JP2008502326A - Methods for predicting and monitoring response to cancer treatment - Google Patents

Abstract

  The present invention provides novel compositions, methods, and uses for the prediction, diagnosis, prognosis, prevention, and treatment of malignancy and breast cancer. The invention further relates to genes that are differentially expressed in breast tissue of breast cancer patients relative to normal “healthy” tissue. Also provided are differentially expressed genes to identify patients who are likely to respond to chemotherapy.

Description

  The present invention relates to methods and compositions for prediction, diagnosis, prognosis, prevention, and treatment of neoplastic disease treatment outcome (eg, tumor response to treatment). The method of the present invention is based on the measurement of the expression level of 65 human genes that are differentially expressed after initiation of anti-cancer chemotherapy.

  The methods and compositions of the present invention are most useful in testing for breast cancer, but are also useful in testing for other types of cancer.

  Cancer is the second most common cause of death in the United States after cardiovascular disease. One in three Americans will have cancer in their lifetime. And one in four Americans will die from cancer. More specifically, in the United States alone, about 40,000 women are killed by breast cancer each year and about 200,000 women are diagnosed with breast cancer. Tumors are usually classified based on various parameters such as tumor size, invasion status, lymph node involvement, metastasis, histopathology, immunohistologic markers, and molecular markers (WHO. International Classification of Diseases ( 1); Sabin and Wittekind, 1997 (2)).

  It is a well established fact that systemic adjuvant treatment after surgery reduces the risk of disease recurrence and death in patients with operable primary breast cancer. As an alternative treatment concept, neoadjuvant therapy or primary systemic therapy (PST) can be provided to patients with larger inoperable breast cancer or those interested in breast-conserving surgery (91). ). In general, PST does not provide a survival advantage over standard adjuvant treatment, but may identify patients with pathologically confirmed complete response (CR). This clinical response to PST is associated with improved survival (92) and reflects a significant benefit to approximately 14% of patients receiving PST treatment. Careful planning for PST demonstrated that early gene expression changes were significantly associated with clinical response (94). Identifying patients who would not benefit from receiving a PST (a given combination of chemotherapy) after the first round of treatment (eg, several hours after iv administration of the drug) Will provide an opportunity to change the regimen to improve patient health.

  In general, all patients in a given cohort receive the same treatment, many of which will not lead to successful treatment. Biomarkers that reflect tumor responses can function as sensitive short-term surrogate markers for long-term outcomes. The use of such biomarkers will make chemotherapy more effective for individual patients and will allow early regimen changes when non-reactive tumors.

  Although much effort has been made to develop an optimal clinical course of treatment for individual breast cancer patients, little progress has been made in predicting an individual's response to a particular treatment. Usually, such predictions are based on standard clinical parameters such as tumor stage and stage, estrogen receptor (ER) and progesterone receptor (PgR) status, growth rate, HER2 / neu and p53 oncogene overexpression. (78). However, evidence regarding the relevance of ER and / or PgR gene expression to predicting outcome of adjuvant endocrine chemotherapy is still controversial. Studies have shown that the level of ER and PgR gene expression in breast cancer patients is prognostic, independent of subsequent adjuvant chemotherapy. From a theoretical point of view, the therapeutic response of patients with breast cancer is not expected to be independent of ER / PgR status. It is more likely that the effect of receptor expression on prognosis depends on other parameters, such as the effects of the ERBB2 receptor. Trying to find such factors using conventional biological techniques raises a problem because these analyzes all investigate one gene at a time.

  Researchers are increasingly paying attention to the classification of tumors based on differences in marker gene expression, and DNA microarray technology is used to simultaneously quantify the expression levels of thousands of genes in a sample. It has become very useful. To date, this technique has been used to classify cancer tissues, eg, breast tumors [(3), (79-83)], predict metastasis and patient outcome [(4], (84-86)], and chemotherapy It has been applied to the tumor response to ([87-90)].

  But nevertheless, chemotherapy remains the cornerstone of the treatment regime offered to patients with breast cancer, particularly those with cancer that has metastasized from their original site [Perez, 1999, (5)]. . There are several chemotherapeutic agents that have been demonstrated to be active in the treatment of breast cancer, and research is continually attempted to determine the optimal drug and regimen. However, different patients tend to respond differently to the same treatment plan. Currently, individual response to a particular treatment can only be assessed statistically based on data from previous clinical trials. There are still many patients who do not benefit from systemic chemotherapy. In particular, breast cancer is extremely heterogeneous in aggressiveness and therapeutic response. They contain different genetic variations and differences that affect the growth characteristics and sensitivity to some drugs. Identification of the molecular fingerprint of each tumor may therefore help to isolate patients with particularly aggressive tumors, or patients in need of treatment with certain beneficial therapies. As genetic research and research related to response to treatment mature, standardized methods will undoubtedly become more personalized. This allows the physician to provide a specific regimen that matches the genetic profile of the tumor to ensure optimal results.

  The present invention provides that 65 human genes are differentially expressed in tumor tissue after initiating anti-cancer chemotherapy compared to tumor tissue or other baseline expression levels prior to initiating anti-cancer chemotherapy. This is based on an unexpected new finding. After initiation of chemotherapy, expression of each of the 65 genes increased in patients responding to chemotherapy, whereas in patients who did not respond to chemotherapy, expression decreased after initiation of chemotherapy (Ie the opposite was found). Thus, differential expression of one or several of these 65 genes after initiating anti-cancer chemotherapy may allow patients to respond to the mode of chemotherapy applied It has been found to provide valuable information on whether or not.

  The present invention further relates to 65 humans that are differentially expressed in tumor tissue after initiating anti-cancer chemotherapy compared to tumor tissue or other baseline expression levels prior to initiating anti-cancer chemotherapy. Regarding genes.

  The present invention relates to a patient's response to anticancer chemotherapy by measuring the differential expression of one or several genes of a group of 65 human genes before and after initiating anticancer chemotherapy in the patient. It also relates to the method of inspecting. The examination of the reaction can be performed immediately after starting chemotherapy. At this stage, other methods, such as measuring tumor size, still cannot provide the necessary information about the patient's response to chemotherapy.

  Therefore, the present invention should determine whether the applied modality of chemotherapy is likely to be beneficial to the patient's health and / or should maintain or modify the applied modality of chemotherapy— Provide a means to determine-immediately after starting anti-cancer chemotherapy.

  The present invention relates to the identification of 65 human genes that are differentially expressed in tumor tissue resulting in altered clinical behavior of neoplastic lesions. The differential expression of these 65 genes is not limited to neoplastic lesions that are specific in a particular tissue of the human body.

  Genes that undergo expression changes in response to chemotherapeutic agents can further serve as markers for monitoring the treatment, and when correlated with clinical outcome, such genes can also serve as biomarkers for efficacy.

  In a preferred embodiment of the invention, the neoplastic lesion with altered expression of these 65 genes is human breast cancer. This cancer is not limited to women and may be diagnosed and analyzed in men.

  The present invention relates to various methods, reagents, and kits for breast cancer diagnosis, staging, prognosis, monitoring, and treatment. As used herein, “breast cancer” includes carcinomas (eg, carcinoma in situ, invasive cancer, regardless of their histological origin (eg, vascular, lobular, medullary, and hybrid origin). Metastatic cancer), premalignant conditions, and neomorphic changes. The compositions, methods, and kits of the present invention may comprise single or multiple (eg, 2, 5, 10, or 50 or more) genes (hereinafter “marker genes”, Table 1, SEQ ID NOs: 1 to 65) in patient samples And the polypeptide sequences encoded by them are listed in SEQ ID NOs: 66-130, see also Table 1) and in samples obtained from control subjects (eg, human subjects without breast cancer) Comparing the average expression level of the marker gene.

  The invention further relates to various compositions, methods, reagents, and kits for predicting a clinically measurable tumor treatment response to a given breast cancer treatment. The compositions, methods, and kits of the present invention provide single or multiple (eg, 2, 5, 10, or 50 or more) breast cancer marker gene mRNA expression levels in unclassified patient samples and breast cancer treatments administered. Comparing the average expression level of the marker gene in a sample cohort containing patients that respond to different intensities. In a preferred embodiment of the present invention, a marker gene specific is used to distinguish between those who respond to an anthracycline-based chemotherapy (eg, multidrug chemotherapy with epirubicin or doxorubicin) and those who do not. Expression can be utilized.

  In a further preferred embodiment, the control level of mRNA expression is obtained from several (eg 2, 3, 4, 5, 8, 10, 12, 15, 20, 30, or 50) control subjects. Mean expression level of the marker gene in each sample. These control subjects may also have breast cancer and be classified according to their clinical profile, and need not necessarily be classified according to their individual expression profiles.

  As detailed below, a significant change in the expression level of one or more marker genes (a set of marker genes) in a patient sample when compared to a control level is related to breast cancer status in the patient and to chemotherapy. Provide important information about responsiveness. In the compositions, methods, and kits of the present invention, the marker genes listed in Table 1 can be used in combination with well-known breast cancer marker genes (eg, CEA, mammaglobin, or CA15-3).

  According to the present invention, the following conditions are present: Stage 0 breast cancer patient, Stage I breast cancer patient, Stage II breast cancer patient, Stage III breast cancer patient, Stage IV breast cancer patient, Grade I breast cancer patient, Grade II breast cancer patient, Grade III breast cancer The positive predictive value of the compositions, methods, and kits of the present invention for any of patients, malignant breast cancer patients, primary breast cancer, and all other types of breast-related cancers, malignant tumors, and transformed patients Marker genes and marker gene sets are selected to be at least about 10%, preferably about 25%, more preferably about 50%, and most preferably about 90%.

  Detection of the marker gene expression is not limited to detection in primary lesions, secondary lesions, or metastatic lesions of breast cancer patients, but may include lymph nodes affected by breast cancer cells, or localized deposition (eg, bone marrow). , Liver, kidney), or detection of minimal residual disease cells floating freely throughout the patient's body.

  In one embodiment of the compositions, methods, reagents and kits of the invention, the sample to be analyzed is aspirated or punctured, by excision, or any other surgical method to obtain biopsy cell material or excised cell material. Tissue material obtained from neoplastic lesions collected by In one embodiment of the compositions, methods and kits of the invention, the sample comprises cells obtained from a patient. These cells are found, for example, in breast cell “smear” specimens collected by nipple aspiration, tube lavage, or fine needle biopsy, or in breast cell smears obtained from induced or spontaneous nipple secretions. It can be. In another embodiment, the sample is a body fluid. Such body fluids include, but are not limited to, for example, blood, lymph, ascites, gynecological body fluids, or urine.

In the compositions, methods, and kits of the present invention, gene expression measurement is not limited to any particular method, nor is it limited to mRNA detection. The presence and / or expression level of a marker gene in a sample can be assessed, for example, by measuring and / or quantifying:
1) A protein encoded by the marker gene (SEQ ID NO: 1 to 65) in Table 1, or a protein comprising a polypeptide selected from SEQ ID NO: 66 to 130, or a polypeptide produced by processing or degradation of the protein (For example, using reagents such as antibodies, antibody derivatives, or antibody fragments that specifically bind to the above proteins or polypeptides)
2) Directly (ie catalyzed) or indirectly by a protein encoded by the marker gene in Table 1 (SEQ ID NO: 1 to 65) or a polypeptide comprising a polypeptide selected from SEQ ID NO: 66 to 130 Produced metabolites 3) RNA transcripts encoded by the marker genes in Table 1 (eg, mRNA, hnRNA), or fragments of the RNA transcript (eg, RNA transcripts obtained from the sample, or the above By contacting the mixture of cDNAs prepared from the transcripts with a substrate having a nucleic acid comprising the sequence of one or more marker genes listed in Table 1 immobilized on the substrate at selected positions). The mRNA expression of these genes can be detected using, for example, DNA microarrays provided by Affymetrix (US Pat. No. 5,556,752) or other manufacturers. In yet another embodiment, the expression of these genes can be detected using bead-based direct fluorescence measurement techniques such as those provided by Luminex (WO 97/14028).

In one aspect, the invention provides compositions, methods, and kits for assessing whether a patient suffers from breast cancer (eg, a patient at particular risk of developing breast cancer (eg, Novel detection or “screening”, detection of recurrence, or response testing) in patients with medical history and patients identified as having a mutant form of an oncogene. For this purpose, the above compositions, methods and kits are:
comparing the expression level of single or multiple marker genes in a patient sample, and b) the normal expression level of the marker gene in a control subject without breast cancer, comprising: One (eg, 2, 5, 10, or 50 or more) is selected from the marker genes in Table 1.

  The expression level of the selected marker gene (eg, 2, 5, 10, or 50 or more) in the patient sample is significantly increased and decreased compared to the normal expression level of each marker gene This is an indicator that the patient has breast cancer.

The compositions, methods, and kits of the invention are also useful for prognosing malignant tumor progression or outcome. For this purpose, the above compositions, methods and kits are:
comparing the expression level of single or multiple marker genes in a patient sample, and b) a control pattern of expression of these marker genes, wherein at least one of the marker genes (eg, 2, 5, 10 or more) are selected from the marker genes in Table 1.

The compositions, methods, and kits of the invention are particularly useful for identifying patients who do not respond to a particular chemotherapy. For this purpose, the above compositions, methods and kits are:
comparing the expression level of a single or multiple marker genes in a patient sample, and b) the expression level of the marker genes in a control subject, wherein at least one of the marker genes (eg, 2, 5 10 or more) are selected from the marker genes in Table 1. Control subjects may be those who do not have breast cancer or have been identified and classified according to their clinical response to a particular chemotherapy.

In another aspect, the present invention provides compositions, methods, and kits for assessing the efficacy of treatment for inhibiting breast cancer in a patient. The compositions, methods, and kits are:
a) expression of a single or marker gene in a first sample obtained from the patient before any treatment is given to the patient, and b) a second sample obtained from the patient after at least one dose of the treatment. Wherein at least one of the marker genes is selected from the marker genes listed in Table 1.

  In this composition, method, and kit, “treatment” includes, but is not limited to, chemotherapy, antihormonal therapy, directed antibody therapy, radiation therapy, and surgical removal of tissues such as breast tumors. It will be understood that it can be a therapy for treating breast cancer, including. Thus, the compositions, methods, and kits of the invention can be used to assess patients before, during, and after treatment, for example, to assess reduction in systemic tumor tissue mass.

In yet another aspect, the present invention provides compositions, methods, and kits for monitoring breast cancer progression in a patient. The compositions, methods, and kits are:
a) detecting the expression of single or multiple marker genes in a patient sample at a first time point;
b) repeating step a) at a later time,
c) comparing the expression level of each marker gene detected in step a) and step b), thereby monitoring the progression of breast cancer in the patient, and at least one of said marker genes comprises a table 1 is selected from the marker genes described in 1.

In another aspect, the present invention provides compositions, methods, and kits for selecting in vitro a treatment regime (eg, type of chemotherapeutic reagent) for suppressing breast cancer in a patient. The compositions, methods, and kits are:
a) obtaining a sample containing cancer cells from a patient;
b) maintaining separate aliquots of the sample in the presence of various test compositions;
c) comparing the expression of single or multiple marker genes selected from the marker genes listed in Table 1 in each aliquot;
d) inducing an expression level lower in the gene of SEQ ID NO: 1-46 in the aliquot containing the test composition compared to the expression level of each marker gene in the aliquot containing the other test composition; and And / or selecting one of said test compositions that induces a higher expression level of the genes of SEQ ID NOs: 47 to 65.

The present invention further provides compositions, methods and kits for assessing the carcinogenic potential of specific biological or chemical compounds. The compositions, methods, and kits are:
a) maintaining separate aliquots of breast cells in the presence and absence of the test compound, and b) comparing the expression of single or multiple marker genes in each of the aliquots, At least one of these is selected from the marker genes listed in Table 1. Of the gene of SEQ ID NOs: 1-46 in an aliquot maintained (ie exposed) in the presence of the test compound compared to the expression level of each marker gene in an aliquot maintained in the absence of the test compound A significant increase in expression level and / or a significant decrease in the genes of SEQ ID NOs: 47-65 is indicative of the ability of the test compound to have breast carcinogenesis.

  The present invention further provides compositions, methods, and kits for treating patients suffering from breast cancer. This composition, method, kit comprises providing to the patient's cells an antisense oligonucleotide complementary to the polynucleotide sequence of the marker gene listed in Table 1.

  The present invention further provides compositions, methods, and kits that inhibit breast cancer cells in patients at risk of developing breast cancer. The compositions, methods, and kits include inhibiting the expression of the marker genes listed in Table 1.

  In yet another embodiment, the present invention provides compositions, methods and kits for conducting screening to obtain agents that modulate the activity of a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66-130. provide. A test compound is contacted with a specific polypeptide. Binding of the test compound to the polypeptide is detected. Thereby, test compounds that bind to the polypeptides are identified as potential therapeutic agents for treating malignant tumors, and more particularly breast cancer.

  In another embodiment of the invention, another composition, method and kit for screening to obtain an agent that modulates the activity of a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66-130 is provided. A test compound is contacted with a specific polypeptide. A biological activity mediated by the polypeptide is detected. Test compounds that reduce the biological activity are identified as therapeutic agents that may thereby reduce the activity of certain polypeptides in malignant tumors, particularly breast cancer. Test compounds that increase the biological activity are identified as therapeutic agents that may thereby increase the activity of certain polypeptides in malignant tumors, particularly breast cancer.

  Accordingly, the present invention provides a polypeptide selected from one of the polypeptides having SEQ ID NOs: 66-130. For example, act as modulators or modulators such as agonists and antagonists of polypeptides, including polypeptides selected from SEQ ID NOs: 66 to 130, partial agonists, inverse agonists, activators, coactivators, and inhibitors Can be used to identify potential compounds. Accordingly, the present invention provides reagents, compositions, methods and kits for modulating a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66 to 130 in malignant tumors, more particularly breast cancer. The above control may be upward control or downward control. Reagents that modulate the expression, stability, or amount of a polynucleotide set forth in Table 1 (SEQ ID NOs: 1-65), or the activity of a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66-130 are proteins, It can be a peptide, peptidomimetic, nucleic acid, nucleic acid analog (eg, peptide nucleic acid, LNA (locked nucleic acid)), or small molecule. Expression, stability, or amount of a polynucleotide comprising a polynucleotide selected from SEQ ID NO: 1 to 65 (listed in Table 1), or a polypeptide selected from SEQ ID NO: 66 to 130 (Table 1) Compositions, methods, and kits that modulate the activity of a polypeptide can be gene replacement therapy, antisense, ribozyme, and triple-stranded nucleic acid approaches.

  The present invention further provides compositions, methods, and kits for making isolated hybridomas that produce antibodies useful for assessing whether a patient suffers from breast cancer. The composition, method, and kit use a step of isolating a protein encoded by the marker gene described in Table 1, or a polypeptide fragment of the protein, and the isolated protein or polypeptide fragment. Immunizing a mammal, isolating spleen cells from the immunized mammal, fusing the isolated spleen cells with an immortal cell line to form a hybridoma, Screening for the production of antibodies that specifically bind to the protein or polypeptide fragment and isolating the hybridoma. The present invention also includes antibodies produced by this method. Such antibodies comprise a polypeptide selected from SEQ ID NOs: 66 to 130 (listed in Table 1) for use in the prediction, prevention, diagnosis, prognosis, and treatment of malignant tumors, and more particularly breast cancer. It specifically binds to full-length or partial polypeptides.

  Yet another aspect of the invention is a polynucleotide comprising a polynucleotide selected from SEQ ID NOs: 1-65, or SEQ ID NO: 66 in the manufacture of a medicament for treating a malignant tumor in a patient, more particularly a breast cancer Through the use of a reagent that specifically binds to a polypeptide comprising a polypeptide selected from (listed in Table 1).

  Yet another embodiment is the activity or stability of a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66 to 130 (listed in Table 1) in the manufacture of a medicament for treating malignant tumors, particularly breast cancer Or the use of a reagent that modulates the expression, amount, or stability of a polynucleotide comprising a polynucleotide selected from SEQ ID NOS: 1 to 65 (Table 1).

  Yet another aspect of the invention is specific for a polynucleotide comprising a polynucleotide selected from SEQ ID NOs: 1 to 65 (Table 1) or a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66 to 130 A pharmaceutical composition comprising a reagent that binds to a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier.

  Yet another embodiment of the present invention comprises a sequence that hybridizes under stringent conditions to a polynucleotide comprising a polynucleotide selected from SEQ ID NOs: 1 to 65, and is shown for each polynucleotide in Table 1. Provided is a pharmaceutical composition comprising a polynucleotide that encodes a polypeptide that exhibits the same biological function, or a polynucleotide that encodes a polypeptide comprising a polypeptide selected from SEQ ID NOs: 66 to 130 . The pharmaceutical composition useful in the present invention may further comprise a polypeptide comprising a polynucleotide selected from SEQ ID NOs: 1 to 65, or a fragment thereof, an antibody, or a fusion protein comprising an antibody fragment.

  The present invention also provides various kits. Such kits include reagents for evaluating the expression of single or multiple genes selected from the marker genes listed in Table 1.

  In one aspect, the invention provides a kit for assessing whether a patient has breast cancer.

  In another aspect, the present invention provides a kit for assessing the suitability of each of a plurality of compounds for inhibiting breast cancer in a patient. This kit contains reagents for evaluating the expression of the marker genes listed in Table 1. The kit may contain a plurality of compounds.

  In another aspect, the present invention provides a kit for assessing the presence of breast cancer cells. This kit comprises an antibody that specifically binds to a protein encoded by the marker gene listed in Table 1, or a polypeptide fragment of the protein. The kit may include a plurality of antibodies that specifically bind to a protein encoded by each marker gene of the marker gene set described in Table 1.

  In yet another aspect, the present invention provides a kit for assessing the presence of breast cancer cells, the kit comprising a nucleic acid probe. This probe specifically hybridizes to an RNA transcript of the marker gene listed in Table 1, or to the cDNA of the transcript. The kit may include a plurality of probes, each of which specifically hybridizes to one RNA transcript of a marker gene in the marker gene set described in Table 2.

  It will be appreciated that the compositions, methods, or kits of the present invention may include known cancer marker genes, including known breast cancer marker genes. It will be further appreciated that these compositions, methods, and kits can also be used to identify cancers other than breast cancer.

Definitions “Differential expression” or “expression”, as used herein, is the expression of a gene in response to different developments of tumor cells, different genetic backgrounds, and / or tumor responses to the tissue environment. It means both quantitative and qualitative differences in expression patterns. Differentially expressed genes can be “marker genes” and / or “target genes”. The expression patterns of differentially expressed genes disclosed herein can be used as part of a prognostic or diagnostic assessment of breast cancer.

  The term “expression pattern” means, for example, a measured level of gene expression compared to a reference gene (eg, housekeeper gene) or a calculated average expression value (eg, DNA chip analysis). The pattern is not limited to comparing two genes, but rather relates to multiple comparisons of multiple genes against a reference gene or sample. A particular “expression pattern” may also arise from the comparison and measurement of several genes disclosed below, and may be determined and present relative amounts of these transcripts to each other.

  Alternatively, the differentially expressed genes disclosed herein can be used in reagents and compounds for the treatment of breast cancer, use of these reagents and compounds, and methods of identifying therapeutic methods. Differential regulation of genes is not limited to specific cancer cell types or clones, but rather, cancer cells, muscle cells, stromal cells, connective tissue cells, other epithelial cells, endothelial cells and blood vessels, and the immune system Represents the interaction of cells (eg, lymphocytes, macrophages, killer cells).

  “Biological activity”, “biological activity”, “activity”, and “biological function” are used interchangeably and are used herein to refer to a polypeptide (its native or denatured configuration). Means an effector function or antigenic function that is exercised directly or indirectly in vivo or in vitro by one at a locus) or any fragment thereof. Biological activity includes binding to polypeptides, binding to other proteins or molecules, enzyme activity, signal transduction, activity as a DNA binding protein, activity as a transcriptional regulator, ability to bind to damaged DNA, etc. Is included, but is not limited thereto. Biological activity can be modulated by directly affecting the polypeptide of interest. Alternatively, the biological activity can be altered by adjusting the level of the polypeptide, ie by adjusting the expression of the corresponding gene.

  The term “marker” or “biomarker” refers to a biological molecule, eg, nucleic acid, peptide, that can detect its presence or concentration and correlate it with a known condition, such as a disease state. , Refers to hormones.

  The term “marker gene”, as used herein, can utilize its expression pattern as part of a predictive, prognostic, or diagnostic process in the assessment of malignant tumors or breast cancers, or malignant It refers to a differentially expressed gene that can be used in methods of identifying compounds useful for the treatment or prevention of tumors, particularly breast cancer. A marker gene may also have properties as a target gene.

  “Target gene”, as used herein, thereby modulating the level of target gene expression or target gene product activity acts to ameliorate the symptoms of malignant tumors, particularly breast cancer. As such, it refers to a differentially expressed gene associated with breast cancer. The target gene may also have properties as a marker gene.

  The term “neoplastic lesion”, “neoplastic disease”, or “tumor” refers to a carcinoma (eg, epithelium), regardless of their histological origin (eg, vascular, lobular, medullary, and hybrid origin). Internal cancer, invasive cancer, metastatic cancer), premalignant state, and cancerous tissue including neoplastic changes. The term “cancer” is not limited to any stage, grade, histomorphological feature, invasiveness, or grade of diseased tissue or cell population. Specifically, stage 0 breast cancer, stage I breast cancer, stage II breast cancer, stage III breast cancer, stage IV breast cancer, grade I breast cancer, grade II breast cancer, grade III breast cancer, malignant breast cancer, primary breast cancer, and all other types Breast-related cancers, malignant tumors, and transformation are included. The terms “neoplastic lesion”, “neoplastic disease”, “tumor”, or “cancer” are not limited to any tissue or cell type. They include primary lesions, secondary lesions, or metastatic lesions in cancer patients, and are lymph nodes affected by cancer cells or are locally deposited (eg, bone marrow, liver, kidney) Also included are microscopic residual diseased cells that float freely throughout the patient's body.

  Furthermore, the term “characterizing the state of a neoplastic disease” refers to the following conditions: tumor type, histomorphological appearance, dependence on external signals (eg hormones, growth factors), invasiveness , Motility, status due to TNM (2), etc., grade, malignancy, metastatic potential, and responsiveness to a given treatment.

  The term “biological sample” as used herein refers to a sample obtained from an organism or a component of an organism (eg, a cell). The sample may be from any biological tissue or body fluid. The sample will often be a “clinical specimen” which is a sample obtained from a patient. Such samples include sputum, blood, blood cells (eg, white blood cells), tissue samples or fine needle biopsy samples, cell-containing body fluids, suspended nucleic acids, urine, peritoneal fluid, and pleural effusions, or cells derived therefrom. However, it is not limited to these. Biological samples may also include tissue sections such as frozen sections or fixed sections taken for histological purposes. The biological sample to be analyzed is tissue material obtained from a neoplastic lesion taken by aspiration or puncture, by excision, or by any other surgical method to obtain biopsy cell material or excised cell material. Such biological samples can also include cells obtained from a patient. These cells are found, for example, in breast cell “smear” specimens collected by nipple aspiration, tube lavage, or fine needle biopsy, or in breast cell smears obtained from induced or spontaneous nipple secretions. It can be. In another embodiment, the sample is a body fluid. Such body fluids include, but are not limited to, for example, blood, lymph, ascites, gynecological body fluids, or urine.

  The terms “treatment modality”, “treatment mode”, “prescription plan”, or “chemotherapy plan”, and the term “treatment plan” are antitumor and / or immunostimulatory and / or blood cells for cancer treatment. By sequential or simultaneous administration of proliferative agents and / or radiation therapy and / or hyperthermia and / or systemic hypothermia. These administrations can be performed in adjuvant and / or neoadjuvant mode. Such "protocol" compositions vary in single agent dosage, time frame of administration, and frequency of administration within a particular treatment window. Currently, various combinations of various drugs and / or physical methods and various schedules are under investigation.

“Array” or “matrix” means an arrangement of addressable locations or “addresses” on a device. These positions can be arranged in a two-dimensional array, a three-dimensional array, or other matrix format. The number of these locations can range from several places to at least hundreds of thousands. Most importantly, each position represents a completely independent reaction position. Arrays include, but are not limited to, nucleic acid arrays, protein arrays, and antibody arrays. “Nucleic acid array” refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides, or larger portions of genes. The nucleic acids on the array are preferably single stranded. An array in which the probes are oligonucleotides is called an “oligonucleotide array” or “oligonucleotide chip”. A “microarray”, also referred to herein as a “biochip” or “biological chip”, is a plurality of regions where the density of individual regions is at least about 100 / cm ² , preferably at least about 1000 / cm ^2. It is an array. The regions in the microarray are of typical dimensions, for example in the range of about 10-250 μm in diameter, and are approximately the same distance away from other regions in the array. “Protein array” refers to an array containing polypeptide probes or protein probes in a non-denatured form or denatured. “Antibody array” refers to an array containing antibodies, which include monoclonal antibodies (eg, from a mouse), chimeric antibodies, humanized antibodies, or phage and single chain antibodies, and these antibodies. Fragments of, but not limited to.

  The term “agonist” as used herein is intended to refer to an agent that mimics or upregulates (eg, enhances or augments) the biological activity of a protein. An agonist can be a wild-type protein or a derivative thereof having at least one biological activity of the wild-type protein. An agonist may be a compound that upregulates expression of a gene or a compound that enhances at least one biological activity of a protein. An agonist can also be a compound that enhances the interaction of a polypeptide with another molecule, such as a target peptide or target nucleic acid.

  The term “antagonist” as used herein is intended to refer to an agent that downregulates (eg, suppresses or inhibits) at least one biological activity of a protein. An antagonist can be a compound that inhibits or reduces the interaction of a protein with another molecule, such as a target peptide, ligand, or enzyme substrate. An antagonist can be a compound that down-regulates expression of a gene or a compound that is expressed and reduces the abundance of a protein.

  “Small molecule” as used herein is intended to refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic molecules (containing carbon), or inorganic molecules. Many pharmaceutical companies have large libraries of chemical and / or biological mixtures that can be screened using any of the assays of the present invention to identify compounds that modulate biological activity. The biological material is often an extract of fungi, bacteria, or algae.

  The terms “moderated” or “moderated” or “modulated” or “modulated” and “differentially regulated” as used herein are up-regulated (ie, activated or By both stimulation (eg, by agonistic action or enhancement) and down-regulation [ie, inhibition or suppression (eg, by antagonism, reduction, or suppression)] is meant.

  “Transcriptional regulatory unit” refers to a DNA sequence that directs or controls transcription of a protein coding sequence to which they are operably linked, such as an initiation signal, enhancer, promoter, and the like. In a preferred embodiment, transcription of one of the genes is under the control of a promoter sequence (or other transcription control sequence) that controls the expression of the recombinant gene in the cell type intended for expression. A recombinant gene can be placed under the control of the same transcription control sequence that controls the transcription of the naturally occurring form of the polypeptide, or under the control of a different transcription control sequence. You will understand that too.

  The term “derivative” means a chemical modification of a polypeptide or polynucleotide sequence. Chemical modification of the polynucleotide sequence can include, for example, replacement of hydrogen with an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one that has been modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it is derived. The term “derivative” also means a phosphorylated form of a polypeptide sequence or protein.

  The term “nucleotide analog” has at least one characteristic that differs from a naturally occurring nucleotide, oligonucleotide, or polynucleotide, but the functional characteristics of each naturally occurring nucleotide (eg, base pairing, high Hybridization, code information) and refers to an oligomer or polymer that can be used for the composition. Nucleotide analogs can be composed of non-naturally occurring bases or polymer backbones, examples of which include LNA, PNA, and morpholino. Nucleotide analogs have at least one molecule that is different from its naturally occurring counterpart or equivalent.

  “BREAST CANCER GENE”, as used herein, is encoded by the polynucleotides of SEQ ID NOs: 1-65 (listed in Table 1), and derivatives, fragments, analogs, and homologs thereof. Polypeptides (SEQ ID NOs: 66-130, see Table 1), and derivatives, fragments, analogs and homologues thereof, and information disclosed in Tables 1-3, are well known in the art Refers to the corresponding genomic transcription unit that can be derived or identified by standard techniques. Table 1 shows the gene names and reference sequences of the polynucleotide sequences of SEQ ID NOs: 1 to 65 and the polypeptides of SEQ ID NOs: 66 to 130.

  The term “chromosomal region” as used herein refers to cytogenetic markers or other genetic markers such as restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP), expressed sequence tag (EST), a base sequence tag site (STS), a microsatellite, a variable tandem sequence number (VNIR), and a continuous DNA region on a chromosome that can be defined by genes and the like. Typically, a chromosomal region consists of up to 2 megabases (MB), up to 4 MB, up to 6 MB, up to 8 MB, up to 10 MB, up to 20 MB, or more MB.

  The term “kit” as used herein refers to at least one marker gene specifically disclosed in the present invention, in particular at least one type for detecting the expression of the genes listed in Table 1. Means any product (eg, diagnostic or research product) containing reagents, eg, probes, which are sold, distributed, and / or promoted as a unit for performing the methods of the present invention. . Reagents (eg, immunoassays) that detect the presence, stability, activity, and complexity of each marker gene product comprising a polypeptide selected from SEQ ID NOs: 66 to 130 can also be considered as components of the kit. In addition, any combination of nucleic acid and protein detection disclosed in the present invention is considered a kit.

  The present invention relates to a polynucleotide sequence and the protein encoded thereby, a probe derived from the polynucleotide sequence, an antibody against the encoded protein, and an individual with or at risk for a malignant tumor, in particular breast cancer Use for the prediction, prevention, diagnosis, prognosis, and treatment of individuals. The sequences disclosed herein have been found to be differentially expressed in samples obtained from breast cancer.

  The present invention is based on the identification of 65 genes that are differentially regulated (up or down regulated) in tumor biopsies of patients with clinical evidence of breast cancer. By characterizing the co-expression of several of these genes, a new role in breast cancer was identified. The gene name, database accession number (gene name, symbol, and reference sequence) and the putative or known function of the encoded protein are shown in Table 1.

  The present invention relates to the following method.

1. A method for testing (preferably ex vivo) response to anti-cancer chemotherapy comprising:
(I) at least 1, 5, 10, 20, 40 included in the group of genes encoded by SEQ ID NO: 1 to SEQ ID NO: 65 in biopsy samples taken from neoplastic lesions after the start of the chemotherapy schedule Measuring the expression level of 65 genes; and (ii) comparing said expression level with a reference expression level;
And thereby examining the response to anti-cancer chemotherapy.

The present invention is a method for testing (preferably ex vivo) response to anti-cancer chemotherapy comprising:
(I) at least 1, 5, 10, 20, 40 included in the group of genes encoded by SEQ ID NO: 1 to SEQ ID NO: 65 in biopsy samples taken from neoplastic lesions prior to the start of the chemotherapy schedule Measuring the expression level of 65 genes, and (ii) comparing the expression level with a reference expression level,
And thereby a method for testing response to anti-cancer chemotherapy.

2. A method for examining responses to anti-cancer chemotherapy ex vivo,
(I) applying ex vivo chemotherapy to a biopsy sample taken from a neoplastic lesion prior to initiating a chemotherapy schedule;
(Ii) measuring the expression level of at least 1, 5, 10, 20, 40, 65 genes contained in the group of genes encoded by SEQ ID NO: 1 to SEQ ID NO: 65 in the biopsy sample; and (Iii) comparing the expression level with a reference expression level;
And thereby examining the response to anti-cancer chemotherapy ex vivo.

  Expression level measurements may include quantification of expression levels and / or purely qualitative determination of expression levels.

  It will be apparent to those skilled in the art that portions and fragments of the gene can be used as specific to measure gene expression.

  The present invention also relates to a method for examining response to anticancer chemotherapy, as described above, wherein patterns of expression levels are measured and compared in steps (i) and (ii). The “expression level pattern” of a single gene should be understood as the expression level of that gene measured in an appropriate manner.

  The present invention also relates to a method for testing response to anti-cancer chemotherapy using a sequence as described above but which is a homologue of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 65. Preferred homologues have 80%, 90%, 95% or 99% sequence identity to the original sequence. The homologue preferably still has the same biological activity and / or function as the original molecule has.

  It will be apparent to those skilled in the art that a reference to a nucleotide sequence is intended to include a reference to a related protein sequence encoded by the nucleotide sequence.

  “% Identity”, “percent identity” of a first sequence relative to a second sequence means, within the meaning of the present invention,% identity calculated as follows. First, an optimal global alignment between the two sequences is represented by the following command line syntax:. / Clustalw-file =. / File. txt -output = -outorder = aligned -pwmatrix = gonnet -pwdnamatrix = clustalw -pwgapopen = 10.0 -pwgapext = 0.1 -matrix = gonnet -gapopen = 10.0 -gapex = gapopen = 10.0 -gap h = GPSNDQERK-maxdiv = 40, and the CLUSTALW algorithm [Thomson JD, Higgins DG, Gibson TJ, 1994, “ClustalW: Improving the sensitivity of progility of sensitivity. weighting, positions-specific gap penalties and weight matrix choices "Nucleic Acids Res, 22: 4673-4680], version 1.8. The CLUSTALW algorithm can be executed, for example, at http: // www. ebi. ac. It can be easily used at many sites on the Internet including uk. The number of matches in the alignment is then determined by counting the number of nucleotides (or amino acid residues) that are identical at each aligned position. Finally, the percent identity of the first sequence relative to the second sequence is calculated by dividing the total number of matches by the number of longer nucleotides (or amino acid residues) of the two sequences and multiplying by 100.

  3. Method of said 1st point or said 2nd point whose said test | inspection of said reaction is prediction of the probability of the success of chemotherapy.

  4). The method of point 3 above, wherein the success of chemotherapy is understood as a reduction in tumor mass.

  5. The method according to any one of the first to fourth points, wherein the neoplastic lesion is breast cancer or ovarian cancer.

6). The chemotherapy is
(I) affect cell proliferation, and / or (ii) affect cell survival, and / or (iii) affect cell motility,
Any method from the first point to the fifth point.

  7. 7. Any method from point 1 to point 6 above, wherein the chemotherapy is an anthracycline-based chemotherapy.

  8). 7. The method of point 7 above, wherein the anthracycline-based chemotherapy is epirubicin or doxorubicin-based chemotherapy.

  9. Any of the first to eighth points above, wherein a prediction algorithm is used.

  Predictive algorithms are well known to those skilled in the field of data analysis and should be understood to be any type of predictive algorithm known in the art. A preferred example of such an algorithm is, for example, the SVM algorithm disclosed in Example 4, k-Nearest Neighbor Analysis, or Linear Discriminant Analysis.

10. The prediction algorithm is
The method of the ninth point, which is (i) a support vector machine algorithm, or (ii) a k-nearest neighbor algorithm, or (iii) a partial minimum discrimination algorithm.

11. The expression level is
(I) using hybridization-based methods, or (ii) using hybridization-based methods using arrayed probes, or (iii) hybridization-based methods using individually labeled probes. Or (iv) by real-time PCR, or (v) by assessing the expression of a polypeptide, protein, or derivative thereof, or (vi) by assessing the amount of a polypeptide, protein, or derivative thereof By
Any method from the first point to the tenth point is determined.

  12 4. A method of selecting an individually optimized anti-cancer treatment for a patient, comprising the method of claim 1 or 2 or 3.

  The term “personalized cancer treatment” or “individually optimized anti-cancer treatment” as used herein means that all persons with cancer have their physique and cancer molecules It means the observation that the repertoire is clearly different from other patients. Patient tumors that are analyzed and molecularly classified as different then receive personalized treatment based on the type of treatment or combination of treatments that maximizes the uptake of chemotherapeutic drugs into the patient's cancer. This allows for the selection of individual patients that may require one type of chemotherapy or different combinations of multiple chemotherapy. Such “personalized cancer treatment” can be reflected in the administration schedule, drug concentration, treatment sequence, and the addition of ancillary interventions (eg, immune stimulation).

Experimental Procedures and Setup The present invention is directed to gene expression in major breast cancer at the time point after the first cycle of chemotherapy treatment given by epirubicin / cyclophosphamide (EC) or epirubicin / taxol (ET) treatment. It relates to the identification of effects and the use of these expression patterns to identify non-reactive / reactive subjects.

  Cyclophosphamide and epirubicin are common treatments for advanced metastatic breast cancer. In addition, epirubicin treatment has the advantage that the anthracycline-induced cardiotoxicity is clinically obvious in contrast to doxorubicin (adriamycin), which has the same mode of action class and is used more frequently. It is expensive. Taxanes are quickly established to be an important chemotherapeutic agent among a set of drugs for treating breast cancer. Expression profiles in 25 pairs of pre- and post-treatment biopsy pairs have been obtained by low density cDNA arrays (Clontech) and by use of oligonucleotide microarrays (Affymetrix).

  After analyzing 25 pairs of data by applying Student's t-test, the inventors found that only a few genes were indefinitely stimulated after breast tumor treatment. A number of more genes were also found to be up / down-regulated 24 hours after treatment in at least some of the patients analyzed. In order to develop a discriminative minimal gene set for subsequent classification, partial minimal discriminant analysis (PLS-DA) and step-down permutation methods were applied to the data set. PLS-DA can identify samples before and after treatment based on about 25 transcripts from the entire data set.

  In the treated sample, 479 genes were found to be upregulated. Major categories of these genes include cytoskeleton / structural proteins such as collagen, elastin, filamin, fibroblast factors and receptors, cell adhesion / extracellular matrix and some growth arrest proteins, and DNA damage induction Proteins (eg, GAS6, GADD45B) are included. Repressed genes are in several major groups, including ribosomal proteins, nucleotide and protein synthesis, processing, transcription factors, and groups of several oncogenes (eg, c-myc, K-ras, bcl-2) Can be subdivided.

  To identify genes that expressed similar to the p21WAF1 / CIP1 expression profile, a k-means clustering analysis was performed.

  To identify genes whose expression is induced by therapy, profiles of pre- and post-treatment breast cancer biopsies from 25 patients were used with Clontech's Atlas ™ human cancer 1.2 low density cDNA array. Acquired.

  Inventor to classify 50 biopsy samples based on their similarity measured for 249 genes scored to be expressed at an average level in at least 6 out of 50 samples A hierarchical clustering algorithm [(65, 66)] was used. Based on these analyses, the following observations were made. First, pre- and post-treatment biopsy pairs were clustered together in over 90% of cases. This reflects that the expression pattern of tissue samples obtained from the same tumor shows a more conserved expression pattern than that observed by inter-individual dispersion obtained from another patient. Second, the expression pattern was significantly different between different tumors. Third, tumor samples are subdivided into three major subclasses presented in Table 4 regardless of their ER status by hierarchical clustering.

  In the expression data analyzed by two-dimensional original gene clustering, genes with similar expression levels are grouped together on the vertical axis, and tumor samples are clustered on the horizontal axis according to their global expression profile.

Characterization and preselected 249 genes formed separate clusters correlated with gene expression. Genes within one cluster reflected the expression of genes typical in endothelial cells and B lymphocytes. This indicates the possibility of lymphocyte infiltration into the tumor tissue. The second gene cluster consists of oncogenes such as MYC, VEGF, and MYB and growth factors. Yet another cluster contains the genes v-erb-b2 / HER2, keratin 19, 18, which are also known to be expressed separately in certain breast tumors. The fourth cluster contains a group of genes with expression correlated with p21 ^{WAF1 / CIP1} and MIC1 (prostate differentiation factor). The fifth distinct gene cluster is genes involved in cell cycle control and cell proliferation. The genes of these five subpopulations reflect the major expression phenomena that are the result of the effects of a given drug.

  The inventors further analyzed 10 pairs of gene expression out of 25 pairs of breast cancer samples using hybridization of the extracted RNA to 22284 sequence Affymetrix oligonucleotide microarrays. An aliquot of the same total RNA previously used in experiments with the Clontech array was analyzed on an Affymetrix array. Expression of 4906 transcripts hybridizing to the chip surface was reliably detected in all biopsy samples. According to the manufacturer's analysis software MAS5.0 and the quality control signal “absolute call” used for all the paired samples, 15122 transcripts can be detected in at least one of the 10 pairs. , 7161 transcript could not be detected (see Example 4).

  An unsupervised analysis of gene expression in 10 pairs of pre- and post-treatment cancer biopsy pairs was performed based on a precision-tested gene set of 6980 sequences, the entire contents of HG-133A GeneChip. Quality control and gene selection were based on signal intensity levels in individual arrays and reliable detection. A hierarchical cluster algorithm ordered pre-treatment and post-treatment biopsies for all 10 patients except those previously observed. This may be due to the fact that a larger number of sequences were used in this analysis as opposed to 249 genes selected from the filter array. Thus, the high coverage of genes and putative cell mechanisms and pathways increases the rate of sample pairs that are clustered together.

Identification of differentially expressed genes. Paired t-tests were used to identify transcripts that were differentially expressed in pre-treatment and post-treatment samples. After selecting 249 expressed genes, a group of 19 genes that showed high frequency enhancement or reduction under EC or ET treatment was identified (Student t test, P <0.05). Table 3 shows genes that were significantly differentially regulated at a ratio of> 1.8 or <0.6 in multiple sample pairs within the first 24 h of chemotherapy for both treatment groups. The range of fold changes in regulated transcripts is broad, probably reflecting diversity among patients. In more than 95% of all post-treatment samples, two genes, cyclin-dependent kinase inhibitor 1 (p21 ^{WAF1 / CIP1} ) and prostate differentiation factor (MIC-1) were up-regulated.

  To identify the effect of treatment on gene expression, the same analysis was performed on expression data obtained using the Affymetrix GeneChip system, and a group of 37 genes that were highly regulated was identified (Table 3). ). As seen in the data obtained from gene hybridization to the filter array, p21WAF1 / CIP1 and MIC-1 were most significantly upregulated in the post-treatment samples. However, the relative signal intensity detected with MIC-1 was lower with the Affymetrix system than with the Clontech array.

  Unlike previous publications that deal with predicting patient disease outcomes, the inventors identified genes that would change expression levels immediately after treatment without applying additional information about patient response to the analysis. Aimed to do. For this study, we selected the difference in expression immediately after the start of treatment. Important cellular processes such as proliferation, DNA repair, and apoptosis often occur up to 48 hours after exposure to chemotherapy [(101, 102)].

  Nevertheless, appropriate statistical analysis can identify differentially expressed genes in pre- and post-treatment biopsies, distinguishing between reactive and non-reactive tumors .

Biologically important genes that are part of the invention Some of the genes listed in Table 1 represent biological cellular processes and are characterized by similar gene regulation. By way of example, but not by way of limitation, some characteristic genes in Table 1 are described in more detail below.

ERCC1
ERCC1 was so named to represent “removal repair that complements repair failures in Chinese hamsters”. The following DNA-mediated gene transfer was performed to mutant Chinese hamster ovary cells that are sensitive to various DNA damaging reagents and have a defect in the cleavage process at the early stage of DNA repair, such as xeroderma pigmentosum cells. The resulting transformants exhibited normal resistance to DNA damaging reagents and independent transformants exhibited a common set of human DNA sequences associated with human DNA repair genes. Homozygous mutant mice ceased to grow at birth and died of liver failure before weaning. Increased p53 levels were detected in liver, brain and kidney. This supports the putative role of p53 in monitoring DNA damage. The ERCC1 gene was not found to be associated with any particular disease and was only indirectly estimated to be essential. XPA, ERCC1, and ERCC4 are proteins that form a ternary complex that is involved in both damage recognition and cleavage activity.

MSH6 (GTBP)
This mismatch binding factor is part of a heterodimer of 100-kD MSH2 and a 160-kD polypeptide called GTBP (representing a G / T binding protein). Sequence analysis recognized that GTBP is a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the new finding that tumor-derived cell lines lacking either protein also lack mismatch binding activity. All homologues of MutS protein contain a strongly conserved region of about 150 amino acids. It includes a helix-turn-helix region that binds to adenine nucleotides and a magnesium-binding motif called the Walker-A motif. This part of the MutS molecule has ATPase activity. The MSH2-MSH6 complex is “on” (binds to a mismatched nucleotide) when it is ADP-bound and “off” when it is ATP-bound. ATP hydrolysis results in mismatch bond recovery, while ADP to ATP substitution results in mismatch dissociation. People have found that BRCAI is part of large multi-subunit proteins, tumor suppressor complexes, DNA damage sensors, and signal transducers. They named this complex BASC after “BRCA1-related genomic surveillance complex”.

  Endometrial cancer is the most common gynecological malignancy in the United States and the most frequent extracolonic tumor in hereditary nonpolyposis colorectal cancer (HNPCC). Sporadic endometrial cancer usually displays microsatellite instability (MSI) associated with methylation of the MLH1 promoter. Germline MSH6 mutations are rare in HNPCC but have been reported in several families with multiple members suffering from endometrial cancer. Form of hereditary non-polyposis colorectal cancer due to mutation of MSH2 is type 1 (HNPCC1); type 2 due to MLH1 (HNPCC2); type 3 due to mutation of PMS1 (HNPCC3); type 4 due to mutation of PMS2 (HNPCC4); and it is proposed here to call the type 5 (HNPCC5) due to the mutation of MSH6 (GTBP).

DDB2
In human cells, the p53 tumor suppressor gene is required for efficient global genome repair of UV-induced DNA damage. The DDB2 (p48) gene is required for the expression of UV-damaged DNA binding activity, and in a subset of xeroderma pigmentosum group E cells lacking this activity, ie, DDB negative XPE, this gene is destroyed by mutation. p48 mRNA levels are strongly dependent on basal levels of p53 expression and are further enhanced in a p53-dependent manner after DNA damage. Furthermore, like p53 − / − cells, xeroderma pigmentosum group E cells lack a global genome repair. Based on these results, p48 was identified as the junction between p53 and the nucleotide excision repair apparatus. Transfection with p48 conferred UV-DDB on hamster cells and facilitated removal of cyclobutane pyrimidine dimer (CPD) from genomic DNA and untranscribed strands of expressed genes. p48 expression suppressed UV-induced mutations resulting from non-transcription but did not affect the UV sensitivity of the cells. Results confirm the role of p48 in DNA repair, demonstrate the importance of CPD in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans did.

MGMT
O (6) -alkylguanine has a tendency to pair with thymine during DNA replication, making it a major mutagenic and carcinogenic lesion in DNA induced by simple alkylating mutagens . This adduct in DNA is removed by O (6) -methylguanine-DNA methyltransferase (EC 2.1.1.63), a widely distributed unique repair protein. Unlike a true enzyme, this protein accepts alkyl groups from the site of damage in a stoichiometric two-dimensional reaction. The methyl acceptor residue is cysteine.

  The MGMT promoter in tumor DNA was analyzed by a methylation-specific PCR assay to determine whether MGMT promoter methylation was related to glioma responsiveness to alkylating agents. The MGMT promoter was methylated in 19 (40%) gliomas out of 47 patients. This new finding is associated with tumor regression and overall and disease-free survival. It was a stronger prognostic predictor independent of age, stage, tumor grade, or performance status.

Polynucleotides “BREAST CANCER GENE” polynucleotides may be single stranded or double stranded and comprise a coding sequence for a “BREAST CANCER GENE” polypeptide or a complement of the coding sequence. A degenerate nucleotide sequence encoding a human “BREAST CANCER GENE” polypeptide, and at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% of the nucleotide sequence of SEQ ID NOs: 1-65 An identical homologous nucleotide sequence is also a “BREAST CANCER GENE” polynucleotide. Percent sequence identity between two polynucleotide sequences is determined using an affine gap search with a gap start penalty of -12 and a gap extension penalty of -2 using a computer program such as ALIGN utilizing the FASTA algorithm. To do. Complementary DNA (cDNA) molecules, species homologues, and variants of “BREAST CANCER GENE” polynucleotides that encode biologically active “BREAST CANCER GENE” polypeptides are also “BREAST CANCER GENE” polynucleotides.

Polynucleotide Preparation Naturally occurring “BREAST CANCER GENE” polynucleotides that are free of other cellular components such as membrane components, proteins, and lipids can be isolated. Polynucleotides can be produced in cells and isolated using standard nucleic acid purification techniques, synthesized using amplification techniques such as polymerase chain reaction (PCR), or using automated synthesizers. You can also. Methods for isolating polynucleotides are routine and well known in the art. Any technique for obtaining a polynucleotide can be used to obtain an isolated “BREAST CANCER GENE” polynucleotide. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments comprising a “BREAST CANCER GENE” nucleotide sequence. An isolated polynucleotide is one that is in a preparation that is free of other molecules in a proportion of at least 70%, 80%, or 90%.

  "BREAST CANCER GENE" cDNA molecules can be generated using standard molecular biology techniques, using "BREAST CANCER GENE" mRNA as a template. Any RNA isolation technique that does not select against mRNA isolation can be used to purify the RNA sample. See, for example, Sambrook et al., 1989 (6); and Ausubel, F .; M.M. Et al., 1989 (7). Both of these are hereby incorporated by reference in their entirety. Further, for example, Chomczynski, P .; Large volume tissue samples can be readily processed using techniques well known to those skilled in the art, such as the one-step RNA isolation method (1989, US Pat. No. 4,843,155). This disclosure is incorporated herein by reference in its entirety.

  The “BREAST CANCER GENE” cDNA molecule can then be replicated using molecular biology techniques known in the art and those disclosed in manuals such as Sambrook et al., 1989 (6). Using either human genomic DNA or cDNA as a template, amplification techniques such as PCR can be used to obtain additional copies of the polynucleotides of the invention.

  Alternatively, “BREAST CANCER GENE” polynucleotides can be synthesized using synthetic chemistry techniques. The degeneracy of the genetic code allows the synthesis of alternative nucleotide sequences encoding “BREAST CANCER GENE” polypeptides or biologically active variants thereof.

Identification of differential expression Transcripts representing RNA produced by differentially expressed genes in collected RNA samples should be identified using a variety of methods well known to those skilled in the art. Can do. To identify polynucleotide sequences derived from differentially expressed genes, for example, differential screening [Tedder, T. et al. F. 1988 (8)], subtractive hybridization [Hedrick, S .; M.M. 1984 (9)], and preferably a differential display (Liang, P., and Pardee, AB, 1993, US Pat. No. 5,262,311), the disclosure of which is hereby incorporated by reference in its entirety. To use).

  In differential screening, a duplicate screening of a cDNA library is performed, with one copy of the library being screened with a whole cell cDNA probe corresponding to an mRNA population of a cell type, and a duplicate copy of the cDNA library being Screen with total cDNA probe corresponding to mRNA population of the second cell type. For example, one cDNA probe may correspond to a whole cell cDNA probe of a cell type obtained from a control subject, and a second cDNA probe may be a whole cell cDNA probe of the same cell type derived from an experimental subject. Corresponding ones are acceptable. Clones that hybridize to one probe but not to the other probe represent clones derived from genes differentially expressed in the cell type of interest in the experimental versus control subjects. There is a possibility to be.

  Subtractive hybridization techniques typically involve the isolation of mRNA taken from two different sources, such as control and experimental tissues, and the high level of mRNA or reverse-transcribed single-stranded cDNA from the isolated mRNA. Hybridization and removal of all sequences that hybridize and are thus double-stranded. The remaining non-hybridized single stranded cDNA may be representative of clones derived from genes that are differentially expressed in the two mRNA sources. Such single stranded cDNA is then used as a starting material to construct a library containing clones derived from differentially expressed genes.

  The differential display technique utilizes the well-known polymerase chain reaction (PCR; experimental embodiments are described in Mullis, KB, 1987, US Pat. No. 4,683,202) and differential expression. Means a procedure that allows the identification of sequences derived from the generated genes. First, the isolated RNA is reverse transcribed into single stranded cDNA using standard techniques well known to those skilled in the art. Primers for the reverse transcriptase reaction may include, but are not limited to, oligo dT-containing primers, preferably reverse primer type oligonucleotides described below. This technique then uses PCR primer pairs that allow amplification of clones representing random subpopulations of RNA transcripts present in any given cell, such as those described below. Utilizing different primer pairs allows amplification of each mRNA transcript present in the cell. Among the transcripts so amplified, it is possible to identify transcripts that are differentially produced from the expressed gene.

  The reverse oligonucleotide primer of the above primer pair has, at its 5 ′ end, an oligo dT nucleotide region that hybridizes to the poly (A) tail of mRNA or the complementary strand of cDNA reverse transcribed from the mRNA poly (A) tail. This oligo dT region is preferably 11 nucleotides in length. Secondly, the reverse primer can contain one or more, preferably 2 additional nucleotides at its 3 'end to enhance its specificity. Of the mRNA-derived sequences present in the sample of interest, those that hybridize to such primers will be statistically only partial, so by adding nucleotides, It is possible to amplify only a part of the mRNA-derived sequence present in the sample. This is preferred in that it allows more accurate and complete visualization and characterization of each band representing the amplified sequence.

  The forward primer may contain a nucleotide sequence that is statistically predicted to have the ability to hybridize to a cDNA sequence derived from the tissue of interest. The nucleotide sequence can be arbitrary and the length of the forward oligonucleotide primer can range from about 9 to about 13 nucleotides, with about 10 nucleotides being preferred. Arbitrary primer sequences make the length of the partial cDNA amplified variable, thus allowing for the separation of different clones by use of standard denaturing sequencing gel electrophoresis. PCR reaction conditions should be chosen to optimize the yield and specificity of the product to be amplified, in addition to producing amplified products of a length that can be separated using standard gel electrophoresis techniques. You should choose as you like. Such reaction conditions are well known to those skilled in the art, and important reaction parameters include, for example, the length and nucleotide sequence of the oligonucleotide primer, as well as the temperature and reaction time of the annealing and extension steps, as described above. Clone patterns resulting from the reverse transcription process and amplification of mRNA of two different cell types are presented and compared via sequencing gel electrophoresis. Differences between the two band patterns indicate genes that may be differentially expressed.

  When screening for full-length cDNAs, it is preferable to use a library that has been size-selected to contain a relatively large cDNA. Libraries primed with random primers are desirable in that they will contain more sequences containing the 5 'region of the gene. In situations where full length cDNA is not produced by an oligo d (T) library, it may be particularly desirable to use a library primed with random primers. Genomic libraries can be useful for extending sequences to the 5 'non-transcribed regulatory region.

  Commercially available capillary electrophoresis systems can be used to analyze or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing using flowable polymers for electrophoretic separation, four different fluorescent dyes activated by laser (per nucleotide), and detection of emission wavelength by a charge coupled device camera It can be performed. The output / luminosity can be converted to an electrical signal using appropriate software (eg, ABI's GENOTYPER and Perkin Elmer's Sequence NAVIGATOR) to complete the entire process from sample addition to computer analysis and electronic data display. Can be computer controlled. Capillary electrophoresis is particularly preferred for sequencing small pieces of DNA, which may be present in a limited amount in a particular sample.

  If gene sequences that may be differentially expressed are identified by high-throughput techniques, such as those described above, demonstration of differential expression of putatively differentially expressed genes can be demonstrated. Should be done. Demonstration can be achieved through well-known techniques such as, for example, Northern analysis and / or RT-PCR. Upon demonstration, the differentially expressed genes can be further characterized and identified as target and / or marker genes as discussed below.

  Alternatively, an amplified sequence of a differentially expressed gene obtained, for example, through a differential display method can be used to isolate a full-length clone of the corresponding gene. The full length coding portion of the corresponding gene can be easily isolated without undue experimentation by molecular biology techniques well known in the art. For example, differentially expressed, amplified, and isolated fragments can be labeled and used to screen cDNA libraries. Alternatively, the labeled fragment can be used to screen a genomic library.

  Standard techniques well known to those skilled in the art can be used to analyze the biodistribution of mRNA produced by the identified gene. Such techniques can include, for example, Northern analysis and RT-PCR. Such an analysis provides information regarding whether the identified gene is expressed in tissues predicted to contribute to breast cancer. Such an analysis provides quantitative information regarding steady state mRNA regulation, and any of the identified genes show a high level of regulation, preferably in tissues that may be expected to contribute to breast cancer. May produce data on

  Such an analysis may be performed on an isolated cell population of a particular cell type derived from a given tissue. In addition, standard in situ hybridization techniques can be used to obtain information regarding which cells in a given tissue express the identified gene. Such an analysis may provide information on the biological function associated with breast cancer of the identified gene if only a subset of the cells in the tissue are considered to be associated with breast cancer.

Polynucleotide Extension In one embodiment of a procedure for identifying and cloning full-length gene sequences, RNA can be isolated from an appropriate tissue or cell source according to standard procedures. Thereafter, a reverse transcription process reaction of RNA can be performed using a first strand synthesis priming oligonucleotide primer complementary to the mRNA corresponding to the fragment to be amplified. Since this primer is antiparallel to the mRNA, the extension will continue towards the 5 'end of the mRNA. The resulting RNA hybrid can then be “tailed” with guanine using a standard terminal transferase reaction. The hybrid can be digested with RNase H and then primed for second strand synthesis using a poly C primer. Two primers are used and PCR is used to amplify the 5 'portion of the gene. The resulting sequence is then isolated and recombined with the previously isolated sequence to produce the full-length cDNA of the differentially expressed gene of the present invention. See, for example, Sambrook et al. (6); and Ausubel et al. (7) for an overview of cloning strategies and recombinant DNA techniques.

  A variety of PCR-based methods can be used to extend the polynucleotide sequences disclosed herein and to detect upstream sequences such as promoters and regulatory elements. For example, restriction site PCR uses universal primers to recover unknown sequences adjacent to a known locus [Sarkar, 1993 (10)]. First, genomic DNA is amplified in the presence of primers for the linker sequence and primers specific for a known region. The amplified sequence is then subjected to a second round of PCR using the same linker primer and another specific primer for the interior of the first. Each round of PCR product is transcribed with the appropriate RNA polymerase and sequenced with reverse transcriptase.

  Inverse PCR can also be used to amplify or extend sequences using a wide variety of primers based on known regions [Triglia et al., 1988 (11)]. Primers can be obtained using commercially available software such as, for example, OLIGO 4.06 primer analysis software (National Biosciences, Plymouth, Minn., USA), for example, having a GC content of 50% so that it is 2230 nucleotides in length. As such, it can be designed to anneal to the target sequence at a temperature of about 68-72 ° C. In this method, several restriction enzymes are used to generate appropriate fragments within a known region of the gene. This fragment is then circularized by intramolecular ligation and used as a PCR template.

  Another method that can be used is capture PCR, which performs PCR amplification of DNA fragments adjacent to known human sequences and yeast artificial chromosomal DNA [Lagerstrom et al., 1991 (12)]. In this method, multiple restriction enzyme digestion and ligation can also be used to place the genetically engineered double-stranded sequence into an unknown fragment of the DNA molecule prior to performing PCR.

  In addition, PCR, nested primers, and the PROMOTERFINDER library (Clontech, Palo Alto, CA, USA) can be used to perform genomic DNA walking (Clontech, Palo Alto, CA, USA). This process avoids the need to screen the library and is useful for finding intron / exon junctions.

  The sequence of the identified gene can be determined using standard techniques, such as the location of the gene in a genetic map such as the mouse [Copeland and Jenkins, 1991 (13)], human genetic map [Cohen et al., 1993 (14)] Can be used to determine Such mapping information can generate information about the importance of that gene to the disease, for example by identifying a gene that maps near the genetic region to which a known genetic breast cancer trend is mapped. is there.

Identification of Polynucleotide Variants and Homologues or Splice Variants Variants and homologues of the “BREAST CANCER GENE” polynucleotides described above are also “BREAST CANCER GENE” polynucleotides. Homologous “BREAST CANCER GENE” polynucleotide sequences are typically identified by hybridizing candidate polynucleotides to known “BREAST CANCER GENE” polynucleotides under stringent conditions, as is known in the art. Can do. For example, the following washing conditions: 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, twice at room temperature for 30 minutes each; then 2 × SSC, 0 Identifying homologue sequences containing up to about 25-30% base pair mismatch using 1% SDS, 50EC once 30 minutes; then 2 × SSC, 2 times 10 minutes at room temperature it can. More preferably, the homologous polynucleotide strand contains 15-25% base pair mismatch, and even more preferably 5-15% base pair mismatch.

  Species homologues of the “BREAST CANCER GENE” polynucleotides disclosed herein can also be made by making appropriate probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Can be identified. Human variants of “BREAST CANCER GENE” polynucleotides can be identified, for example, by screening a human cDNA expression library. It is well known that the Tm of double-stranded DNA decreases by 1 to 1.5 ° C. every time the homology decreases by 1% [Bonner et al., 1973 (15)]. Thus, by hybridizing a polynucleotide having one nucleotide sequence of SEQ ID NOs: 1 to 65 or a complementary sequence thereof with a putative homolog “BREAST CANCER GENE” polynucleotide to form a test hybrid, Variants of human “BREAST CANCER GENE” polynucleotides or “BREAST CANCER GENE” polynucleotides of other species can be identified. The melting temperature of the test hybrid is compared to the melting temperature of a hybrid containing a polynucleotide having a completely complementary nucleotide sequence to calculate the number or percentage of base pair mismatches in the test hybrid.

A nucleotide sequence that hybridizes to a “BREAST CANCER GENE” polynucleotide or a complementary strand thereof according to stringent hybridization and / or wash conditions is also a “BREAST CANCER GENE” polynucleotide. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al. (6). For stringent hybridization conditions, one should typically select a combination of temperature and salt concentration 12 to 20 ° C. lower than the Tm calculated for the hybrid under study. A “BREAST CANCER GENE” polynucleotide having a nucleotide sequence of one of the sequences of SEQ ID NOs: 1 to 65 or its complementary sequence, and at least about 50, preferably about 75, 90, 96, or one of those nucleotide sequences, The Tm of a hybrid between 98% identical polynucleotide sequences is, for example, the following equation:
Tm = 81.5 ° C.-16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) −0.63 (% formamide) −600 / l)
Where l = length of hybrid (base pairs) [Bolton and McCarthy, 1962 (16)].

  Stringent wash conditions include, for example, 4 × SSC, 65 ° C., or 50% formamide, 4 × SSC, 28 ° C., or 0.5 × SSC, 0.1% SDS, 65 ° C. Highly stringent washing conditions include, for example, 0.2 × SSC, 65 ° C.

  The biological function of the identified gene can be more directly assessed by utilizing appropriate in vivo and in vitro systems. In vivo systems include animal systems that exhibit a natural breast cancer constitution, or animal systems that have been genetically engineered to exhibit such symptoms, including but not limited to oncogene overexpression (eg, HER2 / Neu, ras, raf, or EGFR) malignant tumor mice may be included but are not limited to.

  Splice variants are derived from the same genomic region and are encoded by the same pre-mRNA and can be identified by the hybridization conditions described above for homology search. The specific characteristics of variant proteins encoded by splice variants of the same pre-transcript may differ and these can also be assayed as disclosed. Thus, a “BREAST CANCER GENE” polynucleotide having a nucleotide sequence of one of the sequences of SEQ ID NOs: 1 to 65 or a complementary sequence may differ in the entire sequence. Prediction of splicing events and identification of acceptor and donor sites used in the pre-mRNA can be computerized (eg, GRAIL or GenomeSCAN software package) and confirmed by PCR methods by those skilled in the art.

Antisense oligonucleotides Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. When introduced into a cell, complementary nucleotides combine with the natural sequence produced by the cell to form a complex and block either transcription or translation. Antisense oligonucleotides are preferably at least 6 nucleotides in length, but are at least 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or more than 50 nucleotides in length. There may be. Longer sequences can also be used. Antisense oligonucleotide molecules can be prepared in a DNA construct and introduced into a cell as described above to modify the level of the “BREAST CANCER GENE” gene product in the cell.

  Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA, described in US Pat. No. 5,714,331), lock nucleic acids (LNA; described in WO 99/12826), or combinations thereof. Oligonucleotides include alkyl phosphonates, phosphorothioates, phosphorodithioates, alkyl phosphonothioates, alkyl phosphonates, phosphoramidates, phosphate esters, carbamates, acetamidates, carboxymethyl esters, carbonates, and phosphate triesters. Non-phosphodiester internucleotide linkages can be synthesized manually or by an automated synthesizer by covalently attaching the 5 ′ end of one nucleotide to the 3 ′ end of another nucleotide [Brown 1994 (46)].

  By altering the "BREAST CANCER GENE" control, 5 ', or an antisense oligonucleotide that will double-stranded with the regulatory region, modification of the "BREAST CANCER GENE" expression can be made. A transcription start site, for example, an oligonucleotide derived between a position −10 and a position +10 from the start site is preferred. Similarly, inhibition can be performed using the “triple helix” base-pairing method. Triple helix pairing is useful because it causes suppression of the ability of the double helix to undergo sufficient cleavage for polymerase, transcription factor, or chaperone binding. Therapeutic advances using triplex DNA are described in the literature [Gee et al., 1994 (47)]. Antisense oligonucleotides can also be designed to block translation of mRNA by preventing transcription products from binding to ribosomes.

  Exact complementarity is not required to establish a complex between the antisense oligonucleotide and the complementary sequence of the “BREAST CANCER GENE” polynucleotide. For example, 2, 3, 4, or 5 or more consecutive nucleotides exactly complementary to a “BREAST CANCER GENE” polynucleotide, each separated by consecutive nucleotide stretches that are not complementary to adjacent “BREAST CANCER GENE” nucleotides An antisense oligonucleotide comprising a stretch can provide sufficient target specificity for a “BREAST CANCER GENE” mRNA. Each complementary contiguous stretch of nucleotides is preferably at least 4, 5, 6, 7, or 8 nucleotides or more in length, respectively. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily calculate the degree of mismatch allowed between a particular antisense oligonucleotide and a particular “BREAST CANCER GENE” polynucleotide sequence using a calculation of the melting point per pair of antisense-sense pairs. Can be determined.

  Modifications of antisense oligonucleotides can be made without affecting their ability to hybridize to “BREAST CANCER GENE” polynucleotides. These modifications may be within the antisense molecule or at one or both ends thereof. For example, internucleotide phosphate linkages can be modified by adding various numbers of carbon residue cholesteryl or diamine moieties between the amino group and the terminal ribose. Modified bases and / or sugars may also be used in modified antisense oligonucleotides, such as arabinose as an alternative to ribose, or 3 ′, 5 ′ substituted oligonucleotides substituted with a 3 ′ hydroxyl group or a 5 ′ phosphate group. it can. These modified oligonucleotides can be prepared by methods well known in the art [Uhlmann et al., 2000 (48)].

Ribozymes Ribozymes are catalytic RNA molecules [Couture and Stinchcomb, 1996 (49)]. Ribozymes can be used to suppress gene function by cleaving RNA sequences, as is known in the art (eg, Haseloff et al., US Pat. No. 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of ribozyme molecules to complementary target RNA, followed by nucleotide strand breaks. Examples include engineered hammerhead motif ribozyme molecules that can catalyze specific and efficient nucleotide strand breaks of specific nucleotide sequences.

  The “BREAST CANCER GENE” transcription sequence can be used to generate ribozymes that specifically bind to mRNA transcribed from the “BREAST CANCER GENE” genomic locus. Methods for designing and constructing ribozymes that can cleave other RNA molecules in trans with high sequence specificity have been developed and described in the art [Haseloff et al., 1988 (50)]. For example, specific RNAs can be targeted for ribozyme cleavage activity by constructing separate “hybridization” regions in the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target [see, eg, Gerlach et al., European Patent No. 0321201].

  Specific ribozyme cleavage sites within the “BREAST CANCER GENE” RNA target can be identified by scanning the target molecule for ribozyme cleavage sites including the following sequences: GUA, GUU, and GUC. Once identified, a short RNA sequence of 15 to 20 ribonucleotides corresponding to the target RNA region containing the cleavage site can be evaluated for secondary structural properties that can render the target inoperable. The suitability of a candidate “BREAST CANCER GENE” RNA target can also be assessed using a ribonuclease protection assay by testing its accessibility to hybridization with a complementary oligonucleotide. Longer complementary sequences can also be used to enhance the affinity of the target hybridization sequence. The hybridization region and cleavage region of the ribozyme can be integrated so that the catalytic region of the ribozyme can cleave the target when hybridizing to the target RNA via a complementary region.

  Ribozymes can be introduced into cells as part of a DNA construct. Using mechanical methods such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, DNA constructs containing ribozymes can be introduced into cells where reduction of “BREAST CANCER GENE” expression is desired. Alternatively, if it is desired that the cell stably hold the DNA construct, the construct may be supplied on a plasmid and incorporated as a separate element or into the cell genome, as is known in the art. Can be maintained. A DNA construct encoding a ribozyme can include transcriptional control elements such as a promoter element, an enhancer, or a UAS element, and a transcription terminator signal to control transcription of the ribozyme in the cell.

  As taught in Haseloff et al., US Pat. No. 5,641,673, ribozymes can be engineered such that ribozyme expression occurs in response to factors that induce target gene expression. By providing an additional level of regulation, the ribozyme can be engineered such that mRNA destruction occurs only when both the ribozyme and the target gene are introduced into the cell.

Polypeptides A “BREAST CANCER GENE” polypeptide according to the present invention is a polypeptide selected from either a polypeptide selected from SEQ ID NOs: 66 to 130, or a polynucleotide sequence of SEQ ID NOs: 1 to 65, and derivatives thereof, Includes fragments, analogs, and homologs. Thus, a “BREAST CANCER GENE” polypeptide of the invention can be a portion, full length, or fusion protein comprising all or part of a “BREAST CANCER GENE” polypeptide.

Protein Purification “BREAST CANCER GENE” polypeptides can be purified from any cell that expresses the corresponding protein, including host cells transfected with a “BREAST CANCER GENE” expression construct. Purified “BREAST CANCER GENE” polypeptides are derived from other compounds normally associated with “BREAST CANCER GENE” polypeptides in cells, such as certain proteins, carbohydrates, or lipids, by methods well known in the art. Are separated. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A purified “BREAST CANCER GENE” polypeptide preparation is at least 80% pure, and the preparation is preferably 90%, 95%, or 99% pure. The purity of the preparation can be assessed by any method known in the art, such as SDS-polyacrylamide electrophoresis.

Obtaining Polypeptides “BREAST CANCER GENE” polypeptides can be obtained, for example, by purification from human cells, by expression of “BREAST CANCER GENE” polynucleotides, or by direct chemical synthesis.

Biologically Active Variants Biologically active “BREAST CANCER GENE” polypeptide variants, ie, polypeptide variants that retain “BREAST CANCER GENE” activity, can also be considered “BREAST CANCER GENE” polypeptides. A naturally occurring or non-existing “BREAST CANCER GENE” polypeptide variant is a polypeptide encoded by either the amino acid sequence of the polypeptide of SEQ ID NO: 66-130, or the polynucleotide of SEQ ID NO: 1-65, or a fragment thereof Having an amino acid sequence that is at least about 60, 65, or 70%, preferably 75, 80, 85, 90, 92, 94, 96, or 98% identical to any of the above.

  The percent identity difference can be due, for example, to an amino acid substitution, insertion, or deletion. Amino acid substitutions are defined as one to one amino acid substitutions. If substituted amino acids have similar structural and / or chemical properties, their properties are preserved. Substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, or substitution of threonine with serine are examples of conservative substitutions.

  Amino acid insertions or deletions are changes to or within the amino acid sequence. They are usually in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or removed without compromising the biological or immunological activity of the “BREAST CANCER GENE” polypeptide is well known in the art, such as DNASTAR software. Can be obtained using a computer program. Whether an amino acid change results in a biologically active “BREAST CANCER GENE” polypeptide can be readily determined, for example, by assaying for “BREAST CANCER GENE” activity as described in the specific examples below. Larger insertions or deletions can also occur by alternative splicing. Protein domains can be inserted or removed without altering the main activity of the protein.

Fusion Proteins Fusion proteins are useful in generating antibodies against “BREAST CANCER GENE” polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a “BREAST CANCER GENE” polypeptide. For this purpose, protein affinity chromatography or library-based protein-protein interaction assays such as yeast double hybrid methods or phage display systems can be used. Such methods are well known in the art and can also be used as drug screens.

  A “BREAST CANCER GENE” polypeptide fusion protein comprises two polypeptide segments fused together by peptide bonds. The first polypeptide segment is at least 25, 50, 75, 100, encoded by a polynucleotide sequence of any of SEQ ID NOs: 1-65, or a biologically active variant such as those described above. It includes an amino acid sequence consisting of 150, 200, 300, 400, 500, 600, 700, or 750 consecutive amino acids. The first polypeptide segment can also include a full-length “BREAST CANCER GENE”.

  The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in the construction of fusion proteins include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins including blue fluorescent protein (BFP), glutathione-S-transferase ( GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). In addition, epitope tags including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags have been used in fusion protein constructs. Other fusion constructs can include maltose binding protein (MBP), S tag, Lex a DNA binding domain (DBD) fusion, GAL4 DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Fusion proteins can also be constructed to contain a cleavage site located between the “BREAST CANCER GENE” polypeptide coding sequence and the heterologous protein sequence so that the “BREAST CANCER GENE” polypeptide can be cleaved and purified from the heterologous moiety. .

  Fusion proteins can be chemically synthesized as is known in the art. The fusion protein is preferably produced by covalently linking two polypeptide segments or by standard procedures in the field of molecular biology. A DNA construct comprising, using recombinant DNA methods, for example, a coding sequence selected from any of the polynucleotide sequences of SEQ ID NOs: 1 to 65 in a suitable reading frame together with nucleotides encoding a second polypeptide segment And a fusion protein can be prepared by expressing this DNA construct in a host cell as is known in the art. Kits for constructing fusion proteins include Promega (Madison, Wis., USA), Stratagene (La Jolla, CA, USA), Clontech (Mountain View, CA), Santa Cruz Biotechnology (Santa, CA) Cruz), MBL International Corporation (MIC; Watertown, Mass., USA), and Quantum Biotechnologies (Canada, Montreal; 1-888-DNA-KITS).

Identification of Species Homologues Species homologues of the human “BREAST CANCER GENE” polypeptide can be obtained by using the “BREAST CANCER GENE” polynucleotide (described below) to live cDNA expression from other species, such as mouse, monkey, or yeast. Can be obtained by generating probes or primers suitable for screening the library, identifying cDNA encoding homologues of the “BREAST CANCER GENE” polypeptide and expressing the cDNA as is known in the art .

Polynucleotide Expression In order to express a “BREAST CANCER GENE” polynucleotide, the polynucleotide can be inserted into an expression vector containing elements necessary for transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding “BREAST CANCER GENE” polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (6) and Ausubel et al. (7).

  A variety of expression vector / host systems can be used to contain and express sequences encoding a “BREAST CANCER GENE” polypeptide. These include microorganisms such as bacteria transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA expression vectors, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, viral expression vectors (eg, baculo A viral cell-infected insect cell system, a viral expression vector (eg, cauliflower mosaic virus CaMV; or tobacco mosaic disease virus (TMV)), or a bacterial expression vector (eg, Ti or pBR322 plasmid), or Animal cell lines are included, but are not limited to these.

  Control elements or regulatory sequences are enhancer regions, promoter regions, and 5 'and 3' untranslated regions of the vector that interact with host cell proteins for transcription and translation. The strength and specificity of such elements can vary. Depending on the vector system and host utilized, various suitable transcription and translation elements can be used, including constitutive and inducible promoters. For example, when cloning into a bacterial system, an inducible promoter such as BLUESCRIPT phagemid (Stratagene, California, USA) or a hybrid lacZ promoter of pSPORT1 plasmid (Life Technologies) can be used. In insect cells, the baculovirus polyhedrin promoter can be used. Promoters or enhancers derived from the plant cell genome (eg, heat shock, RUBSCO, and storage protein genes) or those derived from plant viruses (eg, viral promoter or leader sequences) can be cloned into a vector. . In mammalian cell systems, promoters from mammalian genes or mammalian viruses are desirable. If it is necessary to generate a cell line containing multiple copies of a nucleotide sequence encoding a “BREAST CANCER GENE” polypeptide, an SV40-based or EBV-based vector with an appropriate selectable marker can be used.

Bacterial and yeast expression systems In bacterial systems, the number of expression vectors can be selected depending on the intended use for the "BREAST CANCER GENE" polypeptide. For example, if a large amount of “BREAST CANCER GENE” polypeptide is required to induce the antibody, vectors that direct high level expression of the easily purified fusion protein can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In the BLUESCRIPT vector, the sequence encoding the “BREAST CANCER GENE” polypeptide is in frame with the sequence encoding the amino terminal Met followed by 7 residues of β-galactosidase so that a hybrid protein is produced. It can be ligated into a vector. The pIN vector [Van Heeke and Schuster (75)] or the pGEX vector (Promega, Madison, Wis., USA) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. . Proteins produced with such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released freely from the GST moiety.

  In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a review, see Ausubel et al. (7).

Plant and Insect Expression Systems When using plant expression vectors, sequences encoding “BREAST CANCER GENE” polypeptides can be expressed by any of a number of promoters. For example, viral promoters such as the CaMV 35S and 19S promoters can be used alone or in combination with the TMV omega leader sequence [Takamatsu, 1987 (22)]. Alternatively, plant promoters such as the small subunit of RUBISCO or the heat shock promoter can be used [Winter et al., 1991 (23)]. The constructs can be introduced into these plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in the publicly accessible overview.

  Insect systems can also be used to express "BREAST CANCER GENE" polypeptides. For example, in one such system, an autographer nuclear polyhedrosis virus (AcNPV) is used as a vector to express a foreign gene in Spodoptera frugiperda cells or nettle worms (Trichoplusia) larvae. A sequence encoding a “BREAST CANCER GENE” polypeptide can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the “BREAST CANCER GENE” polypeptide will inactivate the polyhedrin gene and produce a recombinant virus lacking the coat protein. This recombinant virus can then be used to infect sweet potato cells or nettle larvae larvae capable of expressing a “BREAST CANCER GENE” polypeptide [Engelhard et al., 1994 (24)].

Mammalian Expression Systems A number of viral-based expression systems can be used to express “BREAST CANCER GENE” polypeptides in mammalian host cells. For example, when an adenovirus is used as an expression vector, a sequence encoding a “BREAST CANCER GENE” polypeptide can be linked to an adenovirus transcription / translation complex containing the late promoter and tripartite leader sequence. Insertion into the E1 or E3 region, a non-essential region of this viral genome, can be used to obtain live viruses capable of expressing the “BREAST CANCER GENE” polypeptide in infected host cells [Logan and Shenk, 1984 ( 25)]. If desired, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to enhance expression in mammalian host cells.

  Human artificial chromosomes (HACs) can also be utilized to deliver larger DNA fragments than can be contained and expressed in plasmids. 6M to 10M HAC is constructed and delivered to cells using conventional delivery methods (eg, liposomes, polycation amino polymers, or vesicles).

  Specific initiation signals can also be used to achieve more efficient translation of sequences encoding “BREAST CANCER GENE” polypeptides. Such signals include the ATG start codon and adjacent sequences. If the sequence encoding the “BREAST CANCER GENE” polypeptide and its initiation codon and upstream sequence are inserted into an appropriate expression vector, no other transcriptional or translational control signals are required. Let ’s go. However, if only the coding sequence or fragment thereof is inserted, an exogenous translational control signal (including the ATG start codon) should be provided. The start codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translation elements and initiation codons may be of various origins, natural or synthetic. Inclusion of an enhancer suitable for the particular cellular system used can enhance the efficiency of expression [Scharf et al., 1994 (26)].

Host Cell A host cell line may be chosen for its ability to moderate expression of the inserted sequences or to process the expressed “BREAST CANCER GENE” polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves a “prepro” type of polypeptide can also be used to facilitate correct insertion, folding, and / or function. Various host cells (eg, CHO, HeLa, MDCK, HEK293, and WI38) with specific cellular mechanisms of post-translational activity and unique mechanisms have been identified by ATCC (American Type Culture Collection, VA, USA (10801 University Boulevard, Manado, Mass.). VA 20110-2209)), and can be selected to ensure correct modification and processing of the foreign protein.

  Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, an expression vector in which a cell line stably expressing a “BREAST CANCER GENE” will contain the viral origin of replication and / or endogenous expression elements and a selectable marker gene on the same or separate vectors. Can be used to transform. After introducing the vector, the cells can be grown in concentrated media for 12 days before switching the media to selective media. The purpose of the selective marker is to confer resistance to selection, and its presence allows for the growth and recovery of cells that have successfully expressed the introduced “BREAST CANCER GENE” sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type [Freshney et al., 1986 (27)].

  Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase [Wigler et al., 1977 (28)] and adenine phosphoribosyltransferase [Lowy et al., 1980 (29)] genes, which are each , Tk-cells or aprt-cells. Antimetabolites, antibiotics, or herbicide resistance can also be used as a basis for selection. For example, dhfr confers resistance to methotrexate [Wigler et al., 1980 (30)] and npt confers resistance to aminoglucoside, neomycin, and G418 [Colbere-Garapin et al., 1981 (31)] als and pat confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively. Other selectable genes, such as trpB or hisD, have been described, trpB allows the use of indole by the cell instead of tryptophan, and hisD replaces histinol by the cell instead of histidine. Make available [Hartman and Mulligan, 1988 (32)]. Use visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin to identify transformants and to quantify transient or stable protein expression due to specific vector systems. [Rhodes et al., 1995 (33)].

Expression and Detection of Gene Products The presence of marker gene expression suggests that a “BREAST CANCER GENE” polynucleotide is also present, but its presence and expression may need to be confirmed. For example, when a sequence encoding a “BREAST CANCER GENE” polypeptide is inserted into a marker gene sequence, transformed cells containing sequences encoding a “BREAST CANCER GENE” polypeptide may be identified by the absence of marker gene function. it can. Alternatively, a marker gene can be placed in tandem with a sequence encoding a “BREAST CANCER GENE” polypeptide and placed under the control of a single promoter. Expression of a marker gene in response to induction or selection typically also indicates expression of a “BREAST CANCER GENE” polynucleotide.

  Alternatively, host cells containing “BREAST CANCER GENE” polynucleotides and host cells expressing “BREAST CANCER GENE” polypeptides can be identified by various procedures known to those of skill in the art. These procedures include DNA-DNA or DNA-RNA hybridization and protein bioassay techniques, including membrane, solution, or chip-based techniques for detecting and / or quantifying polynucleotides or proteins. Including, but not limited to, immunoassay techniques. For example, the presence of a polynucleotide sequence encoding a “BREAST CANCER GENE” polypeptide can be determined by DNA-DNA or DNA-RNA hybridization, or amplification, using a probe or fragment of a polynucleotide encoding a “BREAST CANCER GENE” polypeptide. Can be detected. Nucleic acid amplification-based assays use an oligonucleotide selected from sequences encoding a “BREAST CANCER GENE” polypeptide to detect transformants containing a “BREAST CANCER GENE” polynucleotide.

  Various protocols are known in the art for detecting and measuring expression of such polypeptides using polyclonal or monoclonal antibodies specific for “BREAST CANCER GENE” polypeptides. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A monoclonal-based two-site immunoassay utilizing a monoclonal antibody that reacts with two non-interfering epitopes on the “BREAST CANCER GENE” polypeptide can be used, or a competitive binding assay can be used. These and other assays are described in Hampton et al. (34).

  A wide range of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Oligo-labeling, nick translation, end-labeling, or labeled nucleotides are used to generate labeled hybridization probes or PCR probes used to detect sequences associated with polynucleotides encoding "BREAST CANCER GENE" polypeptides. PCR amplification was included. Alternatively, a sequence encoding a “BREAST CANCER GENE” polypeptide can be cloned into a vector to produce an mRNA probe. Such vectors are known in the art and are commercially available, and an RNA probe can be obtained in vitro by adding an appropriate RNA polymerase such as T7, T3, or SP6 and a labeled nucleotide. Can be used to synthesize. These procedures can be performed using various commercial kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules, ie labels that can be used to facilitate detection, include radionuclides, enzymes, fluorescence, chemiluminescence, or chromogenic materials, as well as cofactors, inhibitors, magnetic particles, and the like Are also included.

Polypeptide Expression and Purification Host cells transformed with a nucleotide sequence encoding a “BREAST CANCER GENE” polypeptide can be cultured under conditions suitable for protein expression and recovery of the protein from cell culture. Polypeptides produced by transformed cells may be secreted or sorted within the cell membrane, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, an expression vector containing a polynucleotide encoding a “BREAST CANCER GENE” polypeptide is capable of secreting a soluble “BREAST CANCER GENE” polypeptide across the cell membrane of prokaryotic or eukaryotic cells. It can be designed to contain a signal sequence that directs, or a signal sequence that directs insertion of a membrane-bound “BREAST CANCER GENE” polypeptide into the membrane.

  As discussed above, other constructs may be used to link the sequence encoding the “BREAST CANCER GENE” polypeptide to a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein. Such domains that facilitate purification include metal chelating peptides such as the histidine tryptophan module that allow purification on immobilized metal, proteins that allow purification on immobilized immunoglobulins A domain and the domain used in the FLAGS extension / affinity purification system (Immunex, Seattle, WA, USA). To facilitate purification, a cleavable linker sequence between the purification domain and the “BREAST CANCER GENE” polypeptide, such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) Can also be included. One such expression vector provides for expression of a fusion protein containing a “BREAST CANCER GENE” polypeptide and a nucleic acid encoding a 6 histidine residue preceding the thioredoxin or enterokinase cleavage site. The histidine residue facilitates purification by IMAC (immobilized metal affinity chromatography) [Porath et al., 1992 (35)] and the enterokinase cleavage site provides a method for purifying the "BREAST CANCER GENE" polypeptide from the fusion protein To do. Vectors containing fusion proteins are disclosed in Kroll et al. (36).

Chemical Synthesis Sequences encoding “BREAST CANCER GENE” polypeptides can be synthesized in whole or in part using chemical methods well known in the art [see Caruthers et al. (37)]. Alternatively, chemical methods for synthesizing amino acid sequences, such as direct peptide synthesis using solid phase techniques, can be used to generate the “BREAST CANCER GENE” polypeptide itself [Porath et al., 1992 (35). ]. Protein synthesis can be performed by manual techniques or by automation. Synthesis by automation can be realized using, for example, a Biosystems 431A peptide synthesizer (Perkin Elmer). If desired, fragments of the “BREAST CANCER GENE” polypeptide can be synthesized separately and combined using chemical methods to produce the full length molecule.

  Newly synthesized peptides can be substantially purified by preparative high performance liquid chromatography [Creighton, 1983 (39)]. The composition of the synthesized “BREAST CANCER GENE” polypeptide can be confirmed by amino acid analysis or sequencing (eg, Edman degradation procedures). In addition, any portion in the amino acid sequence of the “BREAST CANCER GENE” polypeptide can be modified directly during synthesis and / or combined with sequences derived from other proteins using chemical methods to provide variant polypeptides or fusions. It is possible to produce proteins.

Production of Modified Polypeptides As will be appreciated by those skilled in the art, it may be advantageous to produce nucleotide sequences encoding “BREAST CANCER GENE” polypeptides having non-naturally occurring codons. Specific prokaryotic cells, for example, to increase the rate of protein expression or to produce RNA transcripts with desirable properties, such as longer half-lives than transcripts produced from naturally occurring sequences. Codons preferred by the host or eukaryotic host can be selected.

  For a variety of reasons, genetically engineered nucleotide sequences disclosed herein to modify a polypeptide encoding a “BREAST CANCER GENE” sequence using methods generally known in the art. Can do. Such modifications include, but are not limited to, modifications that alter the cloning, processing, and / or expression of the polypeptide or mRNA product. To perform genetic manipulation of nucleotide sequences, random shredding of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly can be used. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon usage, produce splice variants, introduce mutations, and the like.

Predictive, Diagnostic, and Prognostic Assays The present invention relates to disclosed malignant tumor biomarkers, in particular breast cancer biomarkers, ie, disclosed polynucleotide markers comprising any of the polynucleotide sequences of SEQ ID NOs: 1-65, And / or a polypeptide marker encoded thereby, or a polypeptide marker comprising any of the polypeptide sequences of SEQ ID NOs: 66-130, at risk of developing a malignant tumor, particularly a breast cancer Compositions, methods, and kits for determining whether there are are provided.

  In clinical applications, biological samples can be screened for the presence and / or absence of the biomarkers identified herein. Such samples are, for example, puncture biopsies, surgical excision samples, or bodily fluids such as serum, fine needle aspirate and urine. For example, these methods include obtaining a biopsy that is optionally fractionated by cryostat slicing to enrich disease cells to about 80% of the total cell population. In certain embodiments, polynucleotides extracted from these samples can be amplified using techniques well known in the art. The detected expression level of the selection label will be compared between a statistically valid disease sample group and a healthy sample group.

  In one embodiment, the composition, method, and kit are obtained by Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation reaction, Western blot hybridization, or immunohistochemistry, etc. Determining whether the mRNA and / or protein level of the disclosed marker in the subject is abnormal. In accordance with the present invention, cells are obtained from a subject and the disclosed biomarker, protein, or mRNA levels are measured and compared to the levels of these markers in a healthy subject. If the level of the biomarker polypeptide or mRNA is abnormal, it is likely to indicate a malignant tumor such as breast cancer.

  In another embodiment, the compositions, methods, and kits described above are used in a subject, such as by Southern blot analysis, dot blot analysis, fluorescence or colorimetric analysis, in situ hybridization, comparative genomic hybridization, or quantitative PCR. Determining whether the DNA content of the genetic DNA or the genomic locus is abnormal. Usually these assays involve the use of probes derived from representative genomic regions. These probes contain at least part of the genomic region, in particular the gene or the intergenic region of the genomic region or a complementary or similar sequence to the region. A probe can consist of a nucleotide sequence that can bind to a target region by hybridization or a sequence of similar function (eg, PNA, morphino oligomer). In general, whether the modified genomic region in the patient sample is compared to an unaffected control sample (normal tissue from the same or another patient, unaffected surrounding tissue, peripheral blood) Or compared to a genomic region of the same sample that has not undergone said modification and can therefore serve as an internal control. In a preferred embodiment, regions located on the same chromosome are used. Alternatively, gonosom areas and / or specific areas with variable amounts in the sample are used. In a preferred embodiment, the DNA content, structure, composition, or modification is compared to that in another genomic region. A particularly preferred method is a method for detecting the DNA content of the sample, wherein the amount of the target region is modified by amplification and / or deletion. In another embodiment, the target region is analyzed for the presence of polymorphisms (eg, single nucleotide polymorphisms or mutations) that clinically affect or cause disease in the cells in the sample. These polymorphisms have diagnostic, prognostic, or therapeutic value. With regard to the clinical aspect, it is preferred to use sequence variation identification to define the haplotype that results in the characteristic behavior of the sample.

In one embodiment, the composition, method and kit for predicting, diagnosing or prognosing malignancy, particularly breast cancer, in a biological sample,
(A) a polynucleotide selected from the polynucleotides of SEQ ID NOs: 1 to 65,
(B) a polynucleotide that encodes a polypeptide that hybridizes to the polynucleotide identified in (a) under stringent conditions and exhibits the same biological function as that identified in each of the sequences in Table 1.
(C) a genetic code that encodes a polypeptide that exhibits the same biological function as that specified for the polypeptide of SEQ ID NOs: 66 to 130, so that the sequence is a polynucleotide specified in (a) and (b) A polynucleotide deviating from,
(D) a specific fragment, derivative or allele of a polynucleotide sequence that encodes a polypeptide that exhibits the same biological function as that specified for each of the sequences in Table 1, and is specified from (a) to (c) Any polynucleotide or analog oligomer identified by (a) through (d), detected by detection of a polynucleotide that represents a genetic mutation, is hybridized to the polynucleotide material in the biological sample, thereby Forming a hybridization complex and detecting the hybridization complex.

  In another embodiment, the method of predicting, diagnosing, or determining prognosis of a malignant tumor is performed as described above, with the polynucleotide material in the biological sample being amplified prior to hybridization.

In another embodiment, a method for predicting, diagnosing or prognosing malignant tumors, particularly breast cancer,
(A) a polynucleotide selected from the polynucleotides of SEQ ID NOs: 66 to 130,
(B) a polynucleotide that encodes a polypeptide that hybridizes to the polynucleotide identified in (a) under stringent conditions and exhibits the same biological function as that identified in each of the sequences in Table 1.
(C) Since a genetic code encoding a polypeptide having the same biological function as that specified for each of the sequences in Table 1 was prepared, the sequence deviated from the polynucleotide specified in (a) and (b). A polynucleotide,
(D) a specific fragment, derivative or allele of a polynucleotide sequence that encodes a polypeptide that exhibits the same biological function as that specified for each of the sequences in Table 1, and is specified from (a) to (c) A polynucleotide representing a genetic mutation,
(E) a polypeptide encoded by the polynucleotide sequence identified from (a) to (d);
(F) a polypeptide comprising any polypeptide of SEQ ID NOs: 66 to 130,
(G)
And a step of contacting the biological sample with a reagent specified by (a) to (d) or a reagent that specifically interacts with the polypeptide specified in (e).

1. DNA Array Technology In one aspect, the invention also provides a method in which polynucleotide probes are immobilized on a DNA chip as an organized array. Oligonucleotides can be bound to a solid support by a variety of methods, including lithography. For example, the chip can hold up to 410000 oligonucleotides (GeneChip, Affymetrix). The present invention provides significant advantages over available tests for malignant tumors such as breast cancer because it increases the reliability of the test by providing an array of polynucleotide markers on a single chip.

  This method includes obtaining a biological sample, the biological sample being a biopsy of the affected person and a body fluid such as fluid containing serum, urine, serum, or cells (eg, derived from fine needle aspirate) Can be used. The affected biopsy is optionally fractionated by cryostat slices so that the diseased cells are enriched to about 80% of the total cell population. DNA or RNA is then extracted, amplified, and analyzed using a DNA chip to determine whether a marker polynucleotide sequence is present or absent. In one embodiment, polynucleotide probes are spotted on a substrate in a two-dimensional original matrix, ie, in the form of an array. A sample of the polynucleotide can be labeled and then hybridized to the probe. Double-stranded polynucleotides comprising labeled sample polynucleotides bound to probe polynucleotides can be detected after the unbound portions in the sample have been washed away.

  The probe polynucleotide can be spotted on a substrate including glass, nitrocellulose and the like. The probe can be bound to the substrate by covalent bonds or by non-specific interactions such as hydrophobic interactions. The sample polynucleotide can be labeled using a radioactive label, a fluorescent dye, a chromophore or the like. Techniques for constructing arrays and methods for using these arrays are described in EP 0799897, WO 97/29212, WO 97/27317, EP 0785280, WO 97/02357. U.S. Pat. No. 5,593,839, U.S. Pat. No. 5,578,832, European Patent No. 0728520, U.S. Pat. In the issue. In addition, arrays can be used to examine differential expression of genes and can be used to measure gene function. For example, this array of polynucleotide sequences can be used to determine whether any of these polynucleotide sequences are differentially expressed, for example, between normal and diseased cells. If the expression of a particular message in a disease sample is high and this is not observed in the corresponding normal sample, it may indicate a breast cancer specific protein.

  Accordingly, in one aspect, the present invention provides probes and primers specific for the polynucleotide sequences of SEQ ID NOs: 1-65.

In one embodiment, the compositions, methods, and kits comprise the use of a polynucleotide probe to determine whether malignant cancer, particularly breast cancer cells, are present in tissue obtained from a patient. Specifically, the above method
1) A length complementary to a portion of the coding sequence of a polynucleotide selected from the polynucleotides of SEQ ID NOs: 1 to 65 is at least 12 nucleotides, preferably at least 15 nucleotides, more preferably 25 nucleotides, most preferably at least 40 Providing a polynucleotide probe comprising nucleotides and a nucleotide sequence up to all or almost all of the coding sequence or a sequence complementary thereto;
2) obtaining a tissue sample from a patient having a malignant tumor;
3) preparing a second tissue sample from a patient having no malignancy;
4) contacting the polynucleotide probe with the RNA of each of the first and second tissue samples under stringent conditions (eg, in a Northern blot or in situ hybridization assay);
5) (a) comparing the amount of hybridization of the probe with RNA of the first tissue sample, and (b) comparing the amount of hybridization of the probe with RNA of the second tissue sample, In the method, if there is a statistically significant difference in the amount of hybridization of the first tissue sample with RNA compared to the amount of hybridization of the second tissue sample with RNA, The presence of malignant tumors in the sample, in particular breast cancer, is indicated.

2. Data analysis Comparison of a reference expression level, eg, an expression level in a diseased breast cancer cell or a normal counterpart cell, with the expression level of one or more “breast cancer genes” is preferably performed using a computer system. In one embodiment, expression levels are obtained in two cells and these two sets of expression levels are introduced into a comparative computer system. In a preferred embodiment, a set of expression levels is introduced into the computer system for comparison with computer-readable form values that already exist in the computer system or are later introduced into the computer system.

  In one aspect, the invention provides a computer readable form of the gene expression profile data of the invention, or a value corresponding to the expression level of at least one “breast cancer gene” in diseased cells. This value may be an mRNA expression level obtained from an experiment, eg, microarray analysis. This value may be a normalized mRNA level compared to a reference gene whose expression is constant in a number of cells under a number of conditions, for example GAPDH. In other embodiments, the value in the computer is the ratio or difference of normal or non-normal mRNA levels between different samples.

  The gene expression profile data may be in the form of a table, such as an Excel table. The data can be alone or can be part of a larger database, such as those containing other expression profiles. For example, the expression profile data of the present invention may be part of a public database. A computer readable form can exist in the computer. In another embodiment, the present invention provides a computer that displays gene expression profile data.

  In one embodiment, the present invention determines the similarity between the expression level of one or more “BREAST CANCER GENE” in a first cell, eg, the subject cell, and the expression level in a second cell. Obtaining a level of expression of one or more “breast cancer genes” in the first cell; and a level of expression of one or more “breast cancer genes” in the second cell. Databases containing records containing values corresponding to and processor instructions that can accept one or more values selected for comparison with data stored in the computer, such as a user interface Inputting the value of. The computer may further include means for converting the comparison data into a diagram or chart or other type of output.

  In another embodiment, a value representing the expression level of a “BREAST CANCER GENE” is entered into a computer system that includes one or more databases having a reference expression level obtained from a plurality of cells. For example, the computer includes expression data for diseased cells and normal cells. Instructions are given to the computer, which compares the data in the computer with the entered data and determines whether the entered data is more similar to normal cell data or diseased cell data be able to.

  In another embodiment, the computer includes expression level values in cells of interest at different stages of breast cancer, and the computer can compare the stored data with the expression data entered into the computer. Producing a result indicating which of the in-computer expression profiles the input data is most similar, such as determining the stage of the subject's breast cancer.

  In yet another embodiment, the in-computer reference expression profile was obtained from breast cancer cells of one or more subjects treated in vivo or in vitro with a drug used to treat breast cancer. Expression profile. By inputting the expression data of the cells of interest processed in vitro or in vivo with the above drugs, the computer was instructed to compare the entered data with the data in the computer and entered into the computer Results are provided that indicate whether the expression data is more similar to the data of the subject's cells responding to the drug or the data of the subject's cells not responding to the drug. Thus, the results indicate whether the subject is likely to respond to treatment with the drug or is less likely to respond to it.

  In one embodiment, the present invention provides means for receiving gene expression data for one or more genes and means for comparing gene expression data obtained from each of the one or more genes with a common reference frame. And a means for displaying the result of the comparison. The system may further include means for clustering the data.

  In one embodiment of the invention, statistical analysis such as principal component analysis (PCA) or partial least square discriminant analysis (PLS-DA) is applied to the expression data. PLS-DA is superior when much more variables (genes) must be taken into account than observations (samples). The inventors will examine a small set of genes to assess the ability of PLS to separate pre- and post-chemotherapy samples, in addition, we will have a counter-sample and two treatment conditions The classic PCA algorithm was challenged with the identification of the major components that separated the.

  We used expression data from all 25 tumor samples in PCA and PLS-DA analyses. The first repetitive level PLS-DA analysis was performed using all of the 249 genes that were positively expressed previously used in the hierarchical clustering analysis. During the analysis, both PLS components were used to select genes that met the cutoff criteria of having a VIP (variable importance in the projection) greater than 1.8. In the second iteration of PLS-DA performed using SIMCA-P analysis software, only genes with VIPs exceeding the 1.8 threshold (n = 25) were individually identified to avoid over-conforming system operation. Reanalyzed (genes are shown in Table X).

  For the statistical extraction of genes described previously, the class definition problem is limited to 2 (98), so the use of a simple t-test is absolutely appropriate, but the VIP used for PLS-DA The evaluation standard is more versatile and has higher discrimination ability. Although the normal distribution of data is an assumption that only applies to standard parametric t-tests, PLS-DA is not disturbed by the problem that the data is not normally distributed. This is very important when working with a relatively small number of specimens as in this study.

In PCA, all samples before and after chemotherapy are mixedly distributed, but they are classified into distinct regions by PLS-DA. Furthermore, a reordering test was performed to test versatility. As a principle of checking whether an existing model is the most predictable option, the Y-axis intercept should be R ² <0.3 and Q ² <0.05. When R ² wire is close to horizontal, the test indicates that the model is over-fit (99). Reordering analysis revealed that the current model has the highest predictive power. Thus, it was possible to identify changes caused by the implementation of chemotherapy within each individual sample pair by use of PLS analysis. However, it is clear that the absolute value varies depending on the patient. The VIP values listed in Table 3 correspond to the model with the 25 genes selected and show lower values than those obtained with the non-optimized model, which is an already reported phenomenon (100).

  In another embodiment, the present invention provides a computer program for analyzing gene expression data, the computer program comprising: (i) computer code that accepts gene expression data of a plurality of genes as input; and (ii) said Computer code for comparing the gene expression data obtained from each of a plurality of genes with a common reference frame.

  The present invention provides (i) a plurality of values corresponding to expression levels of one or more genes characteristic of breast cancer in query cells, reference expression or expression profile data of one or more reference cells, and cell type. A machine comprising program instructions for performing a comparison with a database containing records containing annotations of: and (ii) indicating which cell the query cell is most similar to based on similarity of expression profiles A readable or computer readable medium is also provided. The reference cell may be a cell obtained from a subject at a different stage of breast cancer. The reference cell may be a cell obtained from a subject that is responsive or non-responsive to a particular drug therapy, and can be incubated with the drug in vitro or in vivo, if desired.

  The reference cell may be a cell obtained from a subject that is responsive or non-responsive to a number of different treatments, and the computer system indicates the preferred treatment for that subject. Accordingly, the present invention provides a method of selecting treatment for a patient having breast cancer, which method provides (i) the expression level of one or more genes characteristic of breast cancer in the patient's disease cells. And (ii) providing a plurality of reference profiles, each of the reference profiles being associated with a treatment, wherein the subject expression profile and each reference profile has a plurality of values, each of the above values Representing the expression level of a gene characteristic of breast cancer and (iii) selecting a reference profile most similar to the expression profile of the subject, thereby selecting treatment for said patient. In a preferred embodiment, step (iii) is performed by a computer. The most similar reference profile can be selected by weighting multiple comparison values using a weight value associated with the corresponding expression data.

  The relative amount of mRNA in the two biological samples is perturbed and its magnitude of measurement (ie, the abundance is different at the two sources of mRNA being tested) or unperturbed (ie, the relative amount is the same). Can be recorded as In various embodiments, the coefficient of difference between the two RNA sources is at least about 25% (25% more RNA from one source than RNA from the other source), more usually about If it is 50%, more frequently 2 (2 times more), 3 (3 times more), or 5 (5 times more), the difference is recorded as a perturbation. The computer can use perturbations for calculations and expression comparisons.

  In addition to recognizing perturbations as positive or negative, it is advantageous to measure the magnitude of the perturbations. This can be done by calculating the ratio of the emission of the two fluorescent dyes used for differential labeling, as described above, or by similar methods that will be readily apparent to those skilled in the art.

  The computer readable medium may further include a pointer to a descriptor relating to breast cancer stage or breast cancer treatment.

  Arithmetic includes hardware or hardware and software; a programmed computer logic circuit or other component that performs the operations specified herein in detail, as indicated by a computer program; or Means for accepting gene expression data within the context of the system of the present invention, implemented by a computer memory encoded with executable instructions representing a computer program that causes the computer to function in a specific manner as described in 1. The means for comparing, the means for display, the means for normalization, and the means for clustering can be associated with a programmed computer having the functions described herein.

  Those skilled in the art will appreciate that the systems and methods of the present invention can be applied to a variety of systems, including IBM compatible personal computers running MS-DOS or Microsoft Windows.

  The computer may have an internal component coupled to the external component. The internal component may include a processor element interconnected to the main memory. The computer system may be an Intel Pentium®-based processor with a 200 MHz or higher clock rate with 32 MB or more of main memory. The external component may include a mass storage device, which may be one or more hard disks (usually packaged with a processor and memory). Such hard disks are usually of 1 GB or greater storage capacity. Other external components include a user interface device along with an input device, which may be a monitor and the input device may be a “mouse” or other graphic input device and / or a keyboard. A printing device can also be attached to the computer.

  Usually, the computer system is also coupled to a network link. The network link may be part of an Ethernet link to another localized computer system, a remote computer system, or a wide area network such as the Internet. This network link allows a computer system to share data and processing tasks with other computer systems.

  During the operation of this system, several software components are loaded into memory. Some are standard in the art and others are specific to the present invention. These software components collectively cause the computer system to function according to the method of the present invention. These software components are typically stored on a mass storage device. A software component represents an operating system, which is responsible for managing the computer system and its network interconnections. The operating system may be of the Microsoft Windows family, such as Windows 95, Windows 98, or Windows NT, for example. Software components represent common languages and functions that conveniently exist on this system to assist programs that implement the methods specific to the present invention. A number of high or low level computer languages can be used to program the folding method of the present invention. The instructions may be interpreted during execution time or compiled. Preferred languages include C / C ++ and Java®. Most preferably, the method of the present invention is programmed with a mathematical software package. The math software package allows high-level singularization of processing, including symbolic equations and the algorithms used, thereby freeing the user from the need to program individual equations or algorithms procedurally . Such packages include Matlab of Mathworks (Natick, Massachusetts, USA), Matthematica of Wolfram Research (Champaign, Ill., USA), and S-Plus of Math Soft, Inc. (Camb, Massachusetts, USA). Thus, the software component represents the solution of the present invention programmed in a procedural language or symbolic package. In a preferred embodiment, the computer system also contains a database containing values representing the level of expression of one or more genes characteristic of breast cancer. The database may contain one or more expression profiles of genes characteristic of breast cancer in various cells.

  In an exemplary embodiment, to perform the method of the present invention, the user first loads expression profile data into the computer system. These data can be input directly by the user from the monitor and keyboard, input from another computer system connected by network connection, or input from a removable storage medium such as a CD-ROM or floppy disk. It can also be entered through the network. Next, the user causes, for example, the execution of expression profile analysis software that clusters into co-fluctuating genes and performs the comparison process.

  In another exemplary embodiment, expression profiles are compared using the method described in US Pat. No. 6,203,987. The user first loads expression profile data into the computer system. Geneset profile definitions are loaded into memory from a storage medium or remote computer, preferably from a dynamic geneset database system, over a network. The user then causes execution of projection software that performs the process of converting the expression profile into a projected expression profile. Thereafter, the projected expression profile is displayed.

  In yet another exemplary embodiment, the user first leads the projection profile to memory. The user then causes loading of the reference profile into memory. The user then causes execution of comparison software that performs the process of objectively comparing the profiles.

3. Detection of Variant Polynucleotide Sequences In yet another embodiment, the invention relates to a malignant tumor, such as breast cancer, associated with an abnormal activity of any one of the polypeptides encoded by any of the polynucleotides of SEQ ID NOs: 1-65. Provides a method for determining whether a subject is at risk of developing a disease, such as a predisposition to develop
The presence or absence of genetic damage characterized by at least one of (i) a modification affecting the integrity of the gene encoding the marker polypeptide, or (ii) misexpression of the encoding polynucleotide. Characterized by detecting.

Such genetic damage is, for example,
I. Deletion of one or more nucleotides from the polynucleotide sequence;
II. Addition of one or more nucleotides to a polynucleotide sequence;
III. Substitution of one or more nucleotides of the polynucleotide sequence;
IV. A large-scale chromosomal rearrangement of the polynucleotide sequence,
V. Extensive alteration of the mRNA transcript level of the polynucleotide sequence,
VI. Abnormal modification of polynucleotide sequence, such as abnormal methylation pattern of genomic DNA,
VII. The presence of a non-wild type splicing pattern of the mRNA transcript of the gene,
VIII. A non-wild type level of the marker polypeptide,
IX. Gene allele disappearance,
X. It can be detected by confirming the presence of at least one of the inappropriate post-translational modifications of the marker polypeptide.

  The present invention provides assay techniques for detecting mutations in the encoding polynucleotide sequence. These methods include methods using sequence analysis, Southern blot hybridization, restriction enzyme site mapping, and detection of the absence of nucleotide pairings between the polynucleotide being analyzed and the probe. However, it is not limited to these.

  Certain diseases or disorders, such as genetic diseases or disorders, are associated with particular allelic variants in a polymorphic region of a particular gene, but they do not necessarily encode mutant proteins. Thus, the presence of a particular allelic variant in a polymorphic region of a gene in a subject can make the subject more likely to develop a particular disease or disorder. By determining the nucleotide sequence of a gene in an individual population, polymorphic regions within the gene can be identified. Once a polymorphic region has been identified, an association with a particular disease can be determined by studying a particular population of individuals, such as breast cancer, who have developed a particular disease. A polymorphic region can be located in any region of a gene, eg, an exon, an exon coding or non-coding region, an intron, and a promoter region.

  In exemplary embodiments, a sense or antisense sequence of a gene or naturally occurring variant thereof, or a 5 ′ or 3 ′ flanking sequence or intron sequence that is naturally linked to the gene of interest or naturally occurring variant thereof. A polynucleotide composition comprising a polynucleotide probe containing a region of a nucleotide sequence capable of hybridizing is provided. The cellular polynucleotide is made accessible to hybridization, the probe is contacted with the sample polynucleotide, and hybridization of the probe to the sample polynucleotide is detected. Such techniques can be used to detect damage or allelic variants at either the genomic or mRNA level, including deletions, substitutions, etc., and to determine the level of mRNA transcripts.

  A preferred detection method is allele-specific hybridization using a probe having an overlap with the mutation or polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides surrounding the mutation or polymorphic region. is there. In a preferred embodiment of the present invention, several probes capable of specifically hybridizing to the allelic variant are attached to a solid support, such as a “chip”. Mutation detection analysis using these chips containing oligonucleotides is also referred to as “DNA probe array” and is described, for example, in Cronin et al. (40). In one embodiment, a single chip contains all the allelic variants in at least one polymorphic region of a gene. This solid phase carrier is then contacted with a test polynucleotide and hybridization to a particular probe is detected. Thus, simple hybridization experiments can identify the identities of multiple allelic variants of one or more genes.

  In certain embodiments, the detection of the damaged site is performed by polymerase chain reaction (PCR) such as anchor PCR or RACE-PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202), or alternatively, ligase chain reaction (LCR). Including the use of probes / primers in [Landegran et al., 1988 (41)]. Of these, the latter may be useful for detecting point mutations within genes [Abravaya et al., 1995 (42)]. In an exemplary embodiment, the method includes (i) collecting a sample of cells from a patient and (ii) isolating a polynucleotide (eg, genome, mRNA, or both) from the cells of the sample. And (iii) one or more primers that specifically hybridize to the polynucleotide sequence under conditions such that hybridization and amplification of the polynucleotide (if present) occurs, and a polynucleotide sample Contacting and (iv) detecting the presence or absence of the amplification product or detecting the size of the amplification product and comparing its length to a control sample. PCR and / or LCR are expected to be desirable for use as a preliminary amplification step in conjunction with any technique described herein used to detect mutations.

  An alternative amplification method is 3SR (self-stained sequence replication) [Guatelli, J. et al. C. 1990 (43)], transcription amplification system [Kwoh, D. et al. Y. 1989 (44)], Q-β replicase [Lizardi, P. et al. M, et al., 1988 (45)], or any other polynucleotide amplification method that later detects the amplified molecule using techniques well known to those skilled in the art. These detection methods are particularly useful for detecting such molecules when polynucleotide molecules are present in very low numbers.

  In a preferred embodiment of the invention, a change in a restriction enzyme cleavage pattern identifies a mutation in a gene from a sample cell, or an allelic variant thereof. For example, sample DNA and control DNA are isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length is determined by gel electrophoresis. In addition, sequence specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to test for the presence of specific mutations by the generation or loss of ribozyme cleavage sites.

4). In Situ Hybridization In one aspect, the method comprises in situ using a probe derived from a given marker polynucleotide comprising a sequence selected from any of the polynucleotide sequences of SEQ ID NOs: 1-65 or a complementary sequence thereof. Includes hybridization. The method described above applies labeled hybridization probes to a given type of tissue obtained from a patient who may have a malignant tumor, particularly breast cancer, as well as to normal tissue of a person without malignant tumor. The step of contacting the sample and the labeling of the patient tissue with the probe is significantly different from the degree of labeling of normal tissue (eg, a factor of at least 2, a factor of at least 5, a factor of at least 20, or a factor of at least 50) determining whether or not. In situ hybridization may be performed on DNA in the nuclei of the cells in the tissue or on mRNA in the cytoplasm to stain transcriptional activity.

Polypeptide Detection The present invention further provides a method for determining whether a cell sample obtained from a subject retains an abnormal amount of a marker polypeptide, the method comprising: (a) obtaining a cell sample from the subject (B) measuring the amount of marker polypeptide present in the sample so obtained; and (c) comparing the amount of marker polypeptide so measured with a known standard. And thereby determining whether the cell sample obtained from the subject retains an abnormal amount of the marker polypeptide. Such marker polypeptides can be detected by immunohistochemical assays, dot blot analysis, ELISA, and the like.

Antibodies Any type of antibody known in the art can be generated that specifically binds to an epitope of a “BREAST CANCER GENE” polypeptide. As used herein, an antibody includes a complete immunoglobulin molecule capable of binding to an epitope of a “BREAST CANCER GENE” polypeptide and fragments thereof such as Fab, F (ab) ₂ , Fv. To form an epitope, usually at least 6, 8, 10, or 12 amino acids in succession are required. However, epitopes involving discontinuous amino acids may require more amino acids, such as at least 15, 25, or 50 amino acids.

  Antibodies that specifically bind to an epitope of a “BREAST CANCER GENE” polypeptide are therapeutic and Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitation reactions, or other immunochemistry known in the art. It can be used for immunochemical assays such as assays. Various immunoassays can be used to identify antibodies with the desired specificity. Numerous protocols for performing competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically measure complex formation between an immunogen and an antibody that specifically binds to the immunogen.

  Typically, an antibody that specifically binds to a “BREAST CANCER GENE” polypeptide produces a detection signal that, when used in an immunochemical assay, is at least 5, 10, or 20 times higher than the detection signal produced with other proteins. Is. The antibody that specifically binds to the “BREAST CANCER GENE” polypeptide is preferably one that does not detect other proteins in an immunochemical assay and that can immunoprecipitate the “BREAST CANCER GENE” polypeptide from solution.

  A “BREAST CANCER GENE” polypeptide can be used to immunize a mammal such as a mouse, rat, rabbit, guinea pig, monkey, or human to produce a polyclonal antibody. If desired, the “BREAST CANCER GENE” polypeptide can be linked to carrier proteins such as bovine serum albumin, thyroglobulin, and mussel hemocyanin. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gels (eg, aluminum oxide trihydrate), and surfactants (eg, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, mussel hemocyanin, and Dinitrophenol), but is not limited thereto. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly useful.

  Monoclonal antibodies that specifically bind to a “BREAST CANCER GENE” polypeptide can be prepared using any technique that provides for the production of antibody molecules by passaged cell lines in culture. These techniques include, but are not limited to, hybridoma techniques, human B cell hybridoma techniques, and EBV hybridoma techniques [Kohler et al., 1985 (51)].

  In addition, techniques developed to produce chimeric antibodies and splicing of mouse antibody genes to human antibody genes can be used to obtain molecules with appropriate antigen specificity and biological activity [Takeda et al. 1985 (52)]. When using an antibody for therapy, monoclonal antibodies and other antibodies can also be humanized to prevent the patient from initiating an immune response against the antibody. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy, or may require some major residue modifications. Sequence differences between rodent antibodies and human sequences can be determined by site-directed mutagenesis of individual residues, by substituting residues that differ from those in the human sequence, or by complementarity determination. It can be minimized by transplanting the entire area. Alternatively, humanized antibodies can be produced using the recombinant methods described in British Patent No. 2188638. An antibody that specifically binds to a “BREAST CANCER GENE” polypeptide can contain a partially or fully humanized antigen binding site, as disclosed in US Pat. No. 5,565,332.

  Alternatively, a single chain that specifically binds to a “BREAST CANCER GENE” polypeptide by adapting the techniques described for the production of single chain antibodies using methods known in the art. Antibodies can be produced. Chain shuffling from random combinatorial immunoglobulin libraries can generate antibodies with related specificity but different idiotype composition [Burton, 1991 (53)].

  Single-chain antibodies can also be constructed using hybridoma cDNA as a template and DNA amplification methods such as PCR [Thirion et al., 1996 (54)]. Single chain antibodies can be monospecific or bispecific and can be bivalent or tetravalent. Construction of tetravalent bispecific single chain antibodies is taught, for example, by Coloma and Morrison (55), and construction of bivalent bispecific single chain antibodies is described by Mallender and Voss (56). Is taught.

  Nucleotide sequences encoding single chain antibodies are constructed using manual or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and cells that express the coding sequence as described below. Can be introduced inside. Alternatively, single chain antibodies can be produced directly, eg, using filamentous phage technology [Verhaar et al., 1995 (57)].

  Antibodies that specifically bind to a “BREAST CANCER GENE” polypeptide can be produced by in vivo production in a lymphocyte population, or as disclosed in the literature, an immunoglobulin library or a panel of highly specific binding reagents. Can also be produced by screening [orlandi et al., 1989 (58)].

  Other types of antibodies can be constructed and used therapeutically in the methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Multivalent and multispecific binding proteins derived from immunoglobulins such as the antibodies described in WO 94/13804 can also be prepared.

  According to the present invention, antibodies can be purified by methods well known in the art. For example, an antibody can be affinity purified by passing the antibody through a column to which a “BREAST CANCER GENE” polypeptide is bound. The bound antibody can then be eluted from the column using a high salt buffer.

  Immunoassays are commonly used to quantify protein levels in cell samples, and many other immunoassay techniques are known in the art. The present invention is not limited to a particular assay procedure and therefore encompasses both homogeneous and heterogeneous procedures. Exemplary immunoassays that can be performed in accordance with the present invention include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (ETA), turbidimetric immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA). ), And radioimmunoassay (RIA). The indicator moiety or labeling group can be attached to the antibody of interest and is selected to meet the needs of various uses of the above methods. Such a need is often governed by the availability of assay equipment and compatible immunoassay procedures. The general techniques to be used in performing the various immunoassays described above are known to those skilled in the art.

  Another method for quantifying the level of a particular protein, protein fragment, or modified protein in a particular sample is based on flow cytometry. Flow cytometry uses protein-specific antibodies labeled with fluorescent dyes, or combines unlabeled antibodies with secondary antibodies labeled with fluorescent dyes to identify proteins on the cell surface in the same way as intracellular proteins. enable. The general techniques to be used in performing the flow cytometry assays described above are known to those skilled in the art. A special method based on the same principle is microparticle-based flow cytometry. Label the microbeads with the correct amount of fluorescent dye and a specific antibody. Such a technique is provided by Luminex, WO 97/14028. In another embodiment, the level of a particular protein or protein fragment or modified protein in a particular sample can be measured by 2D gel electrophoresis and / or mass spectrometry. Determination of protein properties, sequence, molecular mass, and charge amount can be achieved in one detection step. Mass spectrometry can be performed by methods known to those skilled in the art as MALDI, ToF, or combinations thereof.

  In another embodiment, the level of the encoded product in the patient's biological fluid (eg, blood or urine), ie, the product encoded by either the polynucleotide sequence of SEQ ID NOs: 1-65 or its complementary sequence. Can be determined by monitoring the expression level of the marker polynucleotide sequence in the cells of the patient. Such methods include the steps of obtaining a sample of biological fluid from a patient, contacting the sample (or protein from the sample) with an antibody specific for the encoded marker polypeptide, and immunization with the antibody. Measuring the amount of complex formation, wherein the amount of immune complex formation indicates the level of product encoded by the marker in the sample. This measurement is particularly beneficial when compared to the amount of immune complex formation by the same antibody in a control sample taken from a normal individual, or one or more samples obtained from the same person, either pre- or post-hoc. It is.

  In another embodiment, the above method can be used to measure the amount of marker polypeptide present in a cell, which can then be correlated with the progression of the disorder, eg, plaque formation. it can. The level of marker polypeptide can be used predictively to assess whether a sample of cells contains cells that are or are likely to become plaque-related cells. Observation of marker polypeptide levels can be used, for example, to determine whether to use more stringent treatments.

  As described above, one aspect of the invention relates to diagnostic assays for determining whether marker polypeptide levels are significantly reduced in sample cells in the context of cells isolated from a patient. The term “significantly reduced” refers to a cell phenotype in which the amount of marker polypeptide carried by the cell is reduced compared to normal cells of similar tissue origin. For example, the cells have less than about 50%, 25%, 10%, or 5% marker polypeptide of normal control cells. In particular, the assay assesses the level of marker polypeptide in the test cell, and preferably the measured level is known to at least one control cell, eg, normal cell and / or phenotype. Compare to marker polypeptide detected in transformed cells.

  Of particular importance to the present invention is the ability to quantify the level of marker polypeptide as measured by the number of cells associated with normal or abnormal marker polypeptide levels. The number of cells with a particular marker polypeptide phenotype can then be correlated with the prognosis of the patient. In one embodiment of the invention, the marker polypeptide phenotype at the site of injury is determined as the percentage of cells in the biopsy where the level of marker polypeptide has been found to be abnormally high or low. Such expression can be detected by immunohistochemical assays, dot blot analysis, ELISA, and the like.

Immunohistochemistry When tissue samples are employed, immunohistochemical staining can be used to determine the number of cells having a marker polypeptide phenotype. For such staining, multiple blocks of tissue are removed from a biopsy or other tissue sample and subjected to proteolytic hydrolysis using agents such as protease K or pepsin. In certain embodiments, it may be desirable to isolate the nuclear fraction from the sample cells and detect the level of the marker polypeptide in the nuclear fraction.

  Tissue samples are fixed by treatment with reagents such as formalin, glutaraldehyde, methanol and the like. The sample is then incubated with an antibody having a binding specificity for the marker polypeptide, preferably a monoclonal antibody. This antibody may be conjugated to a label for later detection of binding. The sample is incubated for a time sufficient for the formation of immune complexes. Antibody binding is detected using a label attached to the antibody. If the antibody is not labeled, a labeled second antibody, eg, an antibody specific for the isotype of the anti-marker polypeptide antibody may be utilized. Examples of labels that can be used include radionuclides, fluorescence, chemiluminescence, and enzymes.

  When an enzyme is employed, a substrate for that enzyme can be added to the sample to produce a colored or fluorescent product. Examples of enzymes suitable for use in the conjugate include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.

  In one embodiment, the assay is performed as a dot blot assay. When a tissue sample is employed, the average amount of marker polypeptide associated with a single cell can be determined by correlating the amount of marker polypeptide in the cell-free extract produced from a given number of cells. As it allows, dot blot assays have special applications.

  In yet another embodiment, the present invention contemplates the use of a panel of antibodies raised against the marker polypeptide of the present invention encoded by any of the polynucleotide sequences of SEQ ID NOs: 1-65. Such a panel of antibodies can be used as a reliable diagnostic probe for breast cancer. The assay of the present invention comprises contacting a biopsy sample containing cells, eg macrophages, with a panel of antibodies against one or more encoded products to determine the presence or absence of a marker polypeptide.

  The diagnostic method of the present invention can also be used as a follow-up of treatment, for example, quantification of marker polypeptide levels is currently or previously employed, the effectiveness of treatment of malignant tumors, particularly breast cancer, and patient It may indicate the impact of these treatments on prognosis.

  The diagnostic assays described above can also be adapted for use as a prognostic assay. Such applications take advantage of the sensitivity of the assay of the present invention to events that occur at characteristic stages in the progression of plaque generation in the case of malignant tumors. For example, a given marker gene may be up- or down-regulated before a cell develops into a foam cell, perhaps at a very early stage, while another marker gene is at a much later stage. Only characteristically may be adjusted up or down. Such a method comprises contacting test cell mRNA with a polynucleotide probe derived from a given marker polynucleotide that is expressed at different characteristic levels in breast cancer tissue cells at different stages of malignant tumor progression. And measuring an approximate amount of hybridization of the probe to the mRNA of the cell, such amount being an indicator of the expression level of the gene in the cell, and thus The assay can be performed using an antibody specific for the gene product of a given marker polynucleotide that is an indicator of the stage of disease progression of the cell or, alternatively, is in contact with the protein of the test cell. A series of such studies not only discloses the presence of a particular neoplastic lesion, but also allows clinicians to select the most appropriate treatment method for the disease and further predict the likelihood of successful treatment. Would also be possible.

  The methods of the invention can also be used to follow the clinical course of a given breast cancer constitution. For example, the assay of the present invention can be applied to a blood sample obtained from a patient, and after the patient is treated for breast cancer, another blood sample is taken and the test is repeated. Effective treatment will result in the removal of the indicated differential expression characteristic of breast cancer tissue cells, possibly approaching or even surpassing normal levels.

Polypeptide Activity In one aspect, the invention provides a method of screening for therapeutic agents that may modulate the activity of one or more “BREAST CANCER GENE” polypeptides, whereby malignant tumors, particularly breast cancer. In comparison with the activity of certain polypeptides in a subject who has or is at risk of having no malignant tumor, no breast cancer, or at risk of being treated with a therapeutic agent, a “BREAST CANCER GENE” If the activity of the polypeptide is enhanced as a result of upregulation of the therapeutic agent, the therapeutic agent will decrease the polypeptide activity. Similarly, in a subject who has or is at risk of having a malignant tumor, in particular breast cancer, the same polypeptide in a subject that is not malignant, has no breast cancer, is not at risk, but has not been treated with a therapeutic agent. If the activity of the polypeptide is reduced as a result of down-regulation of the “BREAST CANCER GENE” as compared to activity, the therapeutic agent will increase polypeptide activity.

  The activity of the “BREAST CANCER GENE” polypeptide shown in Table 1 or 3 can be measured by any method known to those of skill in the art and specific methods for the type of activity exhibited by the particular polypeptide. Examples of specific assays that can be used to measure the activity of a particular polynucleotide are shown below.

a) Ion Channels Ion channels are transmembrane proteins involved in electrical signaling, transmembrane signaling, and electrolyte-loaded solute transport. By forming polymer pores that penetrate the membrane lipid bilayer, the ion channels are responsible for the flow of certain ionic species driven by the electrochemical potential gradient of the permeable ions. At the single molecule level, individual channels undergo a conformational transition between an “open” state (ionic conduction) and a “closed” (non-conducting) state (“gating”). Typical opening of a single channel lasts a few milliseconds, ^resulting in a basic transmembrane currents in the range of 10 ^{9 to 10 -12} amps. Channel opening and closing is controlled by various chemical and / or biophysical parameters such as neurotransmitters and intracellular second messengers (“ligand-gated channels”) or membrane potentials (“voltage-gated channels”) . Ion channels are functionally characterized by their ion selectivity, gating properties, and regulation by hormones and drugs. Because of their central role in signal transduction and transport processes, ion channels have become ideal targets for pharmacological therapeutics in a variety of pathophysiological settings.

  In one embodiment, the “BREAST CANCER GENE” may encode an ion channel. In one embodiment, the present invention provides a method of screening for potential activators or inhibitors of channel activity of a “BREAST CANCER GENE” polypeptide. Screening to obtain compounds that interact with ion channels to suppress or promote their activity can be based on (1) binding assays and (2) functional assays in living cells [Hille (74) ].

  1. For ligand-gated channels, such as ionotropic neurotransmitter / hormone receptors, the assay can be designed to detect binding to the target by competition between the compound and the labeled ligand. it can.

2. Ion channel function can be functionally tested in living cells. The target protein is either endogenously expressed in a suitable reporter cell or has been introduced recombinantly. Channel activity is caused by (2.1) changes in permeant ion concentration (Ca ²⁺ ions are most prominent) caused by target activity, (2.2) by changes in transmembrane potential gradient, and ( 2.3) It can be monitored by measuring cellular responses (eg, reporter gene expression, neurotransmitter secretion).

2.1 Channel activity results in transmembrane ion flux. The ion channels thus activated can be monitored using luminescence or fluorescent indicators by the resulting changes in intracellular ion concentration. This is particularly applicable to changes in intracellular Ca ²⁺ ion concentration ([Ca ²⁺ ] i) because of the wide dynamic range and the availability of appropriate indicators. [Ca ²⁺ ] i can be measured, for example, by aequorin luminescence or fluorescent dye technology (eg, use of Fluo-3, Indo-1, Fura-2). Cell assays that directly measure Ca ²⁺ flux through the target channel itself, or where modulation of the target channel affects membrane potential, thereby affecting the activity of voltage-gated Ca ²⁺ channels that are co-expressed. Can be designed.

2.2 The ion channel current results in a change in electrical membrane potential (Vm), which can be monitored directly using a potentiometric fluorescent probe. These charged indicators (eg, the anionic oxonol dye DiBAC ₄ (3)) redistribute between the extracellular and intracellular compartments in response to potential changes. The equilibrium distribution is governed by the Nernst equation. Thus, changes in membrane potential lead to changes in cell fluorescence associated therewith. Again, the change in V _m is to may be caused directly by the activity of the target ion channel, or may be caused through the amplification and / or prolongation of the signal by channels co-expressed in the same cell.

2.3 Target channel activity can cause cellular Ca ²⁺ entry either directly or through activation of additional Ca ²⁺ channels (see 2.1). The resulting intracellular Ca ²⁺ signal regulates various cellular responses such as secretion or gene transcription. Thus, by monitoring the secretion of a known hormone / transmitter from a target-expressing cell, or a reporter gene (eg, a cyclic AMP / Ca ²⁺ responsive element; CRE) that is controlled by a Ca ²⁺ responsive promoter element (eg, For example, changes in the target channel can be detected through the expression of luciferase).

b) DNA binding proteins and transcription factors In one embodiment, a “BREAST CANCER GENE” may encode a DNA binding protein or transcription factor. For example, the activity of such a DNA binding protein or transcription factor can be measured by a promoter assay that measures the ability of the DNA binding protein or transcription factor to initiate transcription of a test sequence linked to a particular promoter. In one embodiment, the present invention provides an altered expression of a test gene regulated by a promoter that is responsive to the transcription factor to obtain the ability to modulate the activity of such a DNA binding protein or transcription factor. A method is provided for screening test compounds by measuring.

Promoter Assay The promoter assay was set up with human hepatocellular carcinoma cells HepG2 stably transfected with a luciferase gene under the control of a promoter regulated by the gene of interest (eg thyroid hormone). Vector 2xIROLuc was used for transfection. The vector 2xIROluc has a thyroid hormone responsive element (TRE) consisting of two 12 bp reverse palindrome separated by an 8 bp spacer in front of the tk minimal promoter and luciferase gene. Test cultures were seeded in serum-free Eagle's minimum essential medium supplemented with glutamine, tricine, sodium pyruvate, non-essential amino acids, insulin, selenium, and transferrin in a 96-well plate, 10% CO2, 37 ° C humidified atmosphere Incubated in. After 48 hours of incubation, serial dilutions of test or reference compound (eg L-T3, L-T4) and, if appropriate, costimulators (final concentration 1 nM) are added to the cell culture for an optimal period ( Incubation was continued for another 4 to 72 hours, for example. Subsequently, cells were lysed by adding a buffer containing Triton X100 and luciferin, and luminescence of luciferase was induced by T3 or other compounds and measured with a luminometer. Four replicates were tested for each concentration of test compound. EC ₅₀ values for each test compound were calculated by use of Graph Pad Prism Scientific software.

Screening Methods The present invention provides assays for screening for test compounds that bind to or modulate the activity of a “BREAST CANCER GENE” polypeptide or “BREAST CANCER GENE” polynucleotide. Preferably, the test compound binds to a “BREAST CANCER GENE” polypeptide or polynucleotide. More preferably, the test compound reduces or enhances “BREAST CANCER GENE” activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% compared to the absence of the test compound.

Test Compound The test compound may be a drug already known in the art or a compound that was not previously known to have any pharmacological activity. The compounds may be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals or plants and can be produced recombinantly or synthesized by chemical methods known in the art. If desired, test compounds include, but are not limited to, biological libraries, spatially addressable parallel solid phase libraries or solution phase libraries, synthetic library methods that require deconvolution, 1 Can be obtained using any of a number of combinatorial library methods known in the art, including bead single compound library methods and synthetic library methods using affinity chromatography selection. it can. The biological library approach is limited to polypeptide libraries, but the other four approaches are applicable to small molecule libraries of polypeptides, non-peptidic oligomers, or compounds [for review, Lam , 1997 (59)].

  Methods for synthesizing molecular libraries are well known in the art [see, eg, Gallop et al., 1994 (60)]. A library of compounds can be found in solution [eg, Houghten, 1992 (61)], beads [Lam, 1991 (62)], DNA chips [Fodor, 1993 (63)], bacteria or spores (Ladner, US patent). No. 5223409), plasmids [Cull et al., 1992 (64)], or phage [Scott and Smith, 1990 (65)].

High-throughput screening Using high-throughput screening for those that are capable of binding to a “breast cancer gene” polypeptide or polynucleotide, or capable of affecting “breast cancer gene” activity or “breast cancer gene” expression Test compounds can be screened. Using high-throughput screening, a large number of individual compounds can be tested in parallel, so that a large number of test compounds can be rapidly screened. The most widely established technique utilizes 96-well, 384-well, or 1536-well microtiter plates. Microtiter plate wells typically require assay volumes in the range of 5 to 500 μl. In addition to plates, numerous instruments, materials, pipettors, robotics, plate washers, and plate readers that are compatible with the microwell format are commercially available.

  Alternatively, free format assays or assays without physical barriers between samples can be used. For example, a combinatorial peptide library assay, which is a simple homogenous assay using pigment cells (melanocytes), has been described by Jaywickreme et al. (66). Cells are placed under agarose in the culture dish, and then beads holding the combinatorial compound are placed on the surface of the agarose. Combinatorial compounds are partially released from the beads. As the compound diffuses locally into the gel matrix, the active compound changes the color of the cells so that the active compound can be visualized as a dark pigmented region.

  Another example of a free format assay is described by Chelksky (67). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase in an agarose gel so that the enzyme in the gel caused a color change throughout the gel. Thereafter, beads holding the combinatorial compound via a photolinker were placed inside the gel and the compound was partially released by UV light. Compounds that inhibited the enzyme were observed as locally inhibited regions with less discoloration than others.

  In another example, combinatorial library compounds having a cytotoxic effect on cancer cells growing in agar were screened [Salmon et al., 1996 (68)].

  Another high-throughput screening method is described in Beutel et al., US Pat. No. 5,976,813. In this method, a test sample is placed in a porous matrix. The one or more assay components are then placed inside, top, or bottom of a matrix, such as a gel, plastic sheet, filter, or other form of solid support that is easy to manipulate. When samples are introduced into the porous matrix, they diffuse slowly enough so that the assay can be performed without manipulating many test samples simultaneously.

Binding Assay In a binding assay, it is preferred that the test compound is a small molecule, eg, binds to and occupies the active site of an ATP / GTP binding site of an enzyme, or “BREAST CANCER GENE” polypeptide, thereby Normal biological activity is blocked. Examples of such small molecules include but are not limited to small peptides or peptide-like molecules.

  In a binding assay, either the test compound or the “BREAST CANCER GENE” polypeptide contained a detectable label, such as a fluorescent, radioisotope, chemiluminescent, or enzyme label such as horseradish peroxidase, alkaline phosphatase, or luciferase. Can be a thing. The test compound bound to the “BREAST CANCER GENE” polypeptide can then be detected, for example, by direct measurement of radiation emission by scintillation counting or by measuring the conversion of an appropriate substrate to a detectable product.

  Alternatively, the binding of the test compound to the “BREAST CANCER GENE” polypeptide can be measured without labeling any of the interactants. For example, a microphysiometer can be used to detect binding of a test compound to a “BREAST CANCER GENE” polypeptide. A microphysiometer (for example, CytosensorJ) is an analyzer that measures the rate at which cells acidify their environment using a light-addressable potentiometric sensor (LAPS). This change in acidification rate can be used as an indicator of the interaction between the test compound and the “BREAST CANCER GENE” polypeptide [McConnell et al., 1992 (69)].

  Measurement of the ability of a test compound to bind to a “BREAST CANCER GENE” polypeptide can also be achieved using techniques such as real-time BIA (Bimolecular Interaction Analysis) [Szabo et al., 1995 (70)]. BIA is a technique for studying biospecific interactions in real time without labeling any of the interactants (eg, BIAcore ™). Changes in surface plasmon resonance (SPR), which is an optical phenomenon, can be used as an indicator of real-time reactions between biomolecules.

  In another aspect of the invention, a “BREAST CANCER GENE” polypeptide is identified in a two-hybrid assay or a three-hybrid assay to identify other proteins that bind to or interact with a “BREAST CANCER GENE” polypeptide and modulate its activity. As “bait protein” in US Pat. No. 5,283,317; Brent, WO 94/10300.

  The two-hybrid system is based on the fact that most transcription factors consist of separate DNA binding and activation domains and have modular properties. Briefly, this assay utilizes two different DNA constructs. For example, in one construct, a polynucleotide encoding a “BREAST CANCER GENE” polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (eg, GAL4). In the other construct, a DNA sequence encoding an unidentified protein ("prey" or "sample") can be fused to a polynucleotide encoding the activation domain of a known transcription factor. When the “bait” protein and the “prey” protein interact in vivo to form a protein-dependent complex, the transcription factor DNA binding domain and the activation domain are in close proximity. This proximity allows transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site that is responsive to the transcription factor. Reporter gene expression can be detected, thereby isolating cell colonies containing functional transcription factors and used to obtain DNA sequences that encode proteins that interact with "BREAST CANCER GENE" polypeptides can do.

  Immobilize either a “BREAST CANCER GENE” polypeptide (or polynucleotide) or test compound to facilitate separation of bound from unbound forms in one or both interactants and further facilitate assay automation It may be desirable to do so. Thus, either a “BREAST CANCER GENE” polypeptide (or polynucleotide) or a test compound can be bound to a solid support. Suitable solid supports include particles such as, but not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or beads (including but not limited to latex, polystyrene, or glass beads). Is included, but is not limited thereto. To bind a “BREAST CANCER GENE” polypeptide (or polynucleotide) or test compound to a solid support, a covalent and non-covalent bond, passive absorption, or the polypeptide (or polynucleotide) or test compound, respectively, Any method known in the art may be used, including the use of a pair of binding moieties attached to a solid support. Test compounds are preferably bound to the solid support of the array so that the position of individual test compounds can be tracked. Binding of the test compound to the “BREAST CANCER GENE” polypeptide (or polynucleotide) may be performed in any container suitable for containing the reactants. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes.

  In one embodiment, the “BREAST CANCER GENE” polypeptide is a fusion protein comprising a domain that allows the “BREAST CANCER GENE” polypeptide to bind to a solid support. For example, glutathione S-transferase fusion protein can be adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, Mo., USA) or glutathione derivatized microtiter plates, which are then Alternatively, the test compound and non-adsorbed “BREAST CANCER GENE” polypeptide are bound; the mixture is incubated under conditions that contribute to complex formation (eg, salt conditions and pH at physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. The measurement of binding of the interactant can be performed directly or indirectly as described above. Alternatively, the complex can be separated from the solid support prior to measuring binding.

  Other techniques for immobilizing proteins or polynucleotides on a solid support can also be used in the screening assays of the invention. For example, either a “BREAST CANCER GENE” polypeptide (or polynucleotide) or a test compound can be immobilized utilizing the binding of biotin and streptavidin. A biotinylated “BREAST CANCER GENE” polypeptide (or polynucleotide) or test compound is biotinylated using a technique well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, Ill., USA) N-hydroxysuccinimide) and immobilized on streptavidin-coated 96-well plates (Pierce Chemicals). Alternatively, an antibody that specifically binds to a “BREAST CANCER GENE” polypeptide, polynucleotide, or test compound, but does not interfere with a desired binding site, such as an ATP / GTP binding site or an active site of a “BREAST CANCER GENE” polypeptide Can be derivatized and bound to the wells of the plate. Unbound target or protein can be captured in the well by antibody binding.

  Methods for detecting such complexes include immunization of complexes with “BREAST CANCER GENE” polypeptides or antibodies that specifically bind to test compounds in addition to those described above for GST-immobilized complexes. Detection, an enzyme binding assay that relies on detecting activity of the “BREAST CANCER GENE” polypeptide, and SDS gel electrophoresis under non-reducing conditions.

  Screening to obtain a test compound that binds to a “BREAST CANCER GENE” polypeptide or polynucleotide can also be performed in intact cells. Any cell containing a “BREAST CANCER GENE” polypeptide or polynucleotide can be used in a cell-based assay system. “BREAST CANCER GENE” polynucleotides may be naturally occurring in the cell or introduced using techniques such as those described above. Binding of the test compound to the “BREAST CANCER GENE” polypeptide or polynucleotide is measured as described above.

Altering Gene Expression In another embodiment, test compounds that enhance or decrease expression of a “BREAST CANCER GENE” are identified. The “BREAST CANCER GENE” polynucleotide is contacted with a test compound in a suitable expression test system or cellular system described below, and the expression of the RNA or polypeptide product of the “BREAST CANCER GENE” polynucleotide is measured. The expression level of the appropriate mRNA or polypeptide in the presence of the test compound is compared to the expression level of the mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a regulator of expression based on this comparison. For example, if the expression of mRNA or polypeptide is greater in the presence and absence of the test compound, the test compound is identified as a stimulator or promoter of expression of the mRNA or polypeptide. Instead, if the expression of the mRNA or polypeptide is less in the presence of the test compound than in the absence, the test compound is identified as an inhibitor of the expression of the mRNA or polypeptide.

  The expression level of “BREAST CANCER GENE” mRNA or polypeptide in a cell can be measured by methods for detecting mRNA or polypeptide that are well known in the art. Both qualitative and quantitative methods can be used. The presence of a polypeptide product of a “BREAST CANCER GENE” polynucleotide is measured using a variety of techniques known in the art including, for example, immunochemistry, radioimmunoassay, western blotting, immunohistochemistry, etc. be able to. Alternatively, polypeptide synthesis can be measured in vivo in cell culture or by detecting the incorporation of labeled amino acids into the “BREAST CANCER GENE” polypeptide in an in vitro translation system.

  Such screening can be performed in a cell-free assay system or in intact cells. Any cell that expresses a “BREAST CANCER GENE” polynucleotide can be used in a cell-based assay system. “BREAST CANCER GENE” polynucleotides may be naturally occurring in the cell or introduced using techniques such as those described above. Either primary cultures or established cell lines such as CHO or human embryonic kidney 293 cells can be used.

Therapeutic applications and methods Therapies for treating breast cancer have relied primarily on effective chemotherapeutic drugs to intervene in cell proliferation, cell proliferation, or angiogenesis. With the advent of genomics-led molecular target identification, breast cancer specific new targets for use in therapeutic interventions will provide safer and more effective treatment for patients with malignant tumors, particularly breast cancer patients. The possibility to identify was opened. Thus, newly discovered breast cancer-related genes, and their products, can be used as tools for developing innovative therapies. Identification of the Her2 / neu receptor kinase presents a new and exciting opportunity to treat a particular subset of tumor patients, as described above. Genes that play an important role in any of the physiological processes outlined above can be characterized as breast cancer targets. A gene or gene fragment identified by genomics can be readily expressed in one or more heterologous expression systems to produce a functional recombinant protein. With respect to the biochemical properties of these proteins, they are characterized in vitro and used as a tool to identify chemical regulators of their biochemical activity in high-throughput molecular screening programs. In this way, modulators of target gene expression or protein activity can be identified and subsequently tested for therapeutic activity in cellular and in vivo disease models. Lead compound optimization through repeated trials in biological models, as well as detailed pharmacokinetics and toxicology analysis, form the basis for drug development and subsequent human testing.

  The present invention further relates to the use of novel agents identified by the screening assays described above. Accordingly, the use of test compounds identified as described herein in an appropriate animal model is also encompassed by the present invention. For example, an agent identified as described herein (eg, a modulator, antisense polynucleotide molecule, specific antibody, ribozyme, or human “BREAST CANCER GENE” polypeptide binding molecule) in an animal model, The effectiveness, toxicity, or side effects of treatment with such agents can be determined. Alternatively, agents identified as described herein can be used in animal models to determine the mechanism of action of such agents. The present invention further relates to the use of novel agents identified for the treatments described herein by the screening assays described above.

  Reagents that affect the activity of the human “BREAST CANCER GENE” can be administered to human cells either in vitro or in vivo to reduce or enhance the activity of the human “BREAST CANCER GENE”. Preferably, the reagent binds to the expression product of human “BREAST CANCER GENE”. When the expression product is a protein, the reagent is preferably an antibody. To treat human cells ex vivo, antibodies can be added to a preparation of stem cells removed from the body. The cells can then be replaced with the same or another human body, with or without clonal expansion, as is known in the art.

  In one embodiment, liposomes are used to deliver the reagent. The liposome is preferably stable in the animal to which it is administered for at least about 30 minutes, more preferably for at least about 1 hour, even more preferably for at least about 24 hours. Liposomes contain a lipid composition that can target a specific site in an animal, such as a human, to deliver a reagent, particularly a polynucleotide. The lipid composition of the liposome is preferably such that it can target specific organs of the animal, such as lung, liver, spleen, heart, brain, lymph node, and skin.

Liposomes useful in the present invention comprise a lipid composition that can be fused with the plasma membrane of the target cell to deliver its contents to the cell. Preferably, the transfection efficiency of the liposomes is about 0.5 μg of DNA per 16 nmol of liposomes delivered to about 10 ⁶ cells, more preferably about 1.times. DNA per 16 nmol of liposomes delivered to about 10 ⁶ cells. 0 μg, more preferably about 2.0 μg of DNA per 16 nmol of liposomes delivered to about 10 ⁶ cells. Liposomes preferably have a diameter of about 100-500 nm, more preferably about 150-450 nm, and even more preferably about 200-400 nm.

  Liposomes suitable for use in the present invention include, for example, liposomes commonly used in gene delivery methods known to those skilled in the art. More preferred liposomes include liposomes having a polycationic lipid composition and / or liposomes having a cholesterol backbone bound to polyethylene glycol. Optionally, the liposome includes a compound that can target the specific cell type, such as a cell-specific ligand exposed on the external surface of the liposome, to deliver the liposome.

  Liposomes can be complexed with reagents such as antisense oligonucleotides or ribozymes using standard methods in the art (see, eg, US Pat. No. 5,705,151). Preferably, about 0.1 μg to about 10 μg of polynucleotide is mixed with about 8 nmol liposomes, more preferably about 0.5 μg to about 5 μg of polynucleotide is mixed with about 8 nmol liposomes, more preferably about 1.0 μg of polynucleotide is mixed with about 8 nmol of liposomes.

  In another embodiment, receptor-mediated directed delivery can be used to deliver antibodies in vivo to specific tissues. Receptor-mediated DNA delivery techniques are taught, for example, in Findeis et al., 1993 (71); Chiou et al., 1994 (72).

Determination of a therapeutically effective dose Determination of a therapeutically effective dose is within the ability of one skilled in the art. A therapeutically effective dose refers to that amount of active ingredient that enhances or reduces human “BREAST CANCER GENE” activity as compared to that human “BREAST CANCER GENE” activity present in the absence of a therapeutically effective dose.

  For any compound, the therapeutically effective dose can be estimated initially from cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, eg, ED ₅₀ (a dose that is therapeutically effective in 50% of the population) and LD ₅₀ (a dose that is lethal to 50% of the population) are measured in cell culture or laboratory animal standard pharmaceutical procedures. Can be determined by. The ratio between the dose with toxic effect and the dose with therapeutic effect is the therapeutic index and can be calculated as the ratio LD ₅₀ / ED ₅₀ .

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies is used to formulate a range of doses for use in humans. Doses are contained in such compositions is no toxicity with little or without at all, it is preferably in the range of circulating concentrations that include the ED _50. The dosage may vary within this range depending on the dosage form used, patient sensitivity, and route of administration.

  The exact dose will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include severity of disease state, overall subject health, subject age, weight, and gender, diet, time and frequency of administration, combined formulation, response sensitivity, and tolerance / Reaction is included. Long-acting pharmaceutical compositions can be administered once every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

  The normal dose may vary from 0.1 to 10,000 μg, up to a total amount of 1 g, depending on the route of administration. Hands on specific doses and methods of delivery are provided in the literature and are generally available to practitioners in the art. For nucleotides, those skilled in the art will use different formulations than proteins and their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, sites, etc.

  If the reagent is a single chain antibody, construct the polynucleotide encoding the antibody, including, but not limited to, transferrin-polycation mediated DNA transport, transfection with naked or encapsulated nucleic acid, Well-established techniques including liposome-mediated cell fusion, intracellular introduction of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun DEF or calcium phosphate-mediated transfection Can be introduced into cells either ex vivo or in vivo.

  Effective in vivo doses of the antibody range from about 5 μg to about 50 μg / kg patient weight, from about 50 μg to about 5 mg / kg, from about 100 μg to about 500 μg / kg, and from about 200 to about 250 μg / kg patient weight. . Effective in vivo doses for administration of polynucleotides encoding single chain antibodies are about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, about 20 μg to about 100 μg of DNA. Is in the range

  When the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by various methods as described above.

  Preferably, the reagent has an expression of a “BREAST CANCER GENE” gene, or an activity of a “BREAST CANCER GENE” polypeptide at least about 10, preferably about 50, more preferably about 75, as compared to the absence of the reagent. Reduce by 90 or 100%. Evaluation of the expression level of the gene of the “BREAST CANCER GENE” or the effectiveness of the mechanism selected to reduce the activity of the “BREAST CANCER GENE” polypeptide can include hybridization of a nucleotide probe to mRNA specific for “BREAST CANCER GENE”, It can be performed using methods well known in the art, such as quantitative RT-PCR, immunological detection of a “BREAST CANCER GENE” polypeptide, or measurement of “BREAST CANCER GENE” activity.

  In any of the embodiments described above, any of the pharmaceutical compositions of the present invention can be administered in conjunction with other therapeutic agents. Selection of suitable drugs for use in combination therapy can be made by one skilled in the art according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to affect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficiency may be achieved even with small doses of each drug, thereby reducing the potential for adverse side effects.

  Any of the methods of treatment described above require such treatment, including, for example, birds and mammals such as dogs, cats, cows, pigs, sheep, goats, horses, rabbits, monkeys, and most preferably humans. It can be applied to any subject.

  All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are given for purposes of illustration only and are intended to limit the scope of the invention. It is not a thing.

Pharmaceutical Composition The present invention also provides a pharmaceutical composition that can be administered to a patient to achieve a therapeutic effect. The pharmaceutical composition of the present invention includes, for example, “BREAST CANCER GENE” polypeptide, “BREAST CANCER GENE” polynucleotide, ribozyme, or antisense oligonucleotide, antibody that specifically binds to “BREAST CANCER GENE” polypeptide, “BREAST CANCER GENE” It may include mimetics, agonists, antagonists, or inhibitors of peptide activity. The composition can be administered alone or in any sterile and biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, glucose, and water. It can also be administered in conjunction with at least one other agent, such as a stabilizing compound that can be administered. The composition can be administered to a patient alone or in combination with other drugs, drugs, or hormones.

  In addition to the active ingredient, these pharmaceutical compositions contain a suitable pharmaceutically acceptable carrier containing excipients and adjuvants that facilitate the processing of the active compound into pharmaceutically usable preparations. be able to. The pharmaceutical composition of the present invention includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, Administration can be by a variety of routes including topical, sublingual or rectal routes. Pharmaceutical compositions for oral administration can be formulated at dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers may formulate the pharmaceutical composition as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for patient consumption. enable.

  Formulations for oral use combine the active compound with solid excipients and, if desired, grind the resulting mixture, add appropriate adjuvants as necessary, and then treat the granule mixture. Can be formulated through obtaining tablets or dragee cores. Suitable additives include sugars including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose sodium Gums including gum arabic and tragacanth; and carbohydrate or protein excipients such as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

  The dragee core can be used in conjunction with a suitable shell such as a high-concentration sugar solution, which further includes gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide powder, lacquer solution As well as suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active ingredient, ie, dosage.

  Pharmaceutical preparations that can be taken orally include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a skin such as glycerol or sorbitol. Push-fit capsules may contain active ingredients mixed with fillers or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquids, or liquid polyethylene glycols, with or without stabilizers.

  Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers can also be used for delivery. If desired, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  The pharmaceutical composition of the present invention is manufactured by methods known in the art, for example, by conventional mixing, dissolving, grinding, dragee formation, grinding, emulsification, encapsulation, capture, or lyophilization process. Can do. The pharmaceutical composition can be provided as a salt and is formed using a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. be able to. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base form. In other cases, a lyophilized powder drug that may contain 150 mM histidine, 0.1% 2% sucrose, and 27% mannitol, part or all of pH range 4.5-5.5. May be a preferred preparation, which is formulated with a buffer before use.

  A more detailed description of the formulation and administration techniques can be found in the latest edition of “REMINGTON'S PHARMACEUTICAL SCIENCES” (73). After preparing the pharmaceutical compositions, they can be placed in a suitable container and labeled for the treatment of the condition being applied. Such labels will include the amount, frequency and method of administration.

  One strategy for identifying genes involved in breast cancer is to identify differentially expressed genes in non-disease or in the context of a therapeutic response state under conditions related to the disease. Is to detect. The following subsections describe a number of experimental systems that could be used to detect such differentially expressed genes. In general, these experimental systems include at least one experimental condition in addition to at least one experimental control condition, in which the subject or sample is treated in a manner associated with breast cancer, and the control condition Below, treatments associated with such diseases are missing or do not respond to such treatments. Differentially expressed genes are detected by comparing gene expression patterns between experimental conditions and control conditions as described below.

  By using one such experiment, once a particular gene has been identified, its expression pattern can be further characterized by studying its expression in another experiment, It can be confirmed by an independent technique. The use of such multiple experiments can be useful in identifying the role and relative importance of a particular gene in breast cancer and its treatment. A mixed approach that compares the gene expression pattern of cells obtained from breast cancer patients with the expression pattern of an in vitro cell culture model may provide substantial hints regarding the pathways involved in the development and / or progression of breast cancer it can. It can also elucidate the role of such genes in resistance development or insensitivity to certain therapeutic agents (eg chemotherapeutic agents).

  Among these experiments that can be used to identify differentially expressed genes involved in malignancies and in particular breast cancer, some are designed to analyze genes involved in signal transduction. Such experiments may help to identify genes involved in cell growth.

  The following is a method described to identify genes involved in breast cancer. Such genes represent genes that are differentially expressed in breast cancer conditions or during experimental manipulation based on clinical knowledge, compared to their normal, ie, non-breast cancer condition expression. Such differentially expressed genes represent “target” and / or “marker” genes. Methods for further characterizing such differentially expressed genes and methods for identifying them as target and / or marker genes are shown below.

  Alternatively, the expression of a differentially expressed gene may be altered, ie quantitatively enhanced or decreased, under breast cancer status versus normal status, or control versus experimental conditions. If the degree of expression difference between breast cancer vs. normal or control vs. experimental conditions is large enough to be visualized by standard characterization techniques such as differential display techniques described below, for example, That's all. Other such standard characterization techniques by which expression differences can be visualized include, but are not limited to, quantitative RT-PCR and Northern analysis well known to those skilled in the art.

  In addition to the experiments described above, the following describes algorithms and statistical analyzes that can be used to evaluate and classify data and predict responses of biological samples not previously classified in the context of control samples. The predictive algorithms and equations described below already demonstrate their ability to subdivide individual cancers.

Clinical specimen removal and nucleic acid isolation Patients with primary breast cancer who received neoadjuvant epirubicin / cyclophosphamide (EC) or epirubicin / taxol (ET) treatment (Table 4) were selected for analysis. Neoadjuvant chemotherapy consisted of short intravenous epirubicin 90 mg m ² 1 day in infusion, and short cyclophosphamide intravenous infusion 600 mg m ² 1 day or Taxol 175 mg m ² 1 day. The study requirement is that participants have untreated primary breast cancer disease who can accept a continuous core biopsy and treat with EC or ET chemotherapy as the first treatment before surgery. there were. A continuous core biopsy of the primary tumor was performed before treatment and 24 hours after the start of the first series of neoadjuvant chemotherapy. The biopsy is performed using a Bard® MAGNUM ™ biopsy device (CR Bard, Inc., Covington, USA) equipped with a Bard® Magnum biopsy needle (BIP GmbH, Tuerfeld, Germany). Obtained from the area under local anesthesia. Serial biopsies were taken from clear tumor areas and additional skin entry sites using a 90 ° angle. This approach was taken to ensure that a high percentage of tumor material was present in the sample. All biopsy samples were snap frozen in liquid nitrogen and stored at −80 ° C. until further processing. Hematoxylin / eosin stained sections from tumor specimens were examined to assess the relative amount of tumor cells, benign epithelium, stroma, and lymphocytes. Standard clinical parameters such as ER, PgR, ki-67, p53, cerbB2 / HER2neu are routinely defined in the laboratory using biohistochemical and / or immunohistochemical methods (Table 4). ).

  Total RNA from tissue specimens was extracted by the guanidine-isothiocyanate method followed by phenol / chloroform extraction and DNase using the recommended protocol of the Atlas ™ Pure Total RNA Labeling System (BD Biosciences Clontech, Heidelberg, Germany). Processed. Total RNA was quantified using UV spectrophotometry (Photometer ECOM 6122, Eppendorf AG, Hamburg, Germany) and quality and integrity were confirmed by electrophoresis of 0.5 μg of isolated RNA on a 1% formamide agarose gel did. In addition, RNA samples were analyzed by microcapillary electrophoresis on LabChip using an Agilent 2100 bioanalyzer (Agilent Technologies GmbH, Boeblingen, Germany) according to the manufacturer's instructions.

Expression Profiling Using Quantitative Kinetic RT-PCR Detailed analysis of gene expression by quantitative PCR methods utilizes primers adjacent to the genomic region of interest and fluorescently labeled probes that hybridize between them . Using PRISM7700 Sequence Detection System from PE Applied Biosystems (Perkin Elmer, Foster City, Calif., USA) with the technology of fluorescent probes consisting of oligonucleotides labeled with both fluorescent reporter dyes and quencher dyes, measurement of such expression is performed. It can be carried out. Amplification of the probe-specific product causes probe cleavage, resulting in an increase in reporter fluorescence. Primers and probes are selected using Primer Express software and are primarily localized in the 3 'region or 3' untranslated region of the coding sequence. All primer pairs were confirmed for specificity by conventional PCR reactions and gel electrophoresis. To standardize the amount of sample RNA, GAPDH was chosen as a reference because there was no difference in control among the samples analyzed. To perform such expression analysis of genes in biological samples, 25 μl of a 100 μM stock solution “upstream primer”, 25 μl of a 100 μM stock solution “downstream primer”, 12.5 μl of a 100 μM stock solution TaqMan-probe (FAM / probe) Each primer / probe was prepared by mixing with Tamra) and adjusting to 500 μl with distilled water (primer / probe mix). For each reaction, 1.25 μl of patient sample cDNA was mixed with 8.75 μl of nuclease-free water and added to one well of a 96-well Optical Reaction Plate (Applied Biosystems No. 4306737). Thereafter, 1.5 μl of the above primer / probe mix, 12.5 μl Taq Man Universal-PCR mix (2 ×) (Applied Biosystems No. 4318157) and 1 μl water were added. The 96-well plate was closed with 8Caps / Strips (Applied Biosystems No. 4323032) and centrifuged for 3 minutes. PCR reaction measurements were performed under appropriate conditions (50 ° C. for 2 minutes, 95 ° C. for 10 minutes, 95 ° C. for 0.15 minutes) using TaqMan 7900HT from Applied Biosystems (No. 20114) according to the manufacturer's instructions. 1 minute at 40 ° C .; 40 cycles). Prior to measuring previously unclassified biological samples, standardization of experimental conditions can be performed using control experiments, eg, cell lines, healthy control samples, samples of defined therapeutic response.

A TaqMan validation experiment was performed, which showed that the efficiency of target and control amplification was approximately equivalent. This is an essential condition for the relative quantification of gene expression by comparison [Delta] [Delta] _T methods known to those skilled in the art. Therefore, Applied Biosystems software SDS 2.0 can be used according to the respective instructions. The CT values are then further analyzed using the appropriate software (Microsoft Excel ™) from the statistical software package (SAS).

  Roche Inc., capable of real-time detection of RT-PCR reactions, along with the above-described technology provided by Perkin Elmer. From Lightcycler ™ or Stratagene Inc. Other technical means, such as iCycler, can be used.

Expression Profiling Utilizing DNA Microarrays Expression profiling can be performed using Affymetrix array techniques. By hybridizing mRNA to such a DNA array or DNA chip, it becomes possible to identify the expression value of each transcript according to the signal intensity at a specific position of the array. Usually, these DNA arrays are made by spotting cDNA, oligonucleotides or subcloned DNA fragments. In the case of the Affymetrix technique, approximately 400,000 individual oligonucleotide sequences were synthesized at individual locations on the surface of the silicon wafer. The minimum length of the oligomer is 12 nucleotides, preferably 25 nucleotides or the full length of the transcript under investigation. Expression profiling can also be performed by hybridization to DNA or oligonucleotides bound to nylon or nitrocellulose membranes. Detection of signals resulting from hybridization can be obtained by colorimetric, fluorescent, electrochemical, electrical, optical, or radioactive readings. Detailed descriptions of array configurations are mentioned above and in other cited patent documents. In order to determine quantitative and qualitative changes in the chromosomal region to be analyzed, RNA from tumor tissue suspected of containing such genomic changes is analyzed for benign tissue (eg, epithelial) based on the expression profile of the entire transcriptome. It must be compared to RNA extracted from breast tissue or microdissected ductal tissue. With minor changes, the sample preparation protocol followed the Affymetrix GeneChip expression analysis guide (Santa Clara, Calif.). Extraction and isolation of total RNA from cells including tumors or benign tissues, biopsies, cell isolates or body fluids using TRIzol (Life Technologies, Rockville, MD) and Oligotex mRNA Midi kit (Qiagen, Hilden, Germany) An ethanol precipitation step should be performed to achieve a concentration of 1 mg / ml. Double-stranded cDNA is prepared by SuperScript system (Life Technologies) using 5-10 mg of mRNA. First strand cDNA synthesis was initiated using T7- (dT24) oligonucleotide. The cDNA can be extracted with phenol / chloroform and precipitated with ethanol to a final concentration of 1 mg / ml. From the resulting cDNA, cRNA can be synthesized using an in vitro transcription kit from Enzo (Enzo Diagnostics Inc., Farmingdale, NY). Within the same process, cRNA can be labeled with biotin nucleotides Bio-11-CTP and Bio-16-UTP (Enzo Diagnostics Inc., Farmingdale, NY). After labeling and cleanup (Qiagen, Hilden (Germany), cRNA in appropriate fragmentation buffer (eg 40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc, 35 minutes at 94 ° C.) Hybridization at 45 ° C. for 24 hours at 60 rpm on HG_U133 arrays A and B each containing approximately 40,000 probed transcripts of fragmented cRNA as in the Affymetrix protocol After the hybridization step, the surface of the chip is washed and streptavidin phycoerythrin (SAPE; Molecula) in the Affymetrix fluidics station. Probe, Eugene, Oreg.) To enhance staining, a second labeling step can be introduced, which is recommended but not compulsory. Times, should be added with an anti-streptavidin biotin labeled antibody Hybridization to the probe array can be detected by a fluorimetric scan (Hewlett Packard Gene Array Scanner; Hewlett Packard Corporation, Palo Alto, Calif.).

  After hybridization and scanning, microarray images can be analyzed for quality control, looking for major chip defects or hybridization signal anomalies. Thus, either Affymetrix GeneChip MAS5.0 software or other microarray image analysis software can be utilized. Primary data analysis should be performed by software provided by the manufacturer.

  In the case of genetic analysis, in one embodiment of the present invention, the primary data was analyzed by additional bioinformatic means and additional filter criteria. The bioinformatics analysis is detailed below.

Data analysis from expression profiling experiments According to the Affymetrix measurement technique (Affymetrix GeneChip Expression Analysis Guide, Santa Clara, CA), a single gene expression measurement on a single chip yields mean difference values and absolute calls. Each chip contains 16-20 oligonucleotide probe pairs per gene or cDNA clone. These probe pairs include perfectly matched and mismatched sets, both of which are necessary for the calculation of the mean difference value, ie the expression value, which is an indicator of the difference in intensity of each probe pair, This is calculated by subtracting the mismatch strength from the perfect match strength. This takes into account hybridization variations between probe pairs and other hybridization artifacts that may affect fluorescence intensity. The average difference is a numerical value that is considered to represent the expression value of the gene. Absolute calls can take values of “A” (not present), “M” (boundary value), or “P” (present) and represent the quality of a single hybridization. We use both quantitative information given by mean difference and qualitative information given by absolute call to differentiate in biological samples from individuals with breast cancer versus biological samples from normal populations. The genes expressed in For other algorithms other than Affymetrix, the comparison yielded different values representing the same expression value and expression difference.

The differential expression E in one of the breast cancer groups compared to the normal population is calculated as follows: N mean difference values d ₁ , d ₂ ,. . . , M pieces of the average difference value of _{d n} and normal individuals population _{c 1, c} 2,. . . , Assuming a c _m, calculated by the following formula:

If d _j <50 or c _i <50 in one or more values of i and j, then these particular values c _i and / or d _j are set to an “artificial” expression value 50. These specific E calculations allow correct results with TaqMan results.

  A gene is said to be up-regulated in breast cancer versus normal if the absolute col value equal to “P” in the breast cancer population is greater than n / 2, which is the minimal change factor shown in E ≧ Table 2. The minimal fold change factor in Table 3 is the patient population that responds to a given chemotherapy (CR), the patient population that does not respond to the administered chemotherapy (NC), or the tissue that has no pathological signs of tumor (NB). Give about. A fold change of greater than 1 refers to an increase in gene expression in the first named tissue sample compared to the second. This control factor is an average value and can vary individually. Here, the combined profile of all 65 genes listed in Table 1 for cluster analysis or principal component analysis represents such sample taxonomic groups.

  The final list of differentially controlled genes consists of all up-regulated genes and all down-regulated genes in biological samples from individuals with breast cancer versus biological samples from normal populations, Or it consists of individual response patterns. Finally, the genes of interest listed in this list for diagnostic or pharmaceutical use were validated by quantitative real-time RT-PCR (see Example 2).

Identification of EC-controlled genes in a single patient time-course study To identify genes that are differentially expressed after chemotherapy, each gene is identified after the first chemotherapy treatment. At that time, a confidence score (CS) can be calculated. The method reported by Jelinksy et al. (97) can be used with some modifications. CS is defined as the sum of the individual scores given for the fold change (FC), expression level (EL), and current call (PC) as defined by Affymetrix software. A fold change is defined for each gene relative to an untreated control at each time point. The FC score is 5 if the fold change is 2 or more for the up-regulated gene or 0.5 or less for the down-regulated gene, and is the fold change greater than 1.5 for the up-regulated gene? , Or for a down-regulated gene less than 0.66, this is 2, and for each up-regulated or down-regulated gene, the fold change is less than 1.5 or greater than or equal to 0.66, which is −0. 5. The EL score is determined based on the scaled intensity of each gene (the average intensity of each GeneChip is globally scaled to a target intensity of 100). As a result, the EL score is set to 3 when the signal intensity exceeds 10,000, is 2 when the intensity exceeds 500, is 0 for genes with an expression level of 500-50, and the intensity is 50 or less. In the case of -0.5. The PC score is 5 if the gene is present in all 5 samples (defined by Affymetrix software), 4 if the gene is present in 4 samples, and in 3 out of 5 genes. 3 if the gene is present, 2 if the gene is present in at least 2 of the 5 samples, 1 if the gene is present in only 1 of the 4 samples, and the gene is present 0 if not. Based on this scoring, the maximum possible CS for any gene is 35. We considered that the gene was controlled under chemotherapy treatment when CS was 27 or higher.

Identification of genes that are differentially expressed after chemotherapy with strong moderate force (E / C and E / T) Tumor tissue that shows differential expression after the first chemotherapy cycle and does not respond In order to identify genes that are distinct from the response from and can be used in the prediction algorithm described in Example 7, the following analysis can be performed.

  Dividing all applicable post-chemotherapy samples into training and test groups. Within the training set, samples are divided into two groups based on their clinical outcome (CR or NC). From the identification of all statistics, the Student t test or Welch test (p value based on all 22,000 elements shown in Table 2) significantly changed the gene. Isolation of all genes with the p-value <0.05 in both tests, or a combined rank less than 2200 (highest 10% fraction). Analysis of the same gene set in a paired pre-chemotherapy sample for differential expression. Identification of genes that have no discriminatory power in samples prior to chemotherapy. These genes describe biological processes based on the action of drugs. Selection of the gene with the highest mean fold change with respect to the median expression of this gene in a general breast cancer sample, shown in Table 2 for NC and CR samples. Further selection of genes can be made by identifying the highest fold change (FC) in the post-chemotherapy sample compared to the pre-chemotherapy sample by referencing the individual groups of CR and NC . The fold change in NC vs. CR of the group prior to chemotherapy should be <2 as described in Table 2.

Analysis of differential gene expression patterns using support vector machines and class prediction Support vector machines (SVMs) are well suited for two or more classes of pattern recognition (Vapnik, 1995 (76); Burges, 1998 ( 77).

を想定し、これは対応する標識
ｙ_ｉ∈｛＋１，−１｝（ｉ＝１、２、．．．、ｍ）
を有する。 For two classes of classification problems (eg tumor tissue vs. non-tumor tissue, or treatment responsive vs. non-responsive), a set of samples, ie a series of input vectors

, Which corresponds to the corresponding label y _i ε {+ 1, −1} (i = 1, 2,..., M)
Have

  Here, +1 and -1 indicate two classes. To classify gene expression patterns of marker genes from Table 1 or 2 to account for current tumor conditions or possible responses to therapeutic agents, the size of the input vector is different oligonucleotides present on the oligonucleotide array Each input vector unit represents the hybridization value of one specific oligonucleotide type.

  The objective is to build a two-component classification index or derive a decision function from available samples that have a slight probability of misclassifying future samples.

を高次元の特徴空間

へとマッピングして最適分離超平面（ＯＳＨ）を構築し、これは境界、すなわち空間Ｈ内の超平面と各クラスの最も近いデータ点との距離を最大にする。特徴空間中の陽性の例を陰性の例から分離することができるもののうちからＯＳＨを選択することによって、ＳＶＭは過剰適合を回避している。 The SVM implements the following ideas. This is the input vector

A high-dimensional feature space

To the optimal separation hyperplane (OSH), which maximizes the distance between the boundary, ie the hyperplane in space H, and the nearest data point of each class. By selecting OSH from among those that can separate positive examples in the feature space from negative examples, SVM avoids overfitting.

は、核関数

によって行い、これは、空間Ｈにおける内積を定義している。 Different SVVs are built with different mappings. mapping

Is the kernel function

Which defines the inner product in space H.

式中、係数α_ｉは、以下の凸２次計画（ＱＰ）問題を解くことによって得ることができる：

を最大化し、以下の式に供する：０≦α_ｉ≦Ｃ（式３）
および

The decision function performed by the SVM can be written as follows (Burges, 1998 (77):

Where the coefficient α _i can be obtained by solving the following convex quadratic programming (QP) problem:

And subject to the following formula: 0 ≦ α _i ≦ C (Formula 3)
and

は、対応するα_ｉ＞０の場合にのみサポートベクターと呼ばれる。 Regularity parameter C (Equation 3) controls the trade-off between boundaries and misclassification errors.

Is called a support vector only if the corresponding α _i > 0.

前者（式４）はｄ次の多項式核関数と呼ばれ、ｄ＝１の場合に最終的に直線関数に戻り、後者（式５）はラジアル基本関数（Ｒａｄｉａｌ Ｂａｓｉｓ Ｆｕｎｃｔｉｏｎ、ＲＢＦ）核関数と呼ばれる。 Two of the kernel functions used in this example are as follows:

The former (formula 4) is called a d-order polynomial kernel function, and finally returns to a linear function when d = 1, and the latter (formula 5) is called a radial basic function (RBF) kernel function. .

  For the treatment data set, only the kernel function and the regularity parameter C need be selected to identify one SVM. SVM has many attractive features. For example, the solution to the QP problem is totally optimized, but in neural networks, gradient-based training algorithms only guarantee finding local minima. In addition, SVM can handle large feature spaces, and can effectively avoid overfitting by controlling the boundaries (see above), and automatically small subsets of information points, ie support vectors, etc. Can be identified.

  Classification of biological samples based on gene expression data, and thus identification of neoplastic lesions, and the response of such lesions to therapeutic agents is a matter of multi-class classification. Class number k is equal to the number of tumor subclasses (eg histological features, TNM stage, grade, hormonal status) and predicts, ie subgroups of responses to specific therapeutic agents present in the training data set (eg disease Equivalent to complete remission, good remission, partial remission, or no remission, as well as progressive disease). Due to the limited number of different classes in this sample set, we decided to handle the multi-class classification by reducing the multi-class to a series of two-component classifications. For the k classifications, k SVMs were constructed. The i th SVM is trained with all samples in the i th class with a positive label and all other samples with a negative label. Finally, the unknown sample is classified into the class corresponding to the SVM with the highest output value. Using this method, a system for predicting / classifying gene expression patterns of differentially expressed marker genes as shown in Table 1 is constructed.

がユークリッド長１を有するように正規化したものと定義した：

Each data point generated by a microarray hybridization experiment or real-time RT-PCR (see Examples 2 and 3) corresponds to and is determined by the copy number of mRNA present in the analyzed sample, ie, poly Experiments using n oligonucleotide types on the nucleotide array yield a series of n expression level values. These n values are typically stored in a metric file that is the result of an analysis of a “cel file” by Affymetrix® Microarray Suite or the software described above. An expression matrix is constructed using data from a series of m metric files (representing m expression analyzes), where each of the m columns consists of an expression vector of n elements in a single experiment. . To normalize the expression values of m experiments, the x _ij, gene j (the mRNA provides a kind j 'hybridize to either or effective [Delta] [Delta] _T strength, the oligonucleotides present on the microarray) Expression level a _ij is the sum of the logarithms of the expression vector

Is defined as normalized to have Euclidean length 1:

  Initial analysis was performed 11 experiments using 60000 elemental expression vector sets (6 experiments in the training set and 5 tests in the test set) as described in Examples 2 and 3.

  Using the knowledge that eleven experiments represent three different response classes and two different tumor states, and information on tumor and non-tumor tissue, we can recognize these response classes and disease states. The above SVM was trained in a training group. Prediction accuracy was evaluated using a test set. Here, the present inventors performed cross-validation using the “one-out” method, and in a more rigorous test, 4-5 times validation (25% omitted) was repeated n times. (N> 10,000).

In such cross-validation and classification experiments, the neutrality of a subset of marker genes selected from the table was tested with the following affinity levels:

  The response indicated by X represents the five test set samples that were analyzed and predicted for the response, as indicated. One patient sample (after P39_) gave a new class PR (partial response) by prediction, but all other samples were correctly classified.

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Claims (12)

A method for testing a response to anticancer chemotherapy,
(I) at least 1, 5, 10, 20, 40 included in the group of genes encoded by SEQ ID NO: 1 to SEQ ID NO: 65 in biopsy samples taken from neoplastic lesions after the start of the chemotherapy schedule Measuring the expression level of 65 genes; and (ii) comparing said expression level with a reference expression level;
And thereby examining the response to anti-cancer chemotherapy. A method for examining responses to anti-cancer chemotherapy ex vivo,
(I) applying ex vivo chemotherapy to a biopsy sample taken from a neoplastic lesion prior to initiating a chemotherapy schedule;
(Ii) measuring the expression level of at least 1, 5, 10, 20, 40, 65 genes contained in the group of genes encoded by SEQ ID NO: 1 to SEQ ID NO: 65 in the biopsy sample; and (Iii) comparing the expression level with a reference expression level;
And thereby examining the response to anti-cancer chemotherapy ex vivo.   3. A method according to claim 1 or 2, wherein the examination of response is a prediction of the likelihood of successful chemotherapy.   4. The method of claim 3, wherein successful chemotherapy is understood as a reduction in tumor mass.   The method according to any one of claims 1 to 4, wherein the neoplastic lesion is breast cancer or ovarian cancer. The chemotherapy is
(I) affects cell proliferation and / or
(Ii) affects cell survival and / or
(Iii) acts on cell motility,
The method according to claim 1.   7. The method according to any of claims 1 to 6, wherein the chemotherapy is an anthracycline-based chemotherapy.   8. The method of claim 7, wherein the anthracycline-based chemotherapy is epirubicin or doxorubicin-based chemotherapy.   9. A method according to any of claims 1 to 8, wherein a prediction algorithm is used. The prediction algorithm is
The method of claim 9, wherein the method is (i) a support vector machine algorithm, or (ii) a k-nearest neighbor algorithm, or (iii) a partial least discriminant algorithm. The expression level is
(I) using hybridization-based methods, or (ii) using hybridization-based methods using arrayed probes, or (iii) hybridization-based methods using individually labeled probes. Or (iv) by real-time PCR, or (v) by assessing the expression of a polypeptide, protein, or derivative thereof, or (vi) by assessing the amount of a polypeptide, protein, or derivative thereof The method according to claim 1, which is determined by:   4. A method of selecting an individually optimized anti-cancer treatment for a patient, comprising the method of claim 1 or 2 or 3.