Gene expression markers for breast cancer prognosis

(Background of invention) (Field of invention) The present invention provides genes and gene sets, and expression of these genes and gene sets is important in the diagnosis and / or prognosis of breast cancer.

(Explanation of prior art) Oncologists have many treatment options available to oncologists, who claim various combinations with chemotherapeutic drugs characterized as "therapeutic criteria" and label claims for a particular cancer. There are many drugs that are not possessed but have evidence of efficacy in the cancer. To be most likely to have good treatment results, it is necessary for the patient to have the optimal available cancer treatment and that this treatment be done as soon as possible after diagnosis.

At present, the diagnostic test used in clinical diagnosis is a single analysis, so this diagnostic test does not acquire the potential value of knowing the relationships between dozens of different markers. Moreover, diagnostic tests are often not quantitative and rely on immunohistochemistry. This method often produces different results in different laboratories, partly because the reagents are not standardized, and partly because the interpretation is subjective and easily quantified. This is because it cannot be converted. RNA-based trials are rarely used due to the problem of RNA degradation over time and the fact that it is difficult to obtain fresh tissue samples from patients for analysis. Fixed paraffin-embedded tissue is more readily available and methods have been established for detecting RNA in fixed tissue. However, these methods typically do not allow the study of large numbers of genes (DNA or RNA) from small amounts of material. Therefore, traditionally, fixatives are rarely used except for immunohistochemical detection of proteins.

Recently, several groups have published studies on the classification of various cancer types by microarray gene expression analysis (eg, Golub et al., Science 286: 531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. .USA 98: 13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Appendix 1): S316-S322 (2001); Ramaswamy et al., Proc.Natl.Acad.Sci.USA 98:15149-15154 (2001) checking). Specific classifications of human breast cancer based on gene expression patterns have also been reported (Martin et al., Cancer Res. 60: 2232-2238 (2000), West et al., Proc. Natl. Acad. Sci. USA 98: 11462- 11467 (2001); Sorlie et al., Proc.Natl.Acad.Sci.USA 98: 10869-10874 (2001); Yan et al., Cancer Res. 61: 8375-8380 (2001)). However, these studies are mostly focused on improving and subdividing the already established classification of various types of cancer (including breast cancer), and are generally differentially expressed. It does not provide new insights into the relationships between genes that have been used and does not lead to insights into treatment strategies to improve the clinical outcome of cancer treatment.

Modern molecular biology and biochemistry include hundreds of genes whose activity affects the behavior of tumor cells, the state of tumor cell differentiation, and the susceptibility or resistance of tumor cells to specific therapeutic agents. Clearly, with a few exceptions, the status of these genes has not been utilized for the purpose of routine clinical decisions regarding drug treatment. One notable exception is the use of estrogen receptor (ER) protein expression in breast cancer, selecting patients for treatment with antiestrogens (eg, tamoxifen). Another exceptional example is the use of ErbB2 (Her2) protein expression in breast cancer, selecting patients with the Her2 antagonist Herceptin® (Genentech, Inc., South San Francisco, CA).

Despite recent advances, the challenge of cancer treatment is to target specific treatment regimens for tumor types of different pathogenicities, and ultimately to personalize tumor treatment to maximize results. Stay. Therefore, there is a need for trials that simultaneously provide predictive information about a patient's response to different treatment options. This is especially true for breast cancer, whose ecology is poorly understood. Several subgroups (eg ErbB2)⁺The classification of breast cancer into subgroups (Perou et al., Nature 406: 747-752 (2000)) characterized by low to absent gene expression of estrogen receptor (ER) and some additional transcription factors. It is clear that it does not reflect the cellular and molecular heterogeneity of breast cancer and does not allow the design of treatment strategies that maximize patient response.

<p> (Gist of the invention) The present invention provides a set of genes, the expression of which set of genes has prognostic value, especially with respect to disease-free survival.</p><p> The present invention provides the use of paraffin-embedded biopsy materials stored for the assay of all markers in the above set, and thus the present invention is compatible with the most widely available types of biopsy materials. The present invention also adapts to several different methods of tumor tissue collection, eg, via core biopsy or fine needle aspiration. In addition, for each member of the gene set, the invention identifies oligonucleotide sequences that can be used in the test.</p><p> In one aspect, the invention relates to a method of predicting the likelihood of long-term survival of a breast cancer patient without recurrence of breast breasts, which method is for prognostic diagnosis of one or more prognosis in breast tissue samples obtained from the above patients. Expression levels of RNA transcripts or their expression products, with respect to the expression levels of all RNA transcripts or their expression products in this breast cancer tissue sample, or of a reference set of RNA transcripts or their expression products. Including the step of determining the expression level, which is normalized to the expression level, where this prognostic RNA transcript is:</p><p><chemistry num="13"><img file="JP4723472B2_D0001.tif" /></chemistry>A transcript of one or more genes selected from the group consisting of here:</p><p><chemistry num="14"><img file="JP4723472B2_D0002.tif" /></chemistry>Expression of one or more of them indicates a reduced chance of long-term survival without recurrence of breast cancer, and:</p><p><chemistry num="15"><img file="JP4723472B2_D0003.tif" /></chemistry>Expression of one or more of them indicates an increased chance of long-term survival without recurrence of breast cancer.</p><p> In certain embodiments, the expression levels of at least 2, or at least 5, or at least 10, or at least 15 prognostic RNA transcripts or their expression products are determined. In another embodiment, the method comprises determining the expression level of all prognostic RNA transcripts or their expression products.</p><p> In another particular embodiment, the breast cancer is invasive breast cancer.</p><p> In a further embodiment, RNA is isolated from a patient's fixed wax-embedded breast cancer tissue specimen. Isolation can be performed, for example, from core biopsy tissue or needle aspirated cells by any technique known in the art.</p><p> In another aspect, the invention presents the following genes:</p><p><chemistry num="16"><img file="JP4723472B2_D0004.tif" /></chemistry>The present invention relates to an array containing a polynucleotide that hybridizes to two or more of the above.</p><p> In certain embodiments, the array hybridizes to at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or all of the genes listed above. Contains a polynucleotide to be used.</p><p> In another particular embodiment, this array is:</p><p><chemistry num="17"><img file="JP4723472B2_D0005.tif" /></chemistry>Contains a polynucleotide that hybridizes to the gene of.</p><p> The polynucleotide can be a cDNA, or an oligonucleotide, and the solid surface on which they are presented can be, for example, glass.</p><p> In another aspect, the invention relates to a method of predicting the likelihood of long-term survival without recurrence of breast cancer in a patient diagnosed with invasive breast cancer. (1) In the breast cancer tissue sample obtained from the above patients:</p><p><chemistry num="18-1"><img file="JP4723472B2_D0006.tif" /></chemistry></p><p><chemistry num="18-2"><img file="JP4723472B2_D0007.tif" /></chemistry></p><p><chemistry num="18-3"><img file="JP4723472B2_D0008.tif" /></chemistry>Expression levels of RNA transcripts or expression products of genes or gene sets selected from the group consisting of, for expression levels of all RNA transcripts or their expression products in the breast cancer tissue sample, or RNA transcription. The step of determining the expression level, which is normalized to the expression level of a reference set of substances or their expression products; (2) Step (1) The step of subjecting the data obtained in step (1) to statistical analysis; (3) The step of determining whether the possibility of long-term survival has increased or decreased, Including.</p><p> In a further aspect, the invention relates to a method of predicting the likelihood of long-term survival without recurrence of breast cancer in a patient diagnosed with estrogen receptor (ER) positive invasive breast cancer. (1) Below:</p><p><chemistry num="19-1"><img file="JP4723472B2_D0009.tif" /></chemistry>A step of determining the expression level of an RNA transcript or expression product of a gene in a gene set selected from the group consisting of the following genes in ER-positive cancer:</p><p><chemistry num="19-2"><img file="JP4723472B2_D0010.tif" /></chemistry>Expression shows a reduced chance of survival without recurrence of cancer after surgery, and the following genes:</p><p><chemistry num="20"><img file="JP4723472B2_D0011.tif" /></chemistry>Expression indicates a better prognostic diagnosis for survival without recurrence of cancer after surgery, step; (2) Step (1) The step of subjecting the data obtained in step (1) to statistical analysis; (3) The process of determining whether this long-term survival potential has increased or decreased, Including.</p><p> In yet another aspect, the present invention relates to a method of predicting the likelihood of long-term survival without recurrence of breast cancer in a patient diagnosed with estrogen receptor (ER) negative invasive breast cancer. Genes:</p><p><chemistry num="21"><img file="JP4723472B2_D0012.tif" /></chemistry>Including the step of determining the expression level of an RNA transcript or expression product of, where the following genes are:</p><p><chemistry num="22-1"><img file="JP4723472B2_D0013.tif" /></chemistry>Expression shows a reduced chance of survival without cancer recurrence, and the following genes:</p><p><chemistry num="22-2"><img file="JP4723472B2_D0014.tif" /></chemistry>Expression indicates a better prognosis for cancer recurrence-free survival.</p><p> In a different aspect, the present invention relates to a method of creating a personalized genomic profile for a patient, the method of which: (a) A step of subjecting RNA extracted from breast tissue obtained from the above patient to gene expression analysis; (b) A step of determining the expression level of one or more genes selected from the breast cancer gene set listed in any one of Tables 1 to 5, wherein the expression level is a regulatory gene. Normalized against and, if necessary, compared to the amount found in the breast cancer reference tissue set, steps; and (c) The process of preparing a report summarizing the data obtained by this gene expression analysis, Including.</p><p> This report may include, for example, predictions of a patient's long-term survival potential and / or recommendations for treatment modalities for this patient.</p><p> In a further aspect, the invention relates to a method for amplifying the genes listed in Tables 5A and 5B by the polymerase chain reaction (PCR), which method is an amplicon and table listed in Tables 5A and 5B. Includes the steps of performing this PCR by using the primer-probe sets listed in 6A-Table 6F.</p><p> In a further aspect, the invention relates to PCR amplicons listed in Tables 5A and 5B.</p><p> In yet another aspect, the present invention relates to a set of PCR primers-probes listed in Tables 6A-6F.</p><p> The present invention further relates to a prognostic method, which is described below: (a) Samples containing breast cancer cells obtained from patients:</p><p><chemistry num="23"><img file="JP4723472B2_D0015.tif" /></chemistry>A step for quantitative analysis of the expression level of RNA transcripts of at least one gene selected from the group consisting of, or their products, and (b) If the normalized expression levels of the genes or their products rise above the defined expression threshold, the patient is likely to have a reduced chance of long-term survival without recurrence of breast cancer. Identification process, Including.</p><p> In different aspects, the present invention further relates to a prognostic method, which is described below: (a) Samples containing breast cancer cells obtained from patients:</p><p><chemistry num="24"><img file="JP4723472B2_D0016.tif" /></chemistry>A step for quantitative analysis of the expression level of RNA transcripts of at least one gene selected from the group consisting of, and (b) Identify patients as likely to have an increased chance of long-term survival without recurrence of breast cancer if the normalized expression levels of the genes or their products rise above the defined expression threshold. Process, Including.</p><p> The present invention further relates to kits, which are suitable for performing any of the above methods: (1) extraction buffers / reagents and protocols; (2) reverse transfer buffers / reagents and protocols; (3) Provide one or more of qPCR buffers / reagents and protocols.</p><p> (A brief description of the drawing) Table 1 is a list of genes, the expression of these genes relating to breast cancer survival. Results from retrospective clinical trials. Binary statistical analysis.</p><p> Table 2 is a list of genes, the expression of these genes relating to breast cancer survival in estrogen receptor (ER) positive patients. Results from retrospective clinical trials. Dual statistical analysis.</p><p> Table 3 is a list of genes, the expression of these genes relating to breast cancer survival in estrogen receptor (ER) negative patients. Results from retrospective clinical trials. Dual statistical analysis.</p><p> Table 4 is a list of genes, the expression of these genes relating to breast cancer survival. Results from retrospective clinical trials. Cox proportional hazard statistical analysis.</p><p> Tables 5A and 5B list the genes and the expression of these genes relates to breast cancer survival. Results from retrospective clinical trials. This table contains the registration number for the gene and the amplicon sequence used for PCR amplification.</p><p> Tables 6A to 6B show forward and reverse primers (indicated by "f" and "r", respectively) and probes ("f" and "r", respectively) used for PCR amplification of the amplicon listed in Tables 5A to 5B. Contains an array for) (indicated by p ").</p><p> (Detailed description of preferred embodiments) (A. Definition) Unless otherwise specified, the terminology used herein has the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, J. Wiley &amp; Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Chemistries and Structure 4th Edition, John Wiley &amp; Sons (New York, NY, NY) 1992) provides those skilled in the art with general guidance on many of the terms used in this application.</p><p> One of ordinary skill in the art will recognize many methods and substances similar to or equivalent to those described herein, which may be used in the practice of the present invention. In fact, the invention is by no means limited to the methods and substances described. For the purposes of the present invention, the following terms are defined below.</p><p> The term "microarray" refers to an ordered array of array elements (preferably polynucleotide probes) that are hybridizable on a substrate.</p><p> The term "polynucleotide", when used in singles or plurals, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. .. Thus, for example, polynucleotides as defined herein are single-strand and double-strand DNA, DNA containing single-strand and double-strand regions, single-strand and double-strand RNA. , And RNA containing single-strand and double-strand regions, DNA and RNA that can be single-strand or, more typically, double-strand or contain single-strand and double-strand regions. Hybrid molecules including, but are not limited to these. In addition, the term "polynucleotide" as used herein refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The chains within such a region can be from the same molecule or different molecules. These regions may contain all one or more molecules, but more typically only a few regions. One of the molecules in the triple helix region is often an oligonucleotide. Specifically, the term "polynucleotide" includes cDNA. The term includes DNA (including cDNA) and RNA, which contain one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or other reasons is the "polynucleotide" as the term is intended herein. In addition, DNA or RNA containing anomalous bases (eg, inosine) or modified bases (eg, tritylated bases) is included within the term "polynucleotide" as defined herein. Is done. In general, the term "polynucleotide" is characteristic of all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides as well as viruses and cells (including single and complex cells). Includes chemical forms of DNA and RNA.</p><p> The term "oligonucleotide" refers to a relatively short polynucleotide, which includes single-stranded deoxyribonucleotides, single-stranded ribonucleotides or double-stranded ribonucleotides, RNA: DNA hybrids and double-stranded DNA. However, it is not limited to these. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized, for example, by chemical methods using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods (by in vitro recombinant DNA-mediated techniques and expression of DNA in cells and organisms).</p><p> The terms "differential gene expression", "differential gene expression" and their synonyms (these are used interchangeably) are subjects whose gene expression is normal or controlled. It contains genes that have been activated to higher or lower levels in a subject suffering from a disease, particularly cancer (eg, breast cancer) as compared to expression in the body. The term also refers to genes whose gene expression has been activated to higher or lower levels at different stages of the same disease. The differentially expressed gene may either be activated or inhibited at the nucleic acid or protein level, or may be selectively spliced to yield a variety of polypeptide products. Understood. Such differences can be demonstrated, for example, by changes in mRNA levels, surface expression of the polypeptide, secretion or other partitioning. As differential gene expression, comparison of expression between two or more genes or their gene products, comparison of expression ratio between two or more genes or their gene products, or further, comparison of expression between two or more genes or their gene products, or further, of the same gene A comparison of two differently processed products, which may differ between a normal subject and a subject suffering from a disease (particularly cancer), or between different stages of the same disease) can be mentioned. .. Differential expression includes, for example, temporal expression patterns or cellular expression in a gene or its expression product, between normal cells and pathogenic cells, or between cells that have undergone various disease events or stages. Both quantitative and qualitative differences in patterns can be mentioned. For the purposes of the present invention, at least about twice, preferably, between expression of a given gene, in normal and affected subjects, or at various stages of disease development in affected subjects, preferably. If there is a difference of at least about 4-fold, more preferably at least about 6-fold, and most preferably at least about 10-fold, then "differential gene expression" is considered to be present.</p><p> The phrase "gene amplification" refers to the process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. Overlapping regions (stretching of amplified DNA) are often referred to as "amplicon". Normally, the amount of messenger RNA (mRNA) produced (ie, the level of gene expression) also increases in proportion to the copy number made from a particular expressed gene.</p><p> The term "diagnosis" is used to refer to the identification of a molecule or pathological condition, pathological disease or condition (eg, identification of a molecular subtype of head and neck cancer, colon cancer, or other types of cancer). As used herein.</p><p> The term "prognosis" is used to refer to the prediction of the likelihood of cancer-induced death or progression of neoplastic diseases (eg, breast cancer), including recurrence, metastatic spread, and drug resistance. Used in the specification.</p><p> The term "predictive" refers to the likelihood that a patient will respond to a drug or a set of drugs either favorably or unfavorably and the extent of their response, or that the patient will undergo surgical resection or primary tumor and /. Alternatively, it is used herein to refer to the likelihood of survival for a period of time without recurrence of the cancer after chemotherapy. The predictive methods of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment mode for any particular patient. The predictive method of the invention is surgery (eg, surgical intervention, chemotherapy with a given drug or combination of drugs, and / or radiation therapy) if the patient tends to respond favorably to the treatment regimen. After the end of sugery) and / or chemotherapy or other modes of treatment, long-term survival of the patient is a valuable tool in predicting whether it is promising.</p><p> The term "long-term" survival is used herein to refer to survival for at least 3 years, more preferably at least 8 years, most preferably at least 10 years after surgery or other procedure.</p><p> As used herein, the term "tumor" means all growth and proliferation (whether malignant or benign), and all precancerous and cancerous. Refers to sex cells and tissues.</p><p> The terms "cancer" and "cancerous" typically refer to or describe physiological conditions in mammals characterized by disordered cell proliferation. Examples of cancers include breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, cancer tumor ( Cancer), melanoma, and brain cancer, but are not limited to these.</p><p> The "pathology" of cancer includes all phenomena that impair the well-being of a patient. The "pathology" of this cancer includes abnormal or uncontrolled cell proliferation, metastasis, interference with the normal functioning of adjacent cells, release of cytokines or other secretory products at abnormal levels, inflammatory reactions or immunology. Suppression or exacerbation of response, neoplasia, premalignant tumors, malignant tumors, invasion of surrounding or distant tissues or organs (eg, lymph nodes), but not limited to.</p><p> The "stringency" of a hybridization reaction can be readily determined by one of ordinary skill in the art and is generally an empirical computational prediction that depends on probe length, wash temperature, and salt concentration. In general, for proper annealing, longer probes require higher temperatures, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA to reanneal when the complementary strand is present in an environment below the melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures tend not to. For further details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).</p><p> "Stringent conditions" or "high stringent conditions", as defined herein, are typically: (1) low ion intensity and high temperature (eg, for cleaning). , Use 0.015 M Sodium Chloride / 0.0015 M Sodium Citrate / 0.1% Sodium Dodecyl Sulfate at 50 ° C; (2) During hybridization, a denaturant such as formamide (eg 0.1% at 42 ° C) Use bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50% (v / v) formamide containing 50 mM sodium phosphate buffer at pH 6.5 containing 750 mM sodium chloride, 75 mM sodium citrate; or ( 3) 50% formamide, 5 × SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhart solution, ultrasonically treated salmon at 42 ° C. Sperm DNA (50 μg / ml), 0.1% Using SDS, and 10% dextran sulfate, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 55 ° C in 50% formamide, then consisting of 0.1 x SSC containing EDTA at 55 ° C. Perform high stringency cleaning.</p><p> "Medium stringent conditions" can be the same as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and more than those described above. It may include the use of low stringent wash solutions and hybridization conditions (eg, temperature, ionic strength and SDS%). Examples of moderate stringent conditions are 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 mg. An overnight incubation at 37 ° C in a solution containing / ml denatured cleaved salmon sperm DNA followed by washing the filter in 1 × SSC at about 37-50 ° C. Those skilled in the art will recognize adjustment methods such as temperature, ionic strength, etc. when factors such as probe length need to be adapted.</p><p> In the context of the present invention, references to "at least one", "at least two", "at least five", etc. of genes listed in any particular set of genes are any reference to any of the listed genes. Means one or any and all combinations.</p><p> The terms "expression threshold" and "defined expression threshold" are used interchangeably, and the gene or gene product above serves as a predictive marker for patient survival without cancer recurrence. Say the level of. Thresholds are empirically defined from clinical studies (eg, those described in the examples below). This expression threshold can be selected for either maximum sensitivity, maximum selectivity, or minimum error. Determining the expression threshold in any situation is well within the knowledge of one of ordinary skill in the art.</p><p> (B. Detailed explanation) The practice of the present invention is the practice of molecular biology (including recombination techniques), microbiology, cell biology, and biochemistry, unless otherwise indicated (these are within the art of the art). Yes) is used. Such techniques are described in the literature (eg, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, (ed.), 1984); "Animal Cell Culture" (RI Freshney). , (Ed.), 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4th Edition (DM Weir and CC Blackwell, (ed.), Blackwell Science Inc., 1987); "Gene" Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, (ed.), 1987); Current Protocols in Molecular Biology (FM Ausubel et al., (ed.), 1987); and PCR: The Polymerase Chain Reaction , (Mullis et al., (ed.), It is fully explained in 1994) ").</p><p> (1. Gene expression profiling) In general, methods of gene expression profiling can be divided into two major groups: methods based on polynucleotide hybridization analysis and methods based on polynucleotide sequencing. The most commonly used methods known in the art for quantifying mRNA expression in samples are Northern blotting and in situ hybridization (Parker and Barnes, Methods in Molecular Biology 106: 247-283 (1999)). ); RNAse protection assay (Hod, Biotechniques 13: 852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics) 8: 263-264 (1992)). Alternatively, antibodies capable of specifically recognizing double strands, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. Typical methods for sequencing-based gene expression analysis are by Serial Analysis of Gene Expression (SAGE) and massively parallel signature sequencing (MPSS). Gene expression analysis can be mentioned.</p><p> (2. Reverse transcription PCR (RT-PCR)) Of the techniques listed above, the most sensitive and most adaptable quantitative method is RT-PCR, which varies in normal and tumor tissue with or without drug treatment. It can be used to compare mRNA levels in a sample population, to characterize patterns of gene expression, to distinguish closely related mRNAs, and to analyze RNA structure.</p><p> The first step is the isolation of mRNA from the target sample. Initiators are typically human tumors or human tumor cell lines, respectively, and the corresponding total RNA isolated from normal tissues or cell lines. Thus, RNA, along with pooled DNA from healthy donors, can be tumors of various primary tumors (breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc. Can be isolated from (including tumor cell lines). If the source of mRNA is a primary tumor, the mRNA can be extracted, for example, from a frozen or stored paraffin-embedded and fixed (eg, formalin-fixed) tissue sample.</p><p> Common methods of mRNA extraction are well known in the art and are disclosed in standard textbooks on molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley &amp; Sons (1997). Methods for extracting RNA from paraffin-embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56: A67 (1987), and De Andres et al., BioTechniques 18: 42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer set and protease from a commercial manufacturer (eg, Qiagen) and according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using the Qiagen RNeasy mini-column. Another commercially available RNA isolation kit is MasterPure.^TM Examples include the Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WI), and the Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumors can be isolated, for example, by cesium chloride density gradient centrifugation.</p><p> If RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA and its exponential amplification in subsequent PCR reactions. is there. The two most commonly used reverse transcriptases are the avian (avilo) myeloblastosis virus reverse transcriptase (AMV-RT) and the Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription process is typically prepared using specific primers, random hexamers, or oligo-dT primers, depending on the environment and expression profiling objectives. For example, the extracted RNA can be reverse transcribed using the GeneAmp RNA PCR kit (Perkin Elmer, CA, USA) according to the manufacturer's instructions. The cDNA produced can then be used as a template in subsequent PCR reactions.</p><p> The PCR step can use a variety of thermostable DNA-dependent DNA polymerases, which typically use Taq DNA polymerase, which has 5'-3'nuclease activity, but 3'. -5'Lack of proofreading endonuclease activity. Thus, TaqMan® PCR typically utilizes the 5'-nuclease activity of Taq polymerase or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but with an equivalent 5'. Any enzyme with nuclease activity can be used. Two oligonucleotide primers are used to produce amplicon typical of PCR reactions. The third oligonucleotide (ie, probe) is designed to detect the nucleotide sequence located between the two PCR primers. This probe is Taq It is non-extensible by DNA polymerase enzymes and is labeled with reporter and quencher fluorochromes. Any laser-guided luminescence from the reporter dye is quenched by the quenching dye if the two dyes are on the probe and the two dyes are located close together. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resulting probe fragment is dissociated into solution and the signal from the released reporter dye has no quenching effect on the second fluorophore. One molecule of the reporter dye is released for each of the new molecules synthesized, and detection of the unquenched reporter dye provides the basis for a quantitative interpretation of this data.</p><p> TaqMan® RT-PCR is a commercially available facility (eg, ABI PRISM 7700).^TM Sequence Detection System^TMIt can be done using (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany), etc.). In a preferred embodiment, the 5'nuclease procedure is a real-time quantitative PCR device (eg, ABI PRISM 7700).^TM Sequence Detection System^TM). The system consists of a thermocycler, a laser, a charge-coupled device (CCD), a camera and a computer. This system amplifies samples in 96-well format on a thermal cycler. During amplification, the laser-excited fluorescent signal is collected in real-time via fiber optic cable for all 96 wells and detected on a CCD. The system includes software for operating equipment and analyzing data.</p><p> The 5'-nuclease assay data is initially represented as Ct (ie, the threshold cycle). As discussed above, the fluorescence value represents the amount of product recorded during the entire cycle and amplified up to that point in the amplification reaction. The time when this fluorescent signal is first recorded as a statistically significant difference is the threshold cycle (Ct).</p><p> To minimize the error and effect of sample-to-sample variation, RT-PCR is usually performed using internal standards. The ideal internal standard is expressed at constant levels between different tissues and is unaffected by experimental treatment. The most frequently used RNAs for standardizing gene expression patterns are the mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.</p><p> A more recent variation of RT-PCR technology is real-time quantitative PCR, which measures the accumulation of PCR products via a double-labeled fluorescence-generating probe (ie, the TaqMan® probe). Real-time PCR includes quantitative competitive PCR (internal competitors for each target sequence are used for canonicalization) and standardized genes contained in the sample, or housekeeping genes for RT-PCR. Corresponds to both the quantitative comparative PCR used. For more details, see Held et al., Genome Research 6: 986-994 (1996).</p><p> Typical protocol steps for profiling gene expression using fixed, paraffin-embedded tissue as an RNA source include mRNA isolation, purification, primer extension and amplification, and various published journals. Shown in the paper {eg: TE Godfrey et al., J. Molec. Diagnostics 2: 84-91 [2000]; K. Specht et al., Am.J. Pathol. 158: 419-29 [2001]}. Briefly, the typical process begins with cutting an approximately 10 μm thick section of paraffin-embedded tumor tissue sample. RNA is then extracted and protein and DNA are removed. After analysis of RNA concentration, RNA repair and / or amplification steps are optionally included, and RNA is reverse transcribed using a gene-specific promoter, followed by RT-PCR.</p><p> According to one aspect of the invention, PCR primers and probes are designed based on the intron sequence present in the gene to be amplified. In this embodiment, the first step in primer / probe design is depiction of the intron sequence within the gene. This can be done by publicly available software (eg, DNA BLAT software developed by Kent, WJ, Genome Res. 12 (4): 656-64 (2002)), or BLAST software that includes variations thereof. The next step follows a well-established method of PCR primer and probe design.</p><p> To avoid non-specific signals, it is important to mask the repeating sequences in the intron when designing primers and probes. It uses the Repeat Masker program available online through the Baylor College of Medicine, which screens DNA sequences against a library of repeating elements and returns a query sequence in which repeating elements are masked. Can be easily achieved by. The masked intron sequence is then subjected to any commercially available or otherwise publicly available primer / probe design package (Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Krawetz). S, Misener S (ed.) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp It can be used to design primer and probe sequences using Steve Rozen and Helen J. Skaletsky (2000) Primer3) on the WWW for 365-386 general users and biologist programmers.</p><p> The most important factors to consider when designing PCR primers include primer length, melting temperature (Tm), and G / C content, specificity, complementary primer sequences, and 3'-terminal sequences. In general, optimal PCR primers are generally 17-30 bases long and contain about 20% -80% (eg, about 50% -60%) G + C bases. A Tm between 50 ° C and 80 ° C (eg, about 50 ° C to 70 ° C) is typically preferred.</p><p> For further guidelines for PCR primers and probes, see, for example, Dieffenbach, CW et al., PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp.133-155, General Concepts for PCR Primer Design. PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp.5-11 Innis and Gelfand, "Optimization of PCRs"; and Plasterer, TN Primer select: Primer and probe design.Methods Mol. Biol. See 70: 520-527 (1997), the entire disclosure of which is expressly incorporated herein by reference.</p><p> (3. Microarray) Differential gene expression can also be identified or confirmed using microarray technology. Therefore, the expression profile of breast cancer-related genes can be measured in either new tumor tissue or paraffin-embedded tumor tissue using microarray technology. In this method, the polynucleotide sequence of interest, including cDNA and oligonucleotides, is plated or arrayed on a microchip substrate. The arrayed sequence is then hybridized using a specific DNA probe from the cell or tissue of interest. Just like the RT-PCR method, the source of mRNA is typically a human tumor or human tumor cell line and total RNA isolated from the corresponding normal tissue or normal cell line. Therefore, RNA can be isolated from various primary tumors or tumor cell lines. When the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen tissue samples or stored paraffin-embedded and fixed (eg, formalin-fixed) tissue samples, which are usually clinical By convention, it is customarily prepared and preserved.</p><p> In certain embodiments of microarray technology, PCR-amplified inserts of cDNA clones are applied to the substrate of a dense array. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. This microarrayed gene is immobilized on a microchip with 10,000 elements each and is suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be produced by incorporating fluorescent nucleotides and reverse transcribing RNA extracted from the tissue of interest. The labeled cDNA probe applied on the chip hybridizes specifically to each DNA spot on the array. After stringent cleaning to remove non-specific binding probes, the chip is scanned by a confocal laser scanning microscope or another detection method (eg, a CCD camera). The quantification of hybridization of each arrayed element allows evaluation of the corresponding mRNA abundance. Using bicolor fluorescence, separately labeled cDNA probes produced from two sources of RNA are hybridized in pairs to the array. Therefore, the relative abundance of transcripts from the two sources corresponding to each particular gene is determined simultaneously. The miniaturization scale of hybridization provides a convenient and rapid evaluation of expression patterns for large numbers of genes. Such methods are needed to detect rare transcripts, which are expressed in small copies per cell, and to reproducibly detect differences in expression levels of at least about 2-fold. It has been shown to have the same sensitivity (Schena et al., Proc. Natl. Acad. Sci. USA 93 (2): 106-149 (1996)). Microarray analysis can be performed by commercially available equipment according to the manufacturer's protocol (eg, by using Affymetrix GenChip technology, or Incyte's microarray technology).</p><p> The development of microarray methods for large-scale analysis of gene expression makes it possible to systematically search for molecular markers for cancer classification and outcome predictions for various tumor types.</p><p> (4. Continuous analysis of gene expression (SAGE)) Continuous analysis of gene expression (SAGE) is a method that allows simultaneous quantitative analysis of multiple gene transcripts without the need to provide individual hybridization probes for each transcript. First, a short sequence tag (approximately 10-14 bp) is made to contain sufficient information to uniquely identify the transcript, provided that it is obtained from a unique location within each transcript. Many transcripts then form long, contiguous molecules that can be combined and sequenced together, simultaneously revealing the identity of multiple tags. The expression pattern of any set of transcripts can be evaluated quantitatively by determining the abundance of individual tags and identifying the gene corresponding to each tag. For more details, see, for example, Velculescu et al., Science 270: 484-487 (1995); and Velculescu et al., Cell 88: 243-51 (1997).</p><p> (5. Mass ARRAY technology) MassARRAY (Sequenom, San Diego, California) technology is a method of automated, highly processed gene expression analysis using mass spectrometry (MS) for detection. Following this method, the cDNA is subjected to primer extension following RNA isolation, reverse transcription and PCR amplification. This cDNA-derived primer extension product is purified and dispensed onto a chip array preloaded with the components required for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantified by analyzing the peak regions obtained during mass spectrometry.</p><p> (6. Gene expression analysis by massively parallel gene bead clone analysis method (MPSS)) This method, described by Nature Biotechnology 18: 630-634 (2000), combines non-gel-based signature sequencing with in vitro cloning of millions of templates on isolated 5 μm diameter microbeads. It is an approach of sequencing. First, a microbead library of DNA templates is constructed by in vitro cloning. After this, high density (typically 3x10)⁶Microbeads / cm²Followed by a set of planar arrays of template-containing microbeads in the flow cell. The free ends of the cloned template on each microbead are simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragmentation. This method has been shown to provide hundreds of thousands of gene signature sequences from yeast cDNA libraries simultaneously and accurately in a single operation.</p><p> (7. Immunohistochemistry) Immunohistochemical methods are also suitable for detecting the expression levels of prognostic markers of the invention. Therefore, specific antibodies or antisera (preferably polyclonal antisera, and most preferably monoclonal antibodies) for each marker are used to detect expression. These antibodies can be detected by directly labeling the antibodies themselves. Labeling may be, for example, a radioactive label, a fluorescent label, a hapten label (eg, biotin or an enzyme (eg, horseradish peroxidase or alkaline phosphatase), or an unlabeled primary antibody is an anti-primary antibody specific. Used in combination with labeled secondary antibodies, including serum, polyclonal anti-serum or monoclonal antibodies. Immunohistochemical protocols and kits are well known and commercially available in the art.</p><p> (8. Proteomics) The term "proteome" is defined as the whole protein present in a sample (eg, tissue, organ or cell culture) at a particular point in time. Proteomics includes, among other things, the study of overall changes in protein expression in a sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps: (1) Separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) Individuals recovered from the gel. The steps of identifying proteins (eg, by mass spectrometry or N-terminal sequencing) and (3) analyzing the data using bioinformatics. Proteomics methods are useful supplements to other methods of gene expression profiling and can be used alone or in combination with other methods to detect prognostic marker products of the invention.</p><p> (9. Overview of mRNA isolation, purification and amplification) Typical protocol steps for profiling gene expression include mRNA isolation, purification, primer extension and amplification using fixed paraffin-embedded tissue as the RNA source. These steps are provided in the articles of various published journals {eg: TE Godfrey et al. J. Molec. Diagnostics 2: 84-91 [2000]; K.specht et al., Am.J.Pathol.158: 419-29 [2001]}. Briefly, the typical process begins with cutting a section of paraffin-embedded tumor tissue sample about 10 μm thick. RNA is then extracted and protein and DNA are removed. After analysis of RNA enrichment, RNA repair and / or amplification steps can be included, and if necessary, RNA is reverse transcribed using a gene-specific promoter, followed by RT-PCR. Finally, this data is analyzed to identify the best treatment options available for the patient based on the characteristic gene expression patterns identified in the tumor sample being tested.</p><p> (10. Clinical application of breast cancer gene sets, assayed gene subsequences, and gene expression data) An important aspect of the invention is the use of measured expression of a particular gene to provide prognostic information by breast cancer tissue. For this purpose, it is necessary to correct (standardize) both differences in the amount of RNA assayed and in the quality of RNA used. Therefore, this assay typically measures and incorporates specific standardized gene expression, including well-known housekeeping genes (eg, GAPDH and Cyp1). Alternatively, standardization can be based on the mean or median signal (Ct) (overall standardization approach) of all assayed genes or their large subunits. For each gene, the standardized amount of patient tumor mRNA measured is compared to the amount found in the criteria set for breast cancer tissue. The number of breast cancer tissues (N) in this criteria set must be large enough to ensure that different criteria sets behave in essentially the same manner (as a whole). If this condition is met, the identification of individual breast cancer tissues present in a particular set has no significant effect on the relative amount of genes being assayed. Typically, a reference set of breast cancer tissue consists of at least about 30, preferably at least about 40 different FPE breast cancer tissue specimens. Unless otherwise stated, the standardized expression level for each mRNA / tumor / patient tested is expressed as a percentage of the expression level measured in the reference set. More specifically, a sufficiently large number (eg, 40) of tumor reference sets gives a standardized level distribution of each mRNA species. The levels measured in the particular tumor sample being analyzed fall within the range of a few percent points, and this level can be determined by methods well known in the art. Unless otherwise stated, this is not always explicitly stated below, but references to gene expression levels presuppose standardized expression compared to a reference set.</p><p> Further details of the present invention will be described in the following non-limiting examples.</p>

(Phase II study of gene expression in 79 malignant breast cancers) With the primary purpose of molecularly characterizing gene expression in tissue samples of paraffin-embedded and fixed invasive ductal carcinoma in situ, and investigating the correlation between such molecular profiles and disease-free survival. A gene expression study was planned and conducted.

(Research design) Molecular assays were performed on paraffin-embedded, formalin-fixed major breast cancer tissue from 79 individual patients diagnosed with invasive breast cancer. All patients in this study had 10 or more positive nodules. The mean age was 57 years and the mean clinical tumor size was 4.4 cm. Patients were included in this study only if histopathological assessments performed as described in the Materials and Methods section showed an appropriate amount of tumor tissue and homogeneous pathology.

(Materials and methods) Each representative tumor block was characterized by standard histopathology for diagnosis, semi-quantitative tumor volume assessment, and tumor stage. A total of 6 sections (each 10 microns thick) were prepared and placed in 2 Costar Brand Microcentrifuge Tubes (polypropylene, 1.7 mL tubes, clear; 3 sections in each tube). If the tumor constitutes less than 30% of the total specimen area, the entire microdissection can be used by a pathologist to roughly disassemble the sample and place the tumor tissue directly in the Costa tube.

If more than one tumor block was obtained as part of the surgical procedure, the more representative pathological block was used for analysis.

(Gene expression analysis) MRNA was extracted from fixed paraffin-embedded tissue samples, purified, and prepared for gene expression analysis as described in Section 9 above.

ABI PRISM 7900^TM Sequence Detection System^TM(Perkin-Elmer-Applied Biosystems, Foster City, CA, USA) was used to perform a molecular assay of quantitative gene expression by RT-PCR. ABI PRISM 7900^TMConsists of a thermal cycler, a laser, a charge-coupled device (CCD), a camera and a computer. This system amplifies a sample in a 384-well format on a thermal cycler. During amplification, laser-guided fluorescence signals were collected in real-time via fiber optic cables for all 384 wells and detected on a CCD. The system includes software for operating equipment and analyzing data.

(Analysis and results) Tumor tissues were analyzed for 185 cancer-related genes and 7 reference genes. Threshold cycle (CT) values for each patient were standardized based on the median of 7 reference genes for a particular patient. Data on clinical outcomes were available for all patients from a review of the described data and the medical history of the selected patients.

The results were categorized as follows: 0 Death due to breast cancer or unknown cause, or survive with breast cancer recurrence; 1 Survive without breast cancer recurrence or die from causes other than breast cancer.

The analysis was performed as follows: 1. Analysis of the relationship between standardized gene expression and 0 or 1 binary results.

2. Analysis of the relationship between standardized gene expression and time to outcome (0 or 1 as defined above) in patients who survived without breast cancer recurrence or who died from causes other than breast cancer Was done. This approach was also used to assess the prognostic effects of individual genes and the prognostic effects of multiple gene sets.

(Analysis of patients with invasive breast cancer by a dual approach) In the first (dual) approach, analysis was performed on all 79 patients with invasive breast cancer. A t-test was performed on a group of patients who were classified as having no recurrence and no breast cancer-related deaths at 3 years, and a group of patients who were classified as recurrences, or breast cancer-related deaths at 3 years. Then, the p-value was calculated for the difference between the groups for each gene.

Table 1 lists 47 genes with a p-value of &lt;0.10 for differences between groups. The first column of mean expression relates to patients who did not have metastatic recurrence and did not die of breast cancer. The second column of mean expression relates to patients who either had metastatic recurrence or died of breast cancer.

<tables num="1-1"><img file="JP4723472B2_D0017.tif" /></tables>

<tables num="1-2"><img file="JP4723472B2_D0018.tif" /></tables> In Table 1 above, a negative t-value indicates higher expression associated with worse results, and conversely, a higher (positive) t-value indicates higher expression associated with better results. Thus, for example, elevated expression of the CD68 gene (t-value = -3.41, CT mean survival &lt;CT mean death) indicates a possible reduced disease-free survival. Similarly, elevated expression of the BCl2 gene (t-value = 4.00; CT mean survival&gt; CT mean death) indicates an increased likelihood of disease-free survival.

Based on the data shown in Table 1, expression of any of the following genes in breast cancer above the defined expression threshold indicates reduced survival without cancer recurrence after surgery: Grb7, CD68 , CTSL, Chkl, Her2, STK15, AIB1, SURV, EGFR, MYBL2, HIFlα.

Based on the data shown in Table 1, expression of any of the following genes in breast cancer above the defined expression threshold indicates a better prognosis for survival without cancer recurrence after surgery: TP53BP2, PR , Bcl2, KRT14, EstRl, IGFBP2, BAG1, CEGP1, KLK10, β-catenin, GSTM1, FHIT, Rizl, IGF1, BBC3, IGF1, TBP, p27, IRS1, IGF1R, GATA3, CEGP1, ZNF217, CD9, pS2, Erb TOP2B, MDM2, RAD51, and KRT19.

(Analysis of ER-positive patients by a dual approach) Fifty-seven patients with standardized CT&gt; 0 for estrogen receptor (ER) (ie, ER-positive patients) were subjected to separate analyzes. Student's t-test was performed on two groups, one group of patients with no recurrence and no breast cancer-related death at 3 years, and the other with recurrence or breast cancer-related death at 3 years. did. The p-value was then calculated for the differences between the groups for each gene. Table 2 below lists the genes whose p-values for differences between groups were &lt;0.105. The first column of mean expression relates to patients who did not have metastatic recurrence and did not die of breast cancer. The second column of mean expression relates to patients who either had metastatic recurrence or died of breast cancer.

<tables num="2-1"><img file="JP4723472B2_D0019.tif" /></tables>

<tables num="2-2"><img file="JP4723472B2_D0020.tif" /></tables> For each gene, a classification algorithm was used to identify the best threshold (CT) for using each gene alone in predicting clinical outcomes.

Based on the data listed in Table 2, expression of the following genes in ER-positive cancers above defined expression levels indicates a reduced chance of survival without cancer recurrence after surgery: CD68; CTSL FBXO5; SURV; CCNB1; MCM2; Chkl; MYBL2; HIF1A; cMET; EGFR; TS; STK15. Many of these genes (CD68, CTSL, SURV, CCNB1, MCM2, Chkl, MYBL2, EGFR, and STK15) have also been identified as indicators of poor prognosis in previous analyzes. These are not limited to ER-positive breast cancer. Based on the data presented in Table 2, expression of the following genes in ER-positive cancers above defined expression levels indicates a better prognosis for survival without cancer recurrence after surgery: IGFR1; BCl2; HNF3A; TP53BP2; GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; RB1; EPHX1; CEGP1; TRAIL; DR5; p27; p53; MTA; RIZ1; ErbB3; TOP2B; EIF4E. Of the latter genes, IGFR1; BCl2; TP53BP2; GATA3; BBC3; RAD51C; BAG1; IGFBP2; PR; CD9; CEGP1; DR5; p27; RIZ1; ErbB3; TOP2B; EIF4E also have a good prognosis in previous analyzes. Identified as an index. These are not limited to ER-positive breast cancer.

(Analysis of ER-negative patients by a dual approach) Twenty patients with normalized CT &lt;1.6 for estrogen receptor (ER) (ie, ER-negative patients) were subjected to isolation analysis. A t-test was performed on two groups of patients who had no recurrence and were classified as either without breast cancer-related deaths at 3 years or with recurrence or breast cancer-related deaths at 3 years. The p-value was then calculated for the differences between the groups for each gene. Table 3 lists genes with a p-value of &lt;0.118 for differences between these groups. The first column of mean expression values relates to patients who have not had metastatic recurrence and have not died of breast cancer. The second column of mean expression values relates to patients who either had metastatic recurrence or died of breast cancer.

<tables num="3"><img file="JP4723472B2_D0021.tif" /></tables> Based on the data presented in Table 3, expression of the following genes in ER-negative cancers above defined expression levels indicates a reduced chance of survival without cancer recurrence (p &lt;0.05): CCND1; UPA; HNF3A; CDH1; Her2; GRB7; AKT1; STMY3; α-catenin; VDR; GRO1. Only two of these genes (Her2 and Grb7) were also identified as indicators of poor prognosis in previous analyzes. These are not limited to ER-negative breast cancer. Based on the data listed in Table 3, expression of the following genes in ER-negative cancers above defined expression levels indicates a better prognosis for survival without cancer recurrence: KT14; KLK10; Maspin, TGFα , And FRP1. Of the latter genes, only KLK10 was also identified as a good prognostic indicator in previous analyses. This is not limited to ER-negative breast cancer.

(Analysis of multiple genes and indicators of results) Two approaches were taken to determine whether the use of multiple genes provided better discrimination between the results.

First, a discriminant analysis was performed using a forward stepwise approach. A model was created to classify results that have discriminative potential beyond that obtained with any one gene alone.

Following a second approach (time-to-event approach), a Cox proportional hazards model for each gene (eg, Cox, DR, and Oakes, D. (1984), Analysis of Survival Data, Chapman and Hall, London, New York) was defined with the time to recurrence or death as the dependent variable and the gene expression level as the independent variable. In the Cox model, genes with a p-value &lt;0.10 were identified. For each gene, this cox model is a unit of gene expression. For change), provide the relative risk of recurrence or death (RR). Patients may be selected to subgroup at any threshold of measured expression (on CT scale). All patients with above-threshold expression levels are at higher risk, depending on whether the gene has a poor prognosis (RR&gt; 1.01) or an indicator of a good prognosis (RR &lt;1.01). All patients with expression levels below the threshold have a lower risk. The reverse is also true. Thus, any threshold defines a subgroup of patients at increased or decreased risk, respectively. The results are summarized in Table 4. Title: The third column of exp (coefficient) shows the RR value.

<tables num="4"><img file="JP4723472B2_D0022.tif" /></tables> Dual analysis and temporal event analysis identified the same gene as a prognostic marker with few exceptions. For example, a comparison between Table 1 and Table 4 shows that 10 genes represented the top 15 genes in both lists. Furthermore, if both analyzes identified the same gene at [p &lt;0.10] (this happened for 21 genes), they were always consistent with respect to the direction of survival / recurrence correlation (positive or negative sign). Overall, these results reinforce the conclusion that the identified markers have significant prognostic values.

For Cox models (multivariable models) containing 3 or more genes, step-by-step entry into the model of each individual gene was performed. The first gene entered is preselected from genes with a significant univariate p-value. The gene selected for entry into the model in each subsequent step is the gene that best improves the fit of the model to the data. This analysis can be performed on any total number of genes. In the analysis of the results shown below, stepwise entry was performed for up to 10 genes.

Multivariate analysis was performed using the following equation: RR = exp [coef (gene A) x Ct (gene A) + coef (gene B) x Ct (gene B) + coef (gene C) x Ct (gene C) + ...] In this equation, the coefficients for genes that are forecast values for beneficial results are positive numbers, and the coefficients for genes that are forecast values for unfavorable results are negative numbers. The "Ct" value in this equation is ΔCt, that is, it reflects the difference between the average normalized Ct value of the population and the normalized Ct measured in the patient in question. The convention used in this analysis is that ΔCt below and above the population mean have positive and negative signs, respectively (reflecting higher or lower mRNA abundance). The relative risk (RR) calculated by solving this equation indicates whether the patient has an increased or decreased chance of long-term survival without cancer recurrence.

(Multivariate gene analysis of 79 patients with invasive breast cancer) Gene expression data from all 79 patients with invasive breast cancer were subjected to a multivariate stepwise analysis using the Cox proportional hazards model. The following set of 10 genes was identified by this analysis to have particularly strong predictions of patient survival:

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<chemistry num="25-2"><img file="JP4723472B2_D0024.tif" /></chemistry> Although the present invention has been described with reference to what is considered to be a particular embodiment, it is understood that the present invention is not limited to such embodiments. On the contrary, the present invention is intended to cover various modifications and equivalents contained within the spirit and scope of the appended claims. For example, the disclosure focuses on the identification of various breast cancer-related genes and gene sets, as well as the individualized prognosis of breast cancer, but similar genes, gene sets and methods associated with other types of cancer are clear. Is within the scope of the present invention.

All references cited throughout this disclosure are hereby incorporated by reference.

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