast cancer has not developed. The expression level of the expression product in a group of healthy people can be mentioned.
For example, the expression level of the expression product of the target patient's cancer tissue is close to the expression level in a cancer tissue, normal tissue, or tissue derived from a healthy person isolated from a breast cancer patient who has not metastasized or recurred. If it falls within the range or is significantly higher (or lower) than the expression level, the patient's breast cancer can be evaluated as having no metastasis, low metastatic potential, or low risk of recurrence.

Also, the risk of breast cancer metastasis or recurrence in the present invention can be evaluated by increasing / decreasing the expression level of the expression product of the present invention. In this case, as a control level, for example, a standard value (threshold level) is set based on the expression level of the expression product derived from a normal tissue, a cancer tissue separated from a breast cancer patient without metastasis or recurrence, or a healthy tissue. It can be carried out by setting and comparing the expression level of the expression product in the patient-derived biological sample with a standard value (for example, the range of ± 2SD is allowed). For example, when the expression level of the expression product in a patient-derived biological sample is higher (or lower) than a threshold level, the patient's breast cancer can be evaluated as having no metastasis, low metastatic potential, or low recurrence risk.

In accordance with the method of the present invention, the risk of breast cancer metastasis or recurrence is evaluated based on the provided information in combination with other methods (CT, MRI, PET-CT, etc.) as necessary. If it is determined that there is a possibility of metastasis or recurrence, for example, chemotherapy and / or molecular targeted therapy, or radiation therapy or endocrine therapy can be performed. On the other hand, when it is determined that the possibility of metastasis or recurrence is low, it is considered unnecessary to perform these treatments.

The test kit for evaluating the risk of metastasis or recurrence of breast cancer of the present invention contains a test reagent for measuring the expression level of the expression product of the present invention in a biological sample separated from a patient. Specifically, a reagent for nucleic acid amplification or hybridization containing an oligonucleotide that specifically binds (hybridizes) to the expression product (transcription product) or the like of the present invention, or the expression product (translation product) of the present invention. And a reagent for immunological measurement including an antibody that recognizes). Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above.
In addition to the antibody and nucleic acid, the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.

Example 1 Extraction and verification of transcription start region for discriminating between “with recurrence” and “without recurrence”, “with lymph node metastasis” and “without lymph node metastasis” in breast cancer

(1) Obtaining a specimen sample A part of the cancer tissue is removed from the tissue excised by breast cancer surgery, immediately placed in a microtube, frozen in liquid nitrogen, stored in an ultra-low temperature bath at -80 ℃, and analyzed as appropriate. Used for. The samples used are as follows.
<1: Breast cancer patients who received postoperative chemotherapy without preoperative chemotherapy>
10 samples for transcription start region extraction (no recurrence: 5 specimens, recurrence: 5 specimens)
・ Verification sample 4 specimens (no recurrence: 2 specimens, recurrence: 2 specimens)
<2: Breast cancer patients who are TN type and have not undergone preoperative chemotherapy>
・ 16 samples for extracting transcription start region (no recurrence: 14 specimens, recurrence: 2 specimens)
・ Verification sample 3 specimens (no recurrence: 2 specimens, recurrence: 1 specimen)
<3: Breast cancer patient whose disease type is TN type and stage is 2a or 2b>
・ 10 samples for extraction of transcription start region (no lymph node metastasis: 6 samples, lymph node metastasis: 4 samples)
・ Sample 3 sample for verification (no lymph node metastasis: 2 samples, with lymph node metastasis: 1 sample)
<4: Breast cancer patient whose disease type is HER2-positive type and stage is 2a or 2b>
・ Sample 6 for extracting transcription start region (no lymph node metastasis: 3 samples, lymph node metastasis: 3 samples)
・ Sample 3 sample for verification (no lymph node metastasis: 2 samples, with lymph node metastasis: 1 sample)

(2) Storage and preparation of sample The extracted tissue pieces were appropriately frozen and stored at -80 ° C. The preserved tissue pieces were wrapped in aluminum foil in a frozen state, further subjected to freezing treatment with liquid nitrogen, and then crushed with a rubber hammer. The crushed sample was put in a 2 mL tube so as to be 50 mg or less, QIAzol manufactured by Qiagen was added and sealed, and crushed by osmosis treatment using TissueLyser manufactured by Qiagen.

(3) Preparation of RNA The sample subjected to the crushing and extraction treatment was prepared for RNA according to the attached protocol using miRNeasy mini kit manufactured by Qiagen. The prepared RNA was measured for ultraviolet absorption (230, 260, 280 nm) with a spectrophotometer to calculate 260/230, 260/280 ratios, and the quality of the RNA was tested. In addition, electrophoresis was performed using BioAnalyzer RNA nano chip manufactured by Agilent, and the RIN value indicating the degree of RNA degradation was calculated to test the degree of RNA degradation.

(4) CAGE library preparation 5 μg of purified RNA was prepared, and unamplified untagged CAGE method (“Cell Engineering, separate volume, Advanced Method for Next-Generation Sequencer Purpose”, Juno Kanno, Satoshi Suzuki, Gakken Medical Shujunsha, 2012 The CAGE library was prepared by “Chapter 3“ Exhaustive promoter analysis (non-amplified CAGE method using Illumina sequencer) ”in“ Issue 19 Sep ”. Specifically, the purified RNA was subjected to reverse transcription reaction and purified, and then aldehyde was formed by participation of a ribose diol with sodium periodate, and biotin hydrazide was added to add biotin to the aldehyde group. After digesting and purifying the single-stranded RNA portion with RNase I, only the RNA / cDNA double strand biotinylated with avidin magnetic beads was bound to the bead surface, and the cDNA was released and recovered by RNase H digestion and heat treatment. After adapters necessary for sequencing were connected to both ends of the collected cDNA, sequencing was performed using HiSeq 2500 manufactured by Illumina. The standard conditions of AMPure XP (manufactured by Beckman Coulter) used for purification, buffer replacement, etc. in this step are conditions for recovering nucleic acids having a length of 100 bases or more in the case of double strands. The CAGE library produced by this process using this is composed of double-stranded DNA having a chain length of 100 bases or more.

(5) RNA expression analysis i) Preparation of reference transcription initiation region Project to profile the activity of transcription initiation sites measured in genome-wide for a total of about 1000 human samples including human primary cultured cells, cell lines, and tissues. Among the transcription initiation regions identified in “FANTOM5” (submitting paper), about 180,000 transcription initiation regions defined on the human reference genome hg19 were used as reference transcription initiation regions.

ii) Quantification of transcriptional activity The reads obtained by sequencing and the human reference genome (hg19) were aligned using bwa (Bioinformatics. 2009 Jul 15; 25 (14): 1754-60.). Only alignments with a mapping quality of 20 or more and an alignment start position within the reference transcription start region were selected, and the number of reads for each transcription start region was counted. Using the total number of reads for each library and the library size estimated by the RLE (Genome Biol. 2010; 11 (10): R106.) Method, the count is converted to counts per million. To do.

(6) Results (A) Extraction of transcription start regions with different activities Regarding the transcriptional activity in each sample for transcription start region extraction / evaluation quantified above, “with recurrence” and “without recurrence” or “lymph node” The R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40) was used for differential analysis between the clinical specimen-derived profile groups obtained from “with metastasis” and “without lymph node metastasis”. Used. In other words, whether the average expression level is different between the two groups (if the average hypothesis is equal to the null hypothesis and this null hypothesis is true, the probability that the measurement result will occur by chance Is calculated statistically. When FDR (false discovery rate) 5% was set as a threshold, DNAs containing transcription initiation regions smaller than this were identified (Tables 1-1 to 1-2, Tables 2-1 to 2-3, Table 3). -1 to 3-3 and Table 4). This criterion is statistically estimated that 95% of the candidates extracted by the corresponding threshold are truly differential in expression, and the commonly used P value (assuming there is no differential expression) This is a stricter standard than when the probability of accidental occurrence is 5%.

(B) Selection of transfer start region for high-precision prediction (1)
Consider whether it is possible to classify the presence or absence of metastasis or the presence or absence of recurrence using only one expression level among the transcription initiation regions identified in (A) above. By setting some threshold for each, it was confirmed that both the transcription start region extraction sample and the verification sample can be classified with 100% specificity and 100% sensitivity. Examples of the threshold values are shown below (when the maximum value in one group is smaller than the minimum value in the other group, the average of these values is shown in Tables 5 to 7).

1: DNA for assessing the risk of recurrence in breast cancer patients who have not undergone preoperative chemotherapy

3: DNA for evaluating lymph node metastasis of breast cancer patients whose disease type is TN type and stage is 2a or 2b

4: DNA for evaluation of lymph node metastasis in breast cancer patients whose disease type is HER2-positive type and stage is 2a or 2b

(C) Selection of transfer start region for performing highly accurate prediction (2)
It is considered whether the presence or absence of metastasis or the presence or absence of recurrence can be classified using a plurality of the expression levels of the transcription initiation region identified above. As an example, consider the construction of a logistic regression model, which is a kind of generalized linear model. This is to probabilistically predict the dependent variable (Y) indicating the presence or absence of recurrence or lymph node metastasis with an explanatory variable (Xi, logarithm of the above counts per million) that is the expression level of the transcription initiation region. Is one of the simplest models.

For all two combinations selected from the above transcription start regions, using these expression levels as explanatory variables, construct a logistic regression model to estimate the presence or absence of recurrence or lymph node metastasis for the sample for transcription start region extraction, A sample that can be classified with 100% specificity and 100% sensitivity was selected for both the transcription start region extraction sample and the verification sample (Tables 8 to 11).
Here, the logistic regression model, which is one of the simplest of machine learning devices, is used, but the method of measuring the expression of the transcription start region, the expression level and genotype of other genes, etc. By using other mathematical models appropriately by combination or the like, more robust prediction is possible.

1: DNA for assessing the risk of recurrence in breast cancer patients who have not undergone preoperative chemotherapy

2: DNA for assessing the risk of recurrence in breast cancer patients whose type is TN and who have not undergone preoperative chemotherapy

3: DNA for evaluating lymph node metastasis of breast cancer patients whose disease type is TN type and stage is 2a or 2b

4: DNA for evaluation of lymph node metastasis in breast cancer patients whose disease type is HER2-positive type and stage is 2a or 2b

Example 2 Discrimination of the risk of metastasis or recurrence using HLA-DQA1 as a marker (1) Specimen Patients who received comprehensive consent before surgery (breast cancer patients whose disease type is TN type and stage is 2a or 2b) ) Surgical specimens that have been cryopreserved in 3) have been diagnosed as having recurrence (with lymph node metastasis during surgery) or without recurrence (no lymph node metastasis during surgery) 3 years after surgery It was used.

(2) Detection of protein by immunochemical staining A biological sample separated from a patient was quickly frozen, sliced into tissue pieces, attached to a slide glass and fixed in formalin to obtain a section sample. Subsequently, an anti-HLA-DQA1 antibody (abcam, “ab128959”) (primary antibody, concentration 0.5 μg / mL) was reacted for 1 hour (20 ° C.). After sufficiently washing with a buffer solution, an HRP-labeled anti-rabbit antibody (Nichirei Biosciences # 424141) was used as a secondary antibody and reacted for 30 minutes (20 ° C.). After thoroughly washing with a buffer solution, color was developed using DAB. Cell nuclei were weakly stained with hematoxylin, and the specimens were observed for positive and negative under a light microscope. An example is shown in FIG.
As a result, it was confirmed that the staining pattern was different between relapse / metastasis / no relapse / metastasis, and that both could be distinguished using HLA-DQA1 as a marker.

According to the present invention, it is possible to objectively determine and predict the risk of breast cancer metastasis or recurrence by examining the primary lesion of breast cancer collected before or during surgery. This can be expected to contribute to the medical field and clinical laboratory field.

Claims (22)

1. A risk of metastasis or recurrence of breast cancer of a patient, including the step of measuring the expression level of one or more expression products of DNA including a transcription initiation region for a biological sample derived from a cancer tissue isolated from a breast cancer patient How to evaluate.
2. The DNA is a DNA comprising a base at an arbitrary position of a transcription initiation region in the base sequence represented by SEQ ID NOs: 1 to 199 and at least one base continuous downstream thereof, and the transcription initiation region is represented by SEQ ID NOs: 1 to The method according to claim 1, wherein both ends are defined by the first base of the base sequence represented by 199 and the 101st base from the 3 ′ end.
3. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 107 and at least one base continuous downstream thereof, wherein the transcription start region is represented by SEQ ID NOs: 1 to The method according to claim 1 or 2, wherein the risk of recurrence of breast cancer, which is a region defined at both ends by the first base of the base sequence represented by 107 and the 101st base from the 3 'end, is evaluated.
4. The DNA is a DNA comprising a base at an arbitrary position of a transcription start region in the base sequence represented by SEQ ID NOs: 108 to 199 and at least one base continuous downstream thereof, and the transcription start region is SEQ ID NO: 108 The method according to claim 1 or 2, wherein the lymph node metastasis of breast cancer, which is a region defined at both ends by the first base of the base sequence represented by ~ 199 and the 101st base from the 3 'end, is evaluated.
5. The DNA is a DNA comprising a base at an arbitrary position of a transcription start region in the base sequence represented by SEQ ID NOs: 1 to 32 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 1 to Evaluating the risk of recurrence of breast cancer in a breast cancer patient who has not undergone preoperative chemotherapy, which is a region defined at both ends by the first base of the base sequence represented by 32 and the 101st base from the 3 ′ end, Item 4. The method according to Item 3.
6. The DNA is a DNA comprising a base at an arbitrary position of the transcription start region in the base sequence represented by SEQ ID NOs: 33 to 107 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 33 to 33 Breast cancer in a breast cancer patient whose disease type is TN type and not undergoing preoperative chemotherapy, which is a region defined at both ends by the first base of the base sequence represented by 107 and the 101st base from the 3 ′ end The method according to claim 3, wherein the risk of recurrence is evaluated.
7. The DNA is a DNA comprising a base at an arbitrary position in the transcription start region in the base sequence represented by SEQ ID NOs: 108 to 177 and at least one base continuous downstream thereof, and the transcription start region is represented by SEQ ID NOs: 108 to 108 177 is a region defined at both ends by the first base of the base sequence shown by 177 and the 101st base from the 3 ′ end, and is a type of TN type and stage 2a or 2b. The method of claim 4, wherein lymph node metastasis is evaluated.
8. The DNA is a DNA comprising a base at an arbitrary position of a transcription initiation region in the base sequence represented by SEQ ID NOs: 178 to 199 and at least one base continuous downstream thereof, and the transcription initiation region comprises SEQ ID NOs: 178 to 178 In a breast cancer patient whose disease type is HER2-positive type and stage is 2a or 2b, which is defined by the first base of the base sequence shown in 199 and the 101st base from the 3 'end. The method according to claim 4, wherein lymph node metastasis of breast cancer is evaluated.
9. 6. The method according to claim 5, wherein the base sequence represented by SEQ ID NO: 1 to 32 is the base sequence represented by SEQ ID NO: 29.
10. The method according to claim 7, wherein the base sequences represented by SEQ ID NOs: 108 to 177 are the base sequences represented by SEQ ID NO: 150 and SEQ ID NO: 147.
11. The method according to claim 8, wherein the nucleotide sequences represented by SEQ ID NOs: 178 to 199 are the nucleotide sequences represented by SEQ ID NO: 194, SEQ ID NO: 187, SEQ ID NO: 186, SEQ ID NO: 192, and SEQ ID NO: 193.
12. 6. The method according to claim 5, wherein the base sequences represented by SEQ ID NOs: 1 to 32 are combinations of the base sequences represented by the following sequence numbers 1) to 10).
    

    
13. The method according to claim 6, wherein the base sequences represented by SEQ ID NOs: 33 to 107 are combinations of base sequences represented by SEQ ID NOs: 1) to 34) below.
    

    
14. The method according to claim 7, wherein the base sequences represented by SEQ ID NOs: 108 to 177 are combinations of the base sequences represented by SEQ ID NOs: 1) to 24) below.
    

    
15. The method according to claim 8, wherein the base sequence represented by SEQ ID NO: 178 to 199 is a combination of the base sequences represented by SEQ ID NO: 1) below.
    

    
16. The method according to any one of claims 1 to 15, further comprising a step of comparing the expression level of the expression product of the DNA with a control level.
17. The method according to any one of claims 1 to 15, further comprising a step of comparing the expression level of the expression product of the DNA with a threshold level.
18. The method according to any one of claims 1 to 17, wherein the expression level of the expression product is measured by measuring the amount of the transcription product or the amount of the translation product.
19. The risk of metastasis or recurrence of breast cancer used in the method according to any one of claims 1 to 18, comprising an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes a translation of the DNA. Test kit for assessing.
20. Use of one or more expression products of DNA including the transcription initiation region as a marker for evaluating the risk of breast cancer metastasis or recurrence.
21. The DNA is a DNA comprising a base at an arbitrary position in the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 199 and one or more bases continuous downstream thereof,
    The use according to claim 20, wherein the transcription initiation region is a region defined at both ends by the first base of the base sequence represented by SEQ ID NOs: 1 to 199 and the 101st base from the 3 'end.
22. For breast cancer patients whose disease type is TN type and stage 2a or 2b, or from biological tissue derived from breast cancer patients whose disease type is HER2-positive type and stage 2a or 2b A method for evaluating the risk of breast cancer metastasis or recurrence of the patient, comprising the step of measuring the expression level of HLA-DQA1 RNA or protein.
    

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