JP2012085554A - Method for discriminating subtype of breast cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a marker for detecting triple negative breast cancer; to provide a method for detecting or diagnosing the triple negative breast cancer with the marker; and to provide a kit for detecting or diagnosing the triple negative breast cancer.SOLUTION: SCRG1 gene which is low in expression level in Luminal A, Luminal B and HER2 diseases and rises the expression level in the triple negative breast cancer on the other hand among the sub-types of breast cancer, and a polypeptide encoded by the gene are used as markers for detecting the triple negative breast cancer. A probe for detecting the triple negative breast cancer marker gene and an antibody for the polypeptide encoded by the gene are used. Thus, the marker for detecting the triple negative breast cancer can be detected, and the triple negative breast cancer can quickly and surely be detected or diagnosed.

Description

  The present invention relates to a marker for detecting triple negative breast cancer, in particular, a marker for detecting triple negative breast cancer comprising a gene and a polypeptide specifically expressed in triple negative breast cancer, and a method for detecting or diagnosing triple negative breast cancer using the marker.

  Breast cancer is one of the most common cancers in Japanese women and is a complex disease characterized by mutations and changes in expression levels in many genes (Non-patent Document 1).

  Breast cancer treatment is a method of selecting from treatment methods such as local radiation therapy, cytotoxic anticancer agents, and hormonal therapeutic agents in addition to conventional surgery in accordance with the elucidation of the biological characteristics of breast cancer. It has changed to. Among them, hormone therapy plays a central role in breast cancer treatment as a molecular target treatment. In particular, tamoxifen, an anti-estrogen agent, antagonistically inhibits the binding between estrogen, a growth factor, and estrogen receptor (ER), and is widely used for post-relapse treatment, post-operative treatment, and pre-operative treatment. From this point of view, breast cancer has been classified according to the presence or absence of estrogen receptor (ER) expression (Non-patent Document 2).

  On the other hand, in the late 1980s, overexpression of HER2 (human epidermal growth factor receptor 2) protein was discovered in some breast cancers. With this discovery, a monoclonal antibody (trastuzumab) against the HER2 protein, a so-called antibody drug, was developed and used for therapy (Non-patent Document 3).

  Today, in clinical practice, breast cancer is divided into four subtypes by using estrogen receptor (ER) and progesterone receptor (PgR) as indicators of hormone therapy sensitivity and focusing on HER2 protein as an indicator of trastuzumab sensitivity. It is common to classify. (1) ER and / or PgR positive and HER2 negative, (2) ER and / or PgR positive and HER2 positive, (3) ER and PgR negative and HER2 positive, (4) ER and There are four subtypes: PgR negative and HER2 negative.

  On the other hand, since comprehensive gene expression analysis has become possible due to recent technological innovation, attempts to classify subtypes of breast cancer by gene expression analysis are becoming mainstream. The classification based on gene expression analysis is largely divided into four subtypes, namely Luminal A (having a gene expression pattern similar to ductal epithelial cells) and Luminal B (proliferation index evaluated by Ki67 among Luminal A). HER2 disease (hormone receptor expression level is low and HER2 is overexpressed), Basal-like (having a gene expression pattern similar to basal cells of the ductal-lobular structure), (Non-patent Document 4). Further, it is said that Luminal A corresponds to (1), Luminal B corresponds to (2), HER2 disease corresponds to (3), and Basal-like corresponds to (4) (Non-patent Document 4). ).

  The subtype corresponding to (1) or Luminal A is hormone receptor positive and HER2 protein negative breast cancer, which accounts for about 70% of breast cancer cases. Hormonal therapy can be expected for this subtype. The subtype corresponding to (2) or Luminal B is a hormone receptor positive and HER2 protein positive breast cancer, which accounts for about 10% of breast cancer cases. This subtype can be expected to benefit from hormone therapy and trastuzumab. The subtype corresponding to (3) or HER2 disease is hormone receptor negative and HER2 protein positive breast cancer, accounting for about 10% of breast cancer cases. This subtype can be expected to benefit from trastuzumab. The subtype corresponding to the above (4) is called “triple negative breast cancer” because all of ER, PgR and HER2 are negative, and accounts for about 10% of breast cancer cases. This subtype is said to have a poorer outcome than other breast cancer subtypes (Non-patent Document 5), and neither hormonal therapy nor the effect of trastuzumab can be expected, and there is no choice but to rely on chemotherapy. Current status (Non-Patent Documents 5 and 6).

  When looking at breast cancer as a whole, treatment effects and prognosis can be predicted to some extent depending on the expression level of ER, PgR, HER2 gene or protein. However, for triple-negative breast cancers whose gene or protein expression is negative, no clear target for treatment has yet been found, so only cytocidal chemotherapy is indicated, and further, the therapeutic effect of chemotherapy The current situation is that prediction and prognosis are difficult. Therefore, determining whether a breast cancer is a triple negative breast cancer is very important for determining a breast cancer treatment policy and selecting an appropriate therapeutic agent.

JP 2004-233105 A

Nathanson K. L., Wooster R., and Weber B. L. Breast cancer genetics: what we know and what we need. Nat. Med. 7, 552-556 (2001) Jordan V. C. Tamoxifen: a personal retrospective. Lancet Oncol. 1, 43-49 (2000) Hicks D. G. and Kulkarni S. HER2 + breast cancer: review of biologic relevance and optimal use of diagnostic tools. Am. J. Clin. Pathol. 129, 263-273 (2008) Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D., Conway K., Karaca G., Troester MA, Tse CK, Edmiston S., Deming SL, Geradts J., Cheang MC, Nielsen TO, Moorman PG, Earp HS and Millikan RC Race, Breast Cancer Subtypes, and Survival in the Carolina Breast Cancer Study. JAMA. 295, 2492-2502 (2006) van de Rijn M., Perou CM, Tibshirani R., Haas P., Kallioniemi O., Kononen J., Torhorst J., Sauter G., Zuber M., Kochli OR, Mross F., Dieterich H., Seitz R ., Ross D., Botstein, D. and Brown P. Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am. J. Pathol. 161, 1991-1996 (2002) Perou CM, Sorlie T., Eisen MB, van de Rijn M., Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H., Akslen LA, Fluge O., Pergamenschikov A., Williams C., Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO and Botstein D. Molecular portraits of human breast tumours. Nature 406, 747-752 (2000) Sorlie T., Tibshirani R., Parker J., Hastie T., Marron JS, Nobel A., Deng S., Johnsen H., Pesich R., Geisler S., Demeter J., Perou CM, Lonning PE, Brown PO, Borresen-Dale AL and Botstein D. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc. Natl. Acad. Sci. USA. 100, 8418-8423 (2003) Cleator S., Heller W., and Coombes, R. C. Triple-negative breast cancer: therapeutic options. Lancet Oncol. 8, 235-244 (2007) Kennedy R. D., Quinn, J. E., Mullan P. B., Johnston P. G. and Harkin D. P. The role of BRCA1 in the cellular response to chemotherapy. J. Natl. Cancer Inst. 96, 1659-1668 (2004) Rodriguez-Pinilla SM, Sarrio D., Moreno-Bueno G., Rodriguez-Gil Y., Martinez MA, Hernandez L., Hardisson D., Reis-Filho JS and Palacios J. Sox2: a possible driver of the basal-like phenotype in sporadic breast cancer. Mod. Pathol. 20, 474-481 (2007) Sitterding SM, Wiseman WR, Schiller CL, Luan C., Chen F., Moyano JV, Watkin WG, Wiley EL, Cryns VL and Diaz LK AlphaB-crystallin: a novel marker of invasive basal-like and metaplastic breast carcinomas. Diagn. Pathol. 12, 33-40 (2008) Schena, M., Shalon, D., Davis, R. W. and Brown, P. O.Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 270, 467-470 (1995) Pathology / Clinical Breast Cancer Handling Rules (15th edition) The Japan Breast Cancer Society, Kanbara Publishing Co., Ltd. pp14-15 (2004) Elston C. W. and Ellis I. O. Pathological prognostic factors in breast cancer. Histopathology, 19, 403-410 (1991) HER2 Test Guide 3rd Edition, created by Trastuzumab Pathology Committee (2009) Sauter G., Lee J., Bartlett J. M., Slamon D. J., Press M. F. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J. Clin. Oncol. 27, 1323-1333 (2009) Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M., Fitzgibbons PL, Hanna WM, Langer A., McShane LM, Paik S., Pegram MD, Perez EA, Press MF, Rhodes A ., Sturgeon C., Taube S. E, Tubbs R., Vance GH, van de Vijver M., Wheeler TM, Hayes D. F; American Society of Clinical Oncology; College of American Pathologists. American Society of Clinical Oncology / College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J. Clin. Oncol. 25, 118-145 (2007) Hammond ME, Hayes DF, Dowsett M., Allred DC, Hagerty KL, Badve S., Fitzgibbons PL, Francis G., Goldstein NS, Hayes M., Hicks DG, Lester S., Love R., Mangu PB, McShane L ., Miller K., Osborne CK, Paik S., Perlmutter J., Rhodes A., Sasano H., Schwartz JN, Sweep FC, Taube S., Torlakovic EE, Valenstein P., Viale G., Visscher D., Wheeler T., Williams RB, Wittliff JL and Wolff AC American Society of Clinical Oncology / College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J. Clin. Oncol. 28, 2784-2795 (2010) Umemura S., Kurosumi M., Moriya T., Oyama T., Arihiro K., Yamashita H., Umekita Y., Komoike Y., Shimizu C., Fukushima H., Kajiwara H. and Akiyama F. Recommendations for ' adequate evaluation of hormone receptors' a report of the task force of the Japanese Breast Cancer Society. Oncol. Rep. 24, 299-304 (2010)

  Breast cancer subtypes are diagnosed mainly by immunohistochemically staining ER, PgR, and HER2 proteins in pathological tissues. However, when staining pathological tissue immunohistochemically, the subjectivity of the border examiner is entered based on criteria for staining intensity, criteria for the proportion of staining positive cells, etc. In many cases, it was difficult to determine that the cancer was breast cancer. Therefore, in order to discriminate triple negative breast cancer, in addition to the conventional index, more new molecular biological indices (markers) are required.

  A recent report revealed that Sox2 and heat shock protein αβ crystallin are selectively expressed in triple negative (or Basal-like) breast cancer (Non-patent Documents 10 and 11), but are still sufficient. I can't say there is. Therefore, it has been desired to search for further molecular biological indicators for triple negative breast cancer.

  In addition, chemotherapy is the standard therapy for patients diagnosed with triple negative breast cancer, but the treatment procedure has not yet been established. Responsiveness to DNA-damaging platinum preparations (cisplatin, carboplatin, etc.) and alkylating agents (mitomycin, etc.) is expected (Non-Patent Documents 7 and 8). In addition, sensitivity to anthracyclines is expected in breast cancer with BRCA1 mutation or decreased expression in preoperative chemotherapy (Non-patent Document 9). In addition, molecular targeting drugs targeting EGFR, c-kit, src, angiogenesis inhibitors, targeting drugs that inhibit intracellular signaling pathways, or poly (ADP-Ribose) Polymerase (PRAP) involved in DNA repair Inhibitors are being developed. However, at present, these target drugs are not so effective. Thus, it has been desired to search for molecular biological markers for triple negative breast cancer.

  The present invention provides a triple negative breast cancer marker that can objectively determine a triple negative breast cancer marker, in particular, a triple negative breast cancer detection marker comprising a gene and a polypeptide specifically expressed in triple negative breast cancer, and the use of the marker It is to provide a method for detecting or diagnosing triple negative breast cancer.

  To achieve the above object, the triple negative breast cancer marker of the present invention is a genetic marker used for objectively determining triple negative breast cancer among subtypes of breast cancer, and is SCRG1 (stimulator of chondrogenesis 1). It is a genetic marker that determines that a case containing a gene and having a high expression level of the gene is triple negative breast cancer.

  The present inventor isolated mRNA from surgical materials of breast cancer patients and comprehensively obtained and analyzed gene expression profiles by DNA microarray method. As a result, the simulator of chondrogenesis 1 (SCRG1, GenBank accession number NM_007281) gene was triple negative In breast cancer, we found that it is highly expressed compared to other subtypes (Luminal A, Luminal B, or HER2 disease) and found that the gene can be used as a marker for triple negative breast cancer. It came to be completed.

That is, the present invention provides the following.
1. A gene marker for triple negative breast cancer detection, comprising the base sequence of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing.
2. 3. A DNA microarray or DNA for detection of triple negative breast cancer marker gene expression, wherein at least one or more of DNA or cDNA having the base sequence of all or part of the gene marker for triple negative breast cancer detection according to 1 is immobilized. Chip.
3. An oligonucleotide used for detecting the expression of the SCRG1 gene, a triple negative breast cancer detection gene marker having the base sequence of the SCRG1 gene identified by SEQ ID NO: 1 in the sequence listing,
An oligonucleotide comprising a base sequence of the SCRG1 gene or a part of a complementary base sequence thereof, and forming a site-specific base pair with the mRNA of the SCRG1 gene or cDNA thereof.
4). A DNA microarray or DNA chip for detecting expression of a triple negative breast cancer marker gene, wherein at least one of the oligonucleotides described in 3 above is immobilized.
5. A primer used for detecting a gene marker for detecting triple negative breast cancer having the base sequence of SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing,
At least two kinds of primers for forming a site-specific base pair with the mRNA of the SCRG1 gene or its cDNA and amplifying DNA containing all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing by PCR A primer set for detecting a triple negative breast cancer marker gene, comprising:
6). A polypeptide marker for detecting triple negative breast cancer, comprising the whole or part of the amino acid sequence of SCRG1 protein specified by SEQ ID NO: 2 in the sequence listing.
7). 7. A triple negative breast cancer diagnostic method comprising detecting an SCRG1 protein by using an antibody that specifically binds to a polypeptide having an amino acid sequence of the polypeptide marker for triple negative breast cancer detection according to 6.
8). 8. The triple negative breast cancer diagnostic method according to 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
9. It is induced by administering an expression vector for mammalian cells incorporating the SCRG1 gene to a mammal, and specifically binds to a polypeptide consisting of all or part of the amino acid sequence of the SCRG1 protein shown in SEQ ID NO: 2 in the Sequence Listing. A method of diagnosing triple negative breast cancer, characterized by detecting SCRG1 protein by using an antibody.
10. 10. The triple negative breast cancer diagnostic method according to 9, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
11. A triple negative breast cancer diagnostic method characterized by using the expression level of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing as an index.
12 An oligonucleotide used to detect the expression of the SCRG1 gene, a triple negative breast cancer detection gene marker having the base sequence of the SCRG1 gene identified by SEQ ID NO: 1 in the sequence listing, wherein the base sequence of the SCRG1 gene or 12. The triple negative breast cancer according to 11, wherein the expression level index is obtained using an oligonucleotide comprising a part of the complementary base sequence and forming a site-specific base pair with the SCRG1 mRNA or cDNA thereof. Diagnosis method.
13. A triple negative breast cancer diagnostic method for distinguishing between non-triple negative breast cancer and triple negative breast cancer in a breast cancer subtype,
Collecting a specimen sample comprising tissue from a breast cancer patient;
Measuring the expression level or expression level of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence sample or the SCRG1 protein specified by SEQ ID NO: 2 in the sequence table;
Based on the expression level or expression level of the SCRG1 gene or the SCRG1 protein in a non-triple negative breast cancer tissue that has been grasped in advance, and determining that it is triple negative breast cancer when higher than the standard,
A method of diagnosing triple negative breast cancer, comprising:
14 The triple negative breast cancer according to any one of the above 11 to 13, wherein the expression level or expression level of the SCRG1 gene is obtained by one of RT-PCR, real-time PCR, and hybridization. Diagnosis method.
15. 15. The triple negative breast cancer diagnostic method according to 14, wherein the hybridization method is a microarray method or a blot method.
16. 15. The triple negative breast cancer diagnostic method according to the above 14, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.
17. 17. The triple negative breast cancer diagnostic method according to 16, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.
18. A method for diagnosing triple negative breast cancer using the expression level of SCRG1 protein shown in SEQ ID NO: 2 in the sequence listing as an index.
19. 19. The triple negative breast cancer diagnostic method according to the above 18, wherein the SCRG1 protein in the specimen sample is labeled by a fluorescent antibody method and detected.
20. A pharmaceutical composition for treating triple negative breast cancer, comprising an antibody that specifically binds to a polypeptide comprising all or part of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing.
21. A pharmaceutical composition for treating triple negative breast cancer, comprising a polynucleotide comprising all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a derivative thereof.

  In the present invention, the SCRG1 gene or the SCRG1 gene or the SCRG1 gene by preparing a probe for detecting the SCRG1 gene, which is a marker gene for triple negative breast cancer detection, or an antibody against a polypeptide having all or part of the amino acid sequence of the SCRG1 protein. By combining protein expression levels with other indicators (ER, PgR, and HER2), triple negative breast cancer can be detected or diagnosed extensively and reliably.

(Expression level of SCRG1 gene in breast cancer samples by microarray method) Comprehensive gene expression analysis was performed using DNA microarray in 74 cases of Luminal A or Luminal B, 8 cases of HER2 disease, and 11 cases of triple negative. The expression level ratio of SCRG1 gene to reference was plotted as log ₂ ratio. The vertical axis represents the SCRG1 gene expression level ratio (log ₂ ratio), and the horizontal axis represents the breast cancer subtype. In the figure, `` Luminal A or B '' is a case determined as Luminal A or Luminal B among the subtypes of breast cancer, `` HER2 disease '' is a case determined as HER2 disease among the subtypes of breast cancer, “TNBC” represents a case determined to be triple negative among subtypes of breast cancer. The black rhombus represents the ratio of the expression levels of the SCRG1 gene in individual cases. An asterisk (*) indicates that the P value of the difference between both groups is statistically less than 0.005 by t test. Furthermore, an asterisk (**) indicates that the P value of the difference between both groups is statistically less than 0.005 by t test.

  Unless otherwise specified, the terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (those skilled in the art).

  Those skilled in the art will recognize many methods and materials equivalent or similar to those described herein. However, the present invention is not limited to the methods and materials described herein.

  “Measure the expression level of a gene” or “detect the expression of a gene” is not particularly limited as long as the expression level of the gene is detected, measured or quantified. For example, the amount of mRNA or cDNA of the gene is detected. , May be measured or quantified. For these detection, measurement or quantification, it is desirable to use a molecule that specifically binds to the gene or its gene product, peptide or protein. Although it does not restrict | limit especially with the molecule | numerator specifically couple | bonded with a gene or its gene product peptide or protein, It bind | bond | couples specifically with the nucleotide, DNA, cDNA, RNA, peptide, or protein which bind | bond | couples specifically with this gene. An antibody etc. can be illustrated. In addition, for detection, measurement or quantification of the expression level of the gene, mRNA or protein fragments or homologues of the gene may be used.

  "DNA microarray" refers to oligonucleotides and single-stranded or double-stranded DNA arranged on a glass substrate at high density, and "DNA microarray method" refers to fluorescent labeling on the DNA microarray. A technique that qualitatively and quantitatively measures the type and amount of nucleic acid bound to DNA by hybridization with RNA molecules and cDNA molecules.

  “Oligonucleotide” is a general term for molecules in which several nucleotides are polymerized.

  “Polynucleotide”, when used in singular or plural, refers to any polyribonucleotide or polydeoxynucleotide, which may be DNA or RNA, or may be modified DNA or RNA. DNA or RNA may be single-stranded or double-stranded, and may be a hybrid molecule containing DNA and RNA.

  “Gene marker” or “marker gene” refers to an entire gene or an EST derived from that gene whose expression level varies between certain states. If gene expression correlates with a certain state, that gene is a marker for that state.

  “Triple negative breast cancer” is a subtype of breast cancer in which all of estrogen receptor (ER), progesterone receptor (PgR), and HER2 (human epidermal growth factor receptor 2) are negative.

  “Luminal A” is one of the subtypes of breast cancer based on a classification method based on gene expression analysis, and has a gene expression pattern similar to ductal epithelial cells (Non-patent Document 6).

  “Luminal B” is one of the subtypes of breast cancer based on a classification method based on gene expression analysis, and is characterized by a high proliferation index evaluated by Ki67 in Luminal A (non-patented) Reference 6).

  “HER2 disease” is a subtype of breast cancer based on a classification method based on gene expression analysis, characterized by low ER and PgR expression levels and high HER2 expression levels (non- Patent Document 6).

  “Basal-like” is one of the subtypes of breast cancer based on a classification method based on gene expression analysis, and is characterized by having a gene expression pattern similar to the basal cells of the ductal-lobule structure (non- Patent Document 6).

  Specific methods for detecting, measuring or quantifying the expression level of a gene include Northern blotting and dot blotting using labeled nucleotides, labeled cDNA or labeled RNA for probes that specifically bind to the gene. IAFLP (Introduced Amplified Fragment Length Polymorphism) method, LAMP (Loop-Mediated Isothermal Amplification) method, PCR method, method of directly measuring mRNA molecule, or the like can be used. Examples of the PCR method include RT-PCR method, real-time PCR method, and competitive PCR method.

  As the real-time PCR method, for example, cDNA is synthesized from the total RNA or mRNA in a sample using reverse transcriptase, the target region is amplified by the PCR method using the cDNA as a template, and the production process of the amplified product is performed. The method of monitoring in real time is mentioned. Examples of the reagent to be monitored in real time include SYBR (registered trademark: Moleclar Probes) Green I, TaqMan (registered trademark: Applied Biosystems) probe, and the like.

  Examples of the competitive PCR method include, for example, a method of synthesizing cDNA from total RNA or mRNA in a sample using a reverse transcriptase and reacting the cDNA and an internal standard DNA in the same reaction tube. Examples include a method of reacting by adding an RNA internal standard together with mRNA during the reaction. In addition, the sequence of the internal standard gene may be, for example, a sequence homologous to the sequence of the gene to be amplified or a non-homologous sequence.

  Examples of the method for directly measuring mRNA include nCounter Analysis system (Nanostring).

  Furthermore, specific methods for detecting, measuring or quantifying gene expression levels include DNA microarrays, DNA chips, or antibody arrays. A DNA microarray or DNA chip having at least one nucleotide or cDNA of the gene immobilized thereon is used.

  The nucleotide or cDNA may be a part corresponding to a part of the gene, but is preferably 50 mer (base) or more, more preferably 80 mer (base) or more.

  Usually, a DNA microarray or DNA chip is an array or chip in which probes are fixed on a support, and the support of the DNA microarray or DNA chip may be any one that can be used for hybridization. A substrate such as glass, silicon, or plastic, a nitrocellulose film, a nylon film, or the like can be used.

  Probes used for DNA microarrays and DNA chips may be cDNA or synthetic oligo DNA.

  There are no particular restrictions on the method of using the DNA microarray or DNA chip. For example, purify mRNA from the tissue of the test sample, and use labeled primers and labeled nucleotides to obtain labeled cDNA when performing reverse transcription using the mRNA as a template. Can do. Hybridization between the labeled cDNA and the probe in the present invention immobilized on the surface of a DNA microarray or DNA chip, and comparing the results of hybridization with a test sample and hybridization with a control sample, Triple negative breast cancer can be detected or diagnosed by detecting the presence or absence of the SCRG1 gene and measuring the expression level.

As a labeling substance used for producing the labeled cDNA, a radioisotope, a fluorescent substance, an enzyme, or the like can be used. For example, examples of the fluorescent substance include Cy2, Cy3, Cy5, Cy5.5, Cy7, Cyanine3, Cyanaine5, FITC (fluorescein isothiocyanate), Texas Red, rhodamine and the like. Further, for example, radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³² P], [ ³⁵ S] and the like. Furthermore, examples of the enzyme include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase and the like.

  The DNA sequence information of the SCRG1 gene can be approached by the accession number NM_007281 in the gene database of NCBI (National Center for Biotechnology Information) in addition to the sequence number 1 shown in the sequence listing. Further, the sequence information of the amino acid sequence of the polypeptide or protein encoded by the SCRG1 gene is shown in SEQ ID NO: 2 in the sequence listing, and can also be approached by the accession number NP_009212 in the NCBI gene database.

  The method for detecting, measuring or quantifying the polypeptide or protein is not particularly limited as long as it is a method for detecting, measuring or quantifying a predetermined polypeptide or protein. For example, an antibody or aptamer that specifically binds to the polypeptide or protein can be used. As the antibody, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a humanized antibody, a chimeric antibody, two epitopes can be used simultaneously. Examples include bifunctional antibodies that can be recognized. These antibodies can be produced using the polypeptide or protein or a fragment thereof as an antigen using conventional protocols. Aptamers are nucleic acid molecules that specifically bind to molecules such as proteins and amino acids.

  When detecting, measuring or quantifying the polypeptide or protein present in a test sample using an antibody that specifically binds to the polypeptide or protein, immunoprecipitation, electrochemiluminescence, RIA (Radioimmunoassay) Known immunological methods such as the ELISA method, ELISA (Enzyme-liked immunosorbent assay) method, and fluorescent antibody method can be used.

  The criteria for diagnosing triple negative breast cancer are, for example, the expression level of the SCRG1 gene or protein in a previously known control sample (a breast cancer tissue classified as a subtype of Luminal A, Luminal B, or HER2 disease) The reference value is set from the above, and when the expression level of the SCRG1 gene or protein in the breast cancer tissue as the test sample is higher than the reference, it is determined as triple negative breast cancer.

  In addition, the standard may be an average value or a median value of a plurality of control samples (Luminal A, Luminal B, or HER2 disease subtypes of HER2 disease) that have been grasped in advance. It is not limited.

  Furthermore, when diagnosing triple negative breast cancer using the triple negative breast cancer gene marker of the present invention, the expression level of the SCRG1 gene or protein is used as an index. In addition to the expression level, histological analysis of the test sample It can be comprehensively determined from the diagnosis, the expression level of other genes, or other clinical information. Examples of other genes include ER, PgR, and HER2.

  When the determination of triple negative breast cancer using the gene marker of the present invention is performed by detecting the mRNA of the SCRG1 gene or the protein encoded by the gene as described above, the specimen is not particularly limited. The obtained tissue or pathological specimen may be used, or a biopsy material may be used.

  In addition, the kit used for measuring the expression level of the SCRG1 gene for the determination of triple negative breast cancer of the present invention comprises the specific gene comprising the primer for amplifying the gene marker cDNA so that it can be quantified. A kit capable of quantifying the amount. Other consumable reagents included in the kit are not particularly limited, and examples thereof include enzymes, buffers, reaction reagents and the like necessary for amplifying mRNA, cDNA, or DNA.

  The kit used for measuring the expression level of the protein encoded by the SCRG1 gene for the determination of triple negative breast cancer of the present invention comprises a primary antibody that specifically binds to the protein, and a specific antibody for the primary antibody. And a secondary antibody that binds to the antibody and labeled with an enzyme or a chemical substance. Other consumable reagents included in the kit are not particularly limited, and examples thereof include enzymes, buffers, reaction reagents and the like necessary for quantifying proteins.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  The SCRG1 gene used as the triple negative breast cancer marker of the present invention is a DNA microarray in a specimen in which the expression level of a hormone receptor or HER2 protein is detected in a tissue extracted from a breast cancer patient by surgery, and the breast cancer subtype is determined. As a result of obtaining and comparing the comprehensive gene expression profiles by, it was a gene whose expression level was specifically increased in triple negative breast cancer. Here, the “expression level” need not be an absolute amount, but may be a relative amount.

(Production of microarray)
A microarray is prepared using the synthetic DNA for microarray. Although there are no limitations on the microarray production method and conditions, for example, the production method described in Non-Patent Document 12 (Schena, M. et. Al., Science, 270, 467-470 (1995)) can be used.

  Using an ultra-small volume dispenser (Microdiagnostic), slide the human gene fragment library (Microdiagnostic) into a glass slide (Matsunami Glass Industrial Co., Ltd., HA-coated glass slide) according to the recommended protocol. To produce a microarray. The microarray was allowed to stand at 80 ° C. for 1 hour in a gas-phase incubator, and further irradiated with 120 mJ ultraviolet rays using a UV crosslinker (Hoefer, UVC500).

(Post-processing of microarray)
The post-treatment of the microarray was performed by the method described in the published patent publication (Japanese Patent Application Laid-Open No. 2004-233105).

(Preparation of nucleic acid sample)
Sample nucleic acid was prepared by extracting total RNA from surgical tissue using ISOGEN reagent (Nippon Gene) according to the recommended protocol. Furthermore, mRNA was purified using a Poly (A) Pure kit (Ambion) according to the protocol recommended by the company.

(Synthesis of labeled cDNA)
2.0 μg of this mRNA was labeled with a nucleic acid labeling / hybridizing reagent (manufactured by Microdiagnostic), reverse transcriptase SuperScript II (registered trademark: Life Technologies) (manufactured by Invitrogen), cyanine 5-deoxyuridinetriphosphate (Cyanine 5-dUTP) (Perkin Elmer) Was used to prepare labeled cDNA. On the other hand, a human common reference (manufactured by Microdiagnostic) was used as a control. For common human reference, nucleic acid labeling / hybridization reagent (Microdiagnosis), reverse transcriptase SuperScript II (Invitrogen), Cyanine3-deoxyuridinetriphosphate (Cyanine 3-dUTP) (Perkin Elmer) are used. A labeled cDNA was prepared. The production method followed the protocol recommended by each company. Human common references are 22 types of cell lines (A431 cells, A549 cells, AKI cells, HBL-100 cells, HeLa cells, HepG2 cells, HL60 cells, IMR-32 cells, Jurkat cells, K562 cells, KP4 cells, MKN7. Cells, NK-92 cells, Raji cells, RD cells, Saos-2 cells, SK-N-MC cells, SW-13 cells, T24 cells, U251 cells, U937 cells, Y79 cells) They are mixed one by one.

(Preparation of labeled probe)
After mixing these labeled cDNAs, that is, the labeled cDNA derived from the sample labeled with Cyanine5-dUTP and the labeled cDNA derived from the human common reference labeled with Cyanine3-dUTP in the same test tube, Micropure EZ (Millipore) and Microcon The product was purified by YM30 (registered trademark: Millipore) (Millipore). The purification method was performed according to the protocol recommended by the company. Finally, 15 μl was prepared using a hybridization buffer and pure water attached to a nucleic acid labeling / hybridization reagent (manufactured by Microdiagnostic).

(Hybridization)
The solution was heat denatured by heating at 99 ° C. for 5 minutes and then dropped on the microarray and stored in a hybridization cassette (manufactured by Microdiagnostic). The hybridization cassette was placed in a gas-phase incubator (manufactured by Sanyo Electric Biomedica Co., Ltd.) and kept warm at 42 ° C. for 20 hours.

(Washing)
The slide glass was taken out from the hybridization cassette, and the slide glass was washed using a hybridization washing solution attached to a nucleic acid labeling / hybridization reagent (manufactured by Microdiagnostic) according to the protocol recommended by the company.

(Fluorescence intensity detection and digitization)
Fluorescence is measured on the cleaned glass slide using the scanner GenePix4000B (Axon Instrument), optically evaluated using the analysis software GenePixPro (Axon Instrument) attached to the scanner, and the fluorescence intensity is a common human reference. And expressed as a relative ratio (log ₂ ratio).

(Determining the subtype of breast cancer)
The histological grade, HER2 protein expression level, and hormone receptor expression level were detected in 93 breast cancer patients, and the breast cancer subtype was determined (Tables 1 and 2).

  In Tables 1 and 2, “Age” indicates the age at the time of surgery, “Gender” indicates “F” for female, and “M” for male. The “histological atypia” was classified based on the rules of the “Pathology / Clinical Breast Cancer Handling Rules (15th edition) edited by the Japanese Breast Cancer Society” (Non-Patent Documents 13 and 14). “1” in the column of “histological grade” is “low grade (grade 1)”, “2” is “moderate grade (grade 2)”, “3” is “high grade (grade 3)” ".

  In Tables 1 and 2, “HER2 expression” indicates the expression level of HER2 protein. The expression level of HER2 protein is the method for hercepTestII (manufactured by DAKO), the method for histofine HER2 kit MONO (SV2-61γ), or the Ventana I-view pathway HER2 (4B5) device: Ventana XT system benchmark (Roche Diagnostics) The product was detected according to the protocol recommended by each company. Determination of the expression level of HER2 protein was in accordance with the criteria of “Trastuzumab Pathology Committee HER2 Examination Guide” (Non-Patent Documents 15 to 17). “1” in the column of “HER2 expression” in Table 1 and Table 2 is “negative (score0)”, “2” is “weak positive (score1 + or score2 + with score2 +)”, and “3” is It represents “strongly positive (score2 + and FISH positive, or score3 +)”.

  In Tables 1 and 2, “hormone receptor expression” indicates the expression level of estrogen receptor (ER) and progesterone receptor (PgR). The expression level of estrogen receptor (ER) is determined by ChemMate ENVISION kit / HRP (DAB) -ER, 1D5 (DAKO) or Ventana I-view Confirm ER (SP1) automatic staining device: Ventana XT system benchmark (Roche Detection was performed according to a protocol recommended by each company. The expression level of progesterone receptor (PgR) is determined by ChemMate ENVISION kit / HRP (DAB) -PgR (DAKO) or Ventana I-view confirm PgR (1E2) automatic staining device: Ventana XT system benchmark (Roche Detection was performed according to a protocol recommended by each company. In addition, the expression level was determined according to the guidelines and the criteria recommended by each company (Non-Patent Documents 18 and 19). In addition, “1” in the column of “hormone receptor expression” in Tables 1 and 2 is “ER positive and PgR positive”, “2” is “positive for either ER or PgR”, “3 "Represents" ER negative and PgR negative ".

In Tables 1 and 2, “Intrinsic subtype” means a subtype of breast cancer, and “TNBC” represents triple negative breast cancer. The subtype classification was determined according to the following criteria.
Luminal A: “hormone receptor expression” score is 1 or 2, and “HER2 expression” score is 1 or 2
Luminal B: Score of “hormone receptor expression” is 1 or 2, and score of “HER2 expression” is 3.
HER2 disease: “hormone receptor expression” score of 3 and “HER2 expression” score of 3
TNBC: “hormone receptor expression” score of 3 and “HER2 expression” score of 1 or 2

  In Tables 1 and 2, “expression level ratio” is a value obtained by dividing the expression level of the SCRG1 gene in the test sample (specimen) by the expression level in the human common reference and converting it to a logarithmic ratio with a base of 2. “0” represents the detection limit or less.

(SCRG1 gene expression level)
As a result of determining subtypes for 93 cases of breast cancer, there were 74 cases of Luminal A or Luminal B, 8 cases of HER2 disease, and 11 cases of triple negative. Using the surgical materials in these cases, comprehensive gene expression profiles were obtained and the expression levels of the SCRG1 gene were compared. As a result, it was found that the expression level of the SCRG1 gene was significantly high in triple negative breast cancer (FIG. 1).

  SCRG1 gene, a novel triple negative breast cancer marker of the present invention, has a low expression level in Luminal A, Luminal B, and HER2 disease among breast cancer cases, and it is difficult to objectively determine subtypes. The expression level is remarkably increased. Therefore, it is possible to make a definitive diagnosis of triple negative breast cancer by incorporating objective molecular criteria using the triple negative breast cancer marker of the present invention and making a comprehensive judgment in combination with other indicators. . In particular, a definitive diagnosis can be made even with a small amount of tissue such as pathological cytology before surgery. Furthermore, it can also be used as a marker for follow-up after surgery, and can be an objective judgment material when determining a postoperative treatment policy.

Claims (21)

  A gene marker for triple negative breast cancer detection, comprising the base sequence of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing.   A DNA microarray for detecting expression of a triple negative breast cancer marker gene, wherein at least one DNA or cDNA having the whole or a part of the base sequence of the gene marker for triple negative breast cancer detection according to claim 1 is immobilized. DNA chip. An oligonucleotide used for detecting the expression of the SCRG1 gene, a triple negative breast cancer detection gene marker having the base sequence of the SCRG1 gene identified by SEQ ID NO: 1 in the sequence listing,
An oligonucleotide comprising a base sequence of the SCRG1 gene or a part of a complementary base sequence thereof, and forming a site-specific base pair with the mRNA of the SCRG1 gene or cDNA thereof.   A DNA microarray or DNA chip for triple negative breast cancer marker gene expression detection, wherein at least one oligonucleotide according to claim 3 is immobilized. A primer used for detecting a gene marker for detecting triple negative breast cancer having the base sequence of SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing,
At least two kinds of primers for forming a site-specific base pair with the mRNA of the SCRG1 gene or its cDNA and amplifying DNA containing all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing by PCR A primer set for detecting a triple negative breast cancer marker gene, comprising:   A polypeptide marker for detecting triple negative breast cancer, comprising the whole or part of the amino acid sequence of SCRG1 protein specified by SEQ ID NO: 2 in the sequence listing.   A method for diagnosing triple negative breast cancer, comprising detecting an SCRG1 protein by using an antibody that specifically binds to a polypeptide having the amino acid sequence of the polypeptide marker for triple negative breast cancer detection according to claim 6.   The triple-negative breast cancer diagnostic method according to claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.   It is induced by administering an expression vector for mammalian cells incorporating the SCRG1 gene to a mammal, and specifically binds to a polypeptide consisting of all or part of the amino acid sequence of the SCRG1 protein shown in SEQ ID NO: 2 in the Sequence Listing. A method of diagnosing triple negative breast cancer, characterized by detecting SCRG1 protein by using an antibody.   10. The triple negative breast cancer diagnosis method according to claim 9, wherein the antibody is a monoclonal antibody or a polyclonal antibody.   A triple negative breast cancer diagnostic method characterized by using the expression level of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence listing as an index.   An oligonucleotide used to detect the expression of the SCRG1 gene, a triple negative breast cancer detection gene marker having the base sequence of the SCRG1 gene identified by SEQ ID NO: 1 in the sequence listing, wherein the base sequence of the SCRG1 gene or 12. The triple negative according to claim 11, wherein the expression level indicator is obtained using an oligonucleotide comprising a part of the complementary base sequence and forming a site-specific base pair with the SCRG1 mRNA or its cDNA. Breast cancer diagnosis method. A triple negative breast cancer diagnostic method for distinguishing between non-triple negative breast cancer and triple negative breast cancer in a breast cancer subtype,
Collecting a specimen sample comprising tissue from a breast cancer patient;
Measuring the expression level or expression level of the SCRG1 gene specified by SEQ ID NO: 1 in the sequence sample or the SCRG1 protein specified by SEQ ID NO: 2 in the sequence table;
Based on the expression level or expression level of the SCRG1 gene or the SCRG1 protein in a non-triple negative breast cancer tissue that has been grasped in advance, and determining that it is triple negative breast cancer when higher than the standard,
A method of diagnosing triple negative breast cancer, comprising:   The triple negative according to any one of claims 11 to 13, wherein the expression level or expression level of the SCRG1 gene is obtained by one of an RT-PCR method, a real-time PCR method, and a hybridization method. Breast cancer diagnosis method.   The triple-negative breast cancer diagnostic method according to claim 14, wherein the hybridization method is a microarray method or a blot method.   The triple negative breast cancer diagnostic method according to claim 14, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   The triple negative breast cancer diagnosis method according to claim 16, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.   A method for diagnosing triple negative breast cancer using the expression level of SCRG1 protein shown in SEQ ID NO: 2 in the sequence listing as an index.   19. The triple negative breast cancer diagnostic method according to claim 18, wherein the SCRG1 protein in the specimen sample is labeled by a fluorescent antibody method and detected.   A pharmaceutical composition for treating triple negative breast cancer, comprising an antibody that specifically binds to a polypeptide comprising all or part of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing. A pharmaceutical composition for treating triple negative breast cancer, comprising a polynucleotide comprising all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a derivative thereof.