Patent Publication Number: US-2005142573-A1

Title: Gene segregation and biological sample classification methods

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
      This application is a continuation of co-pending U.S. application Ser. No. 10/660,434, filed Sep. 10, 2003, which claims the benefit of U.S. Provisional Application 60/410,018 filed Sep. 10, 2002; U.S. Provisional Application 60/411,155, filed Sep. 16, 2002; U.S. Provisional Application 60/429,168, filed Nov. 25, 2002; U.S. Provisional Application 60/444,348, filed Jan. 31, 2003; and U.S. Provisional Application 60/460,826, filed Apr. 3, 2003, each of which is incorporated by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
      This invention was made using federal funds awarded by the National Institutes of Health, National Cancer Institute under contract number 1RO1CA89827-01. The government has certain rights to this invention. 
    
    
     FIELD OF THE INVENTION  
      The present invention relates to methods for gene segregation to identify clusters of genes associated with biological sample phenotypes and for classifying biological samples on the basis of gene expression patterns derived from those samples.  
     BACKGROUND OF THE INVENTION  
      For many years established human cancer cell lines have been used as models to study human cancers because, to a large degree, they faithfully recapitulate many biological features of human tumors. Established human cancer cell lines maintained in vitro are not expected to fully recapitulate the gene expression patterns of human clinical cancers. This essentially precludes their use as model systems for global gene expression analysis of human tumors. It is likely that the longer that cancer cell lines are maintained in vitro, the more they degrade as models for transcription changes in human clinical cancers.  
      Recent experiments using established human prostate and breast cancer cell line models indicate that this degradation may be at least partly reversed by using established cancer cell lines to generate experimental tumors in mice and to develop xenograft-derived cell lines from these experimental tumors (Glinsky, G. V., Glinskii, A. B., McClelland, M., Krones-Herzig, A., Mercola, D., Welsh, J. 2002. Microarray gene expression analysis of tumor progression in the nude mouse model of human prostate cancer. In Proceedings of the 93 rd  Annual Meeting of the American Association for Cancer Research, April 6-10, San Francisco, Calif., 43: 462 (Abstract#4480), incorporated herein by reference). Furthermore, the study of differential gene expression observed using cell lines maintained in vitro and in cell line-induced experimental tumors in mice avoids many of the problems associated with cellular heterogeneity and experimental manipulation of clinical samples. It appears that the in vitro and in vivo human prostate cancer progression models partially recapitulate gene expression behavior of clinical prostate tumor samples, at least with respect to the consensus differentially regulated gene class that has been recently defined for multiple xenograft-derived human prostate cancer cell lines (Glinsky, G. V., Glinskii, A. B., McClelland, M., Krones-Herzig, A., Mercola, D., Welsh, J. 2002. Microarray gene expression analysis of tumor progression in the nude mouse model of human prostate cancer. In Proceedings of the 93rd Annual Meeting of the American Association for Cancer Research, April 6-10, San Francisco, Calif., 43: 462 (Abstract#4480), incorporated herein by reference).  
      While several useful methods of classification of human and other tumors are known, these methods tend to be a highly subjective in nature and at best semi-quantitative. Recent advances in global gene expression analysis of human tumors using cDNA or oligonucleotide microarray technologies set the stage for the development of improved quantitative methods for human tumor classification (see, e.g., Magee, J. A., Araki, T., Patil, S., Ehrig, T., True, L., Humphrey, P. A., Catalona, W. J., Watson, M. A., Milbrandt, J. Expression profiling reveals hepsin overexpression in prostate cancer. Cancer Res., 61: 5692-5696, 2001; Dhanasekaran, S. M., Barrette, T. R., Ghosh, D., Shah, R., Varambally, S., Kurachi, K., Pienta, K. J., Rubin, M. A., Chinnalyan, A. M. Delineation of prognostic biomarkers in prostate cancer. Nature, 412:822-826, 2001; Welsh, J. B., Sapinoso, L. M., Su, A. I., Kern, S. G., Wang-Rodriguez, J., Moskaluk, C. A., Frierson, H. F., Jr., Hampton, G. M. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res., 61: 5974-5978, 2001; Luo, J., Duggan, D. J., Chen, Y., Sauvageot, J., Ewing, C. M., Bittner, M. L., Trent, J. M., Isaacs, W. B. Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling. Cancer Res., 61: 4683-4688, 2001; Stamey, T A, Warrington, J A, Caldwell, M C, Chen, Z, Fan, Z, Mahadevappa, M, McNeal, J E, Nolley, R, Zhang, Z. Molecular genetic profiling of Gleason grade 4/5 prostate cancers compared to benign prostatic hyperplasia. J. Urol., 166: 2171-2177, 2001; Luo, J., Dunn, T, Ewing, C, Sauvageot, J., Chen, Y, Trent, J, Isaacs, W. Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis. Prostate, 51: 189-200, 2002; Singh, D., Febbo, P. G., Ross, K., Jackson, D. G., Manola, C. L., Tamayo, P., Renshaw, A. A., D&#39;Amico, A. V., Richie, J. P., Lander, E. S., Loda, M., Kantoff, P. W., Golub, T. R., Sellers, W. R. Gene expression correlates of clinical prostate cancer behavior. Cancer Cell, 1: 203-209, 2002; Rhodes, D. R., Barrette, T. R., Rubin, M. A., Ghosh, D., Chinnaiyan, A. M. Meta-analysis of microarrays: interstudy validation of gene expression profiles reveals pathways dysregulation in prostate cancer. Cancer Res., 62: 4427-4433, 2002; Pollack, J. R., Perou, C. M., Alizadeh, A. A., Eisen, M. B., Pergamenschikov, A., Williams, C. F., Jeffrey, S. S., Botstein, D., Brown, P. O. Genome-wide analysis of DNA-copy number changes using cDNA microarrays. Nature Genetics. 1999. 23: 41-46; Forozan, F., Mahlamaki, E. H., Monni, O., Chen, Y., Veldman, R., Jiang, Y., Gooden, G. C., Ethier, S. P., Kallioniemi, A., Kallioniemi, O-P. Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data. Cancer Res. 2000. 60: 4519-4525; Perou C M, Jeffrey S S, van de Rijn M, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci USA. 1999. 96:9212-9217; Perou C M, Sorlie T, Eisen M B, et al. Molecular portrait of human breast tumors. Nature. 2000. 406:747-752; Clark, EA, Golub T R, Lander E S, Hynes R O. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000. 406:532-535; Welsh, J. B., Zarrinkar, P. P., Sapinoso, L. M., Kern, S. G., Behling, C. A., Monk, B. J., Lockhart, D. J., Burger, R. A., Hampton, G. M. Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc Natl Acad Sci USA. 2001. 98:1176-1181, incorporated herein by reference). However, direct attempts to identify genes differentially regulated in tumors that are useful for tumor classification, clinical management and prognosis have produced limited success, in part, because of intrinsic cellular heterogeneity and variability in cellular composition of clinical samples, the statistically underdetermined nature of the problem in which the number of variables (e.g., expression data points) exceeds the number of observations (i.e., independent samples from which the data are gathered), and the absence of a uniform, readily accessible and reproducible reference standard against which differential expression can be evaluated.  
      In the context of clinical tumor samples, an acceptable reference standard against which differential gene expression can be evaluated should meet the following requirements: 
          Individual clinical tumors should display different degrees of resemblance between their gene expression patterns as compared to the gene expression pattern exhibited by the reference standard samples;     The degree of resemblance between the gene expression patterns in individual clinical samples and that of the reference standard samples should be susceptible to quantitative measurement; and     Quantitative measurements of the degree of resemblance between clinical samples and the reference standard samples should correlate with biological, clinical, and pathohistological features of individual human tumors enabling their use as a basis for classification of clinical tumor samples.        

      In a more general sense, gene expression drives the acquisition of cellular phenotypes during differentiation of precursor or stem cells. Identification of genes that are differentially expressed between precursor cells and differentiated cells, or between different types of differentiated cells is an important step for understanding the molecular processes underlying differentiation. The ability to control differentiation of precursor or stem cells so as to direct the cells down a desired differentiation pathway is an important goal, as it represents a tissue engineering solution to the problem of alleviating the shortage of tissue and organs useful for grafting and transplantation. Furthermore, normal and transformed cell-type specific markers, useful for, e.g., molecular-recognition-based targeting of therapeutics such as e.g., rituximab and other recognition based therapeutics, can be identified from sets of genes concordantly regulated in particular normal and transformed cell types.  
      Attempts to identify directly genes that are differentially regulated in various cell lines suffer from some of the same difficulties referenced above for tumor samples. One of the most common problems for the array-based study is that they usually generate vast data sets. For example, gene expression analysis of a single tumor cell line and a single normal epithelial counterpart typically identifies many thousands of transcripts as differentially expressed at a statistically significant level. Up to 40-50% of the surveyed genes will be identified as differentially expressed when one compares gene expression profiles of normal epithelial and stromal cells. Obviously, any meaningful design of follow-up clinical and/or experimental validation experiments would require an application of further data reduction steps. Our work makes contribution to the solution of this problem by providing a convenient and simple data reduction technique. Two possible approaches seem to be appropriate: one can narrow a set of candidate genes identified in cell lines to those that maintain similar transcript abundance (or other type of gene expression) behavior in a relevant set of clinical tumor samples and design a hypothesis-driven study aimed at identifying potential biologically important genes and/or pathways using cell lines as a model system. Alternatively, one can identify or design cell lines that recapitulate gene expression behavior identified in clinical samples and again use the model system for the assessment of the biological relevance of the gene expression changes. During the last two years or so a third approach is rapidly emerging. It is based on simultaneous analysis of gene expression and DNA copy number changes with an aim to identify the genes that acquired mRNA abundance changes due to the amplification or deletion of the corresponding genes. The cancer cell lines are certainly attractive model systems to undertake such validation study. Suitable reference standards also are needed against which gene expression patterns can be evaluated in normal (i.e., not tumor) cells and/or tissues. Here again, acceptable reference standards would be expected to have the following properties:  
      Different types of normal cells and/or tissues should display different degrees of resemblance between their gene expression patterns as compared to the gene expression pattern exhibited by the reference standard samples; 
          The degree of resemblance between the gene expression patterns in individual normal cells and that of the reference standard samples should be susceptible to quantitative measurement; and     Quantitative measurements of the degree of resemblance between normal cells and the reference standard samples should correlate with biological features of different normal cell types so as to provide a basis for the classification of differentiation state and cell type.        

      There thus exist in the art a need for improved methods of biological sample classification, for improved methods of identifying genes that are differentially expressed or regulated in biological samples such as tumors and normal cells, for reference standards that can be used in accordance with these methods, and for identified sets of coordinately regulated genes, the expression patterns of which can be used for classifying samples and for developing cell- or tissue-specific markers. The present invention addresses these and other shortcomings of the art.  
     BRIEF SUMMARY OF THE INVENTION  
      Broadly, it is an object of the invention to provide improved quantitative methods for classifying tumor and normal samples.  
      It is a further object of the invention to provide useful reference standards for classifying tumors and normal samples.  
      It is a still further object of the invention to provide methods for classifying tumor and normal samples on the basis of gene expression data.  
      Thus, in one aspect, the invention provides a method for classifying a sample in which a first reference set of expressed genes is identified, the first reference set consisting of genes that are differentially expressed between a first set of tumor cell lines and a set of control cell lines, a second reference set of expressed genes is identified, the second reference set consisting of genes that are differentially expressed between a first set of samples and a second set of samples, wherein the first and second samples differ with respect to a sample classification, a concordance set of expressed genes is identified, the concordance set consisting of genes that are common to the first and second reference sets and wherein, preferably, the direction of the differential expression is the same in the first and second reference sets, identifying a minimum segregation set of expressed genes within the concordance set, the minimum segregation set consisting of a subset of expressed genes within the concordance set selected so that a first correlation coefficient between an average fold-change or difference of the gene expression data from the lines and an average fold-change or difference of the gene expression data from the samples exceeds a pre-determined value, calculating for the expressed genes within the minimum segregation set a second correlation coefficient between the average fold-change or difference of the gene, expression data from the cell lines and a fold-change or difference of the gene expression data from an unclassified sample, and classifying the unclassified sample as belonging to the first set of samples or the second set of samples according to the sign of the second correlation coefficient.  
      In a preferred embodiment, the first set of samples and the second set of samples comprise tumor cells and/or tissues containing tumor cells, that differ with respect to a tumor classification such as, e.g., benign versus malignant growth, local and/or systemic recurrence, invasiveness, metastatic propensity, metastatic tumors versus localized primary tumors, degree of dedifferentiation (poor, moderate, or well differentiated tumors), tumor grade, Gleason score, survival prognosis, disease free survival, lymph node status, patient age, hormone receptor status, PSA level, and histologic type.  
      In another embodiment, reference sets are obtained without the use of cell lines, but instead rely solely on the use of clinical samples. In this embodiment, a first reference set is obtained by looking at differential expression among two or more sets of clinical samples, preferably using average expression values, wherein the two or more sets differ with respect to a known phenotype. A concordance set is then obtained by determining concordance between the differentially expressed genes established using the two or more clinical sample groups and one or more individual samples within the group that demonstrate the best fit (highest correlation coefficient) between the individual sample(s) and the average group measurements.  
      In other preferred embodiments, the gene expression data is selected from the group consisting of mRNA quantification data, cDNA quantification data, cRNA quantification data, and protein quantification data.  
      In another aspect, the invention provides for a method for identifying a set of genes in which a first reference set of expressed genes is identified, the first reference set consisting of genes that are differentially expressed between a first set of tumor cell lines and a set of control cell lines, a second reference set of expressed genes is identified, the second reference set consisting of genes that are differentially expressed between a first set of samples and a second set of samples, wherein the first and second samples differ with respect to a sample classification, a concordance set of expressed genes is identified, the concordance set consisting of genes that are common to the first and second reference sets and wherein, preferably, the direction of the differential expression is the same in the first and second reference sets, and identifying a minimum segregation set of expressed genes within the concordance set, the minimum segregation set consisting of a subset of expressed genes within the concordance set selected so that a first correlation coefficient between an average fold-change or difference of the gene expression data from the lines and an average fold-change or difference of the gene expression data from the samples exceeds a pre-determined value.  
      In another embodiment, the minimum segregation set is determined without use of cell line data. This embodiment is preferred when no appropriate cell lines are available. In this embodiment, two or more groups of clinical samples, differing with respect to a known phenotype are used to generate a first reference set. Preferably, this is accomplished by determining average fold expression changes (optionally log transformed), and identifying a set of differentially expressed genes that are consistently (i.e., up- or down-regulated) in one group as compared to another group. The second reference set is obtained by determining for individual sample(s) within a group, fold-expression changes for genes within the first reference set, and finding those genes concordantly over- or under-expressed, in the individual sample(s) cf. the first reference set, and identifying those individual samples for which the individual gene expression values are most highly correlated with the expression of the genes in the first reference set. This essentially consists of calculating phenotype association indices for the individual gene expression measurements within the sample, and selecting as the second reference those genes identified as being concordantly expressed in the most highly correlated individual sample(s).  
      In yet another preferred embodiment, the invention provides minimum segregation sets of expressed genes. Such sets have utility as tools for, e.g., sample classification or prognostication, and as sources of cell- or tissue-specific markers. The markers can be used as, e.g., targets for delivery of cell- or tissue-specific reagents or drugs, or to monitor drug effects on a molecular scale.  
      In yet another preferred embodiment, the invention provides a kit comprising a set of reagents useful for determining the expression of a subset of genes identified using the methods of the invention, along with instructions for their use. The reagents can be affixed to a solid support and used in a hybridization reaction, or alternatively can be primers for use in nucleic acid amplification reactions.  
      Additional advantages and aspects of the present invention are now described with reference to the detailed description and drawings, below. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 8 recurrent versus 13 non-recurrent human prostate tumors for 19 genes of the concordance set.  
       FIG. 2  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 8 recurrent versus 13 non-recurrent human prostate tumors for 9 genes of the PC3/LNCap recurrence minimum segregation set (recurrence cluster).  
       FIG. 3  is a graph showing phenotype association indices for 9 genes of the recurrence cluster in individual human prostate tumors exhibiting recurrent (samples 1-8) or non-recurrent (samples 12-24) clinical behavior.  
       FIG. 4  is a graph showing phenotype association indices for 54 genes of the prostate cancer/normal tissue discrimination minimum segregation set (i.e., cluster) in 24 individual prostate tumors (samples 1-25 [one tumor sample run in duplicate]), 2 normal prostate stroma (NPS) samples (samples 28 and 29), and 9 adjacent normal tissue samples (samples 32-40).  
       FIG. 5  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 24 prostate cancer tissue samples versus 9 adjacent normal prostate samples for 54 genes of the concordance set.  
       FIG. 6  is a graph showing phenotype association indices for 10 genes of the prostate cancer/normal tissue minimum segregation set (i.e. cluster) in 24 prostate tumors (samples 1-25 [one tumor sample run in duplicate]), and 9 adjacent normal tissue samples (samples 29-37).  
       FIG. 7  is a graph showing phenotype association indices for 5 genes of the prostate cancer/normal tissue minimum segregation set (i.e., cluster) in 24 prostate tumors (samples 1-25 [one tumor sample run in duplicate]), and 9 adjacent normal tissue samples (samples 29-37).  
       FIG. 8  is a graph showing phenotype association indices for 10 genes of the prostate cancer/normal tissue minimum segregation set (i.e., cluster) in 47 prostate tumors (samples 1-47), and 47 adjacent normal tissue samples (samples 51-97).  
       FIG. 9  is a graph showing phenotype association indices for 5 genes of the prostate cancer/normal tissue minimum segregation set (i.e., cluster) in 47 prostate tumors (samples 1-47), and 47 adjacent normal tissue samples (samples 51-97).  
       FIG. 10  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 14 invasive versus 38 non-invasive human prostate cancer tissue samples for 104 genes of the concordance set.  
       FIG. 11  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 14 invasive versus 38 non-invasive human prostate cancer tissue samples for 20 genes of the invasion minimum segregation set 1 (i.e., invasion cluster 1).  
       FIG. 12  is a graph showing phenotype association indices for 20 genes of invasion cluster 1 in 14 invasive (samples 1-14) and 38 non-invasive (samples 20-57) human prostate tumor samples.  
       FIG. 13  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 12 invasive versus 17 non-invasive (surgical margins 1+) human prostate cancer tissue samples for 12 genes of the invasion minimum segregation set 2 (i.e., invasion cluster 2).  
       FIG. 14  is a graph showing phenotype association indices for 12 genes of invasion cluster 2 in 12 invasive (samples 1-12) and 17 non-invasive (samples 17-33) human prostate tumor samples.  
       FIG. 15  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 11 invasive versus 7 non-invasive (invasion clusters 1&amp;2+) human prostate cancer tissue samples for 10 genes of the invasion minimum segregation class 3 (i.e., invasion cluster 3).  
       FIG. 16  is a graph showing phenotype association indices for 10 genes of invasion cluster 3 in 11 invasive (samples 1-11) and 7 non-invasive (samples 16-22) human prostate tumor samples.  
       FIG. 17  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 3 invasive versus 21 non-invasive human prostate cancer tissue samples for 13 genes of the invasion minimum segregation class 4 (i.e., invasion cluster 4).  
       FIG. 18  is a graph showing phenotype association indices for 13 genes of invasion cluster 4 in 3 invasive (samples 1-3) and 21 non-invasive (samples 8-28) human prostate tumor samples.  
       FIG. 19  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 46 low Gleason grade human prostate cancer tissue samples for 58 genes of the concordance set.  
       FIG. 20  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 46 low Gleason grade human prostate cancer tissue samples for 17 genes of the high grade minimum segregation set 1 (high grade cluster 1).  
       FIG. 21  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 20 low Gleason grade human prostate cancer tissue samples for 12 genes of the high grade minimum segregation set 2 (high grade cluster 2).  
       FIG. 22  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 16 low Gleason grade human prostate cancer tissue samples for 7 genes of the high grade minimum segregation set 3 (high grade cluster 3).  
       FIG. 23  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 46 low Gleason grade human prostate cancer tissue samples for 38 genes of the ALT high grade minimum segregation set (ALT high grade cluster).  
       FIG. 24  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 17 low Gleason grade human prostate cancer tissue samples for 5 genes of the high grade minimum segregation set 4 (high grade cluster 4).  
       FIG. 25  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 17 low Gleason grade human prostate cancer tissue samples for 4 genes of the high grade minimum segregation set 5 (high grade cluster 5).  
       FIG. 26  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 17 low Gleason grade human prostate cancer tissue samples for 7 genes of the high grade minimum segregation set 6 (high grade cluster 6).  
       FIG. 27  is a scatter plot showing correlation of the expression profiles in 5 xenograft-derived human prostate carcinoma cell lines and 6 high Gleason grade versus 17 low Gleason grade human prostate cancer tissue samples for 13 genes of the high grade minimum segregation set 7 (high grade cluster 7).  
       FIG. 28  is a graph showing phenotype association indices for 54 genes of the BPH minimum segregation class (i.e. cluster) in 8 patients with benign prostatic hypertrophy (BPH) (samples 1-8) and 9 patients with prostate cancer (samples 13-21).  
       FIG. 29  is a graph showing phenotype association indices for 14 genes of the BPH minimum segregation class (i.e. cluster) MAGEA1 in 8 patients with benign prostatic hypertrophy (BPH) (samples 1-8) and 9 patients with prostate cancer (samples 12-20).  
       FIG. 30  is a graph showing phenotype association indices for 17 genes of the metastasis minimum segregation class 1 (i.e. metastasis cluster 1) in 5 patients with benign prostatic hypertrophy (BPH) (samples 7-11), 3 adjacent normal prostate (ANP) samples (samples 1-3), 1 patient with prostatitis (sample 5), 10 patients with localized prostate cancer (samples 13-22), and 7 patients with metastatic prostate cancer (MPC)(samples 24-30).  
       FIG. 31  is a graph showing phenotype association indices for 19 genes of the metastasis minimum segregation class 2 (i.e. metastasis cluster 2) in 5 patients with benign prostatic hypertrophy (BPH) (samples 7-11), 3 adjacent normal prostate (ANP) samples (samples 1-3), 1 patient with prostatitis (sample 5), 10 patients with localized prostate cancer (samples 13-22), and 7 patients with metastatic prostate cancer (MPC)(samples 24-30).  
       FIG. 32  is a graph showing phenotype association indices for 17 genes of the metastasis minimum segregation class 1 (i.e. metastasis cluster 1) in 14 patients with benign prostatic hypertrophy (BPH) (samples 1-14), 4 adjacent normal prostate (ANP) samples (samples 17-20), 1 patient with prostatitis (sample 23), 10 patients with localized prostate cancer (LPC) (samples 26-39), and 20 patients with metastatic prostate cancer (MPC)(samples 42-61).  
       FIG. 33  is a graph showing phenotype association indices for 19 genes of the metastasis minimum segregation class 2 (i.e. metastasis cluster 2) in 14 patients with benign prostatic hypertrophy (BPH) (samples 1-14), 4 adjacent normal prostate (ANP) samples (samples 17-20), 1 patient with prostatitis (sample 23), 14 patients with localized prostate cancer (LPC) (samples 26-39), and 20 patients with metastatic prostate cancer (MPC)(samples 42-61).  
       FIG. 34  is a graph showing phenotype association indices for 6 genes of the Q-PCR-based poor prognosis predictor minimum segregation set (i.e. cluster) in 34 patients with breast cancer who developed distant metastases within 5 years of diagnosis (samples 1-34) and in 44 patients who continued to be disease-free for at least five years (samples 37-80).  
       FIG. 35  is a graph showing phenotype association indices for 14 genes of the Q-PCR-based good prognosis predictor minimum segregation set (i.e. cluster) in 34 patients with breast cancer who developed distant metastases within 5 years of diagnosis (samples 1-34) and in 44 patients who continued to be disease-free for at least five years (samples 37-80).  
       FIG. 36  is a graph showing phenotype association indices for 13 genes of the Q-PCR-based good prognosis predictor minimum segregation set (i.e. cluster) in 34 patients with breast cancer who developed distant metastases within 5 years of diagnosis (samples 1-34) and in 44 patients who continued to be disease-free for at least five years (samples 37-80).  
       FIG. 37  is a graph showing phenotype association indices for 13 genes of the Q-PCR-based good prognosis predictor minimum segregation set (i.e. cluster) in 11 patients with breast cancer who developed distant metastases within 5 years of diagnosis (samples 1-11) and in 8 patients who continued to be disease-free for at least five years (samples 14-21).  
       FIG. 38  is a graph showing phenotype association indices for 11 genes of the ovarian cancer poor prognosis predictor minimum segregation set (i.e. cluster) in 3 poorly differentiated tumors (samples 1-3) and in 11 tumors of well and moderate differentiation (samples 6-16).  
       FIG. 39  is a graph showing phenotype association indices for 10 genes of the ovarian cancer good prognosis predictor minimum segregation set (i.e. cluster) in 3 poorly differentiated tumors (samples 1-3) and in 11 tumors of well and moderate differentiation (samples 6-16).  
       FIG. 40  is a scatter plot showing correlation of the expression profiles in non small cell lung carcinoma (“NSCLC”) cell lines and normal bronchial epithelial cells versus 139 human adenocarcinoma tissue samples versus 17 normal human lung samples for 13 genes of the human lung adenocarcinoma minimum segregation set 1 (lung adenocarcinoma cluster 1).  
       FIG. 41  is a scatter plot showing correlation of the expression profiles in non small cell lung carcinoma (“NSCLC”) cell lines and normal bronchial epithelial cells and 139 human adenocarcinoma tissue samples versus 17 normal human lung samples for 26 genes of the human lung adenocarcinoma minimum segregation set 2 (lung adenocarcinoma cluster 2).  
       FIG. 42  is a graph showing phenotype association indices for 13 genes of the lung adenocarcinoma minimum segregation set 1 (lung adenocarcinoma cluster 1) in 17 normal lung specimens (samples 1-17) and 139 patients with lung adenocarcinoma (samples 20-158).  
       FIG. 43  is a graph showing phenotype association indices for 26 genes of the lung adenocarcinoma minimum segregation set 2 (lung adenocarcinoma cluster 2) in 17 normal lung specimens (samples 1-17) and 139 patients with lung adenocarcinoma (samples 20-158).  
       FIG. 44  is a scatter plot showing correlation of the expression profiles in non small cell lung carcinoma (“NSCLC”) cell lines and normal bronchial epithelial cells and 34 human NSCLC patients with poor prognosis tissue samples versus 16 human NSCLC patients with good prognosis tissue samples for 38 genes of the lung adenocarcinoma poor prognosis minimum segregation set 1 (poor prognosis cluster 1).  
       FIG. 45  is a graph showing phenotype association indices for 38 genes of the lung adenocarcinoma poor prognosis minimum segregation set 1 (poor prognosis cluster 1) in 34 human NSCLC patients with poor prognosis (samples 1-34) 16 human NSCLC patients with good prognosis (samples 37-52).  
       FIG. 46 . Xenografts of human prostate cancer derived from the PC-3M-LN4 highly metastatic cell variant and growing in a metastasis promoting orthotopic setting exhibit pro-invasive and pro-angiogenic gene expression profiles. Expression profiling of the 12,625 transcripts in the orthotopic (“OR”) and subcutaneous (“s.c.” or “SC”) xenografts derived from the cell variants of the PC-3 lineage was carried out. (A1-A4) Expression pattern of the matrix metalloproteinases (MMPs). (B1-B4) Expression pattern of the components of plasminogen/plasminogen activator system. (C1-C4) Pro-angiogenic switch in PC-3M-LN4 orthotopic xenografts: increased levels of expression of interleukin 8, angiopoietin-2, and osteopontin and decreased level of expression of a protease and angiogenesis inhibitor maspin. (D1-D4) Cadherin switch in PC-3M-LN4 orthotopic xenografts: increased level of expression of non-epithelial cadherins (OB-cadherin-2 and VE-cadherin) and decreased level of expression of epithelial E-cadherin.  
       FIG. 47 . Correlation of gene expression profiles 8-gene prostate cancer recurrence signature cluster (A) in highly metastatic orthotopic xenografts and the recurrent versus non-recurrent prostate tumors or 5-gene prostate cancer invasion signature in invasive versus non-invasive human prostate tumors (B).  
       FIG. 48 . Correlation of expression profiles in orthotopic xenografts and clinical samples for 131-gene prostate cancer metastasis signature cluster (A), 37-gene prostate cancer metastasis signature (B), 12-gene prostate cancer metastasis signature (C), 9-gene prostate cancer metastasis signature (D).  
       FIG. 49 . Gene expression patterns of selected gene clusters in highly metastatic orthotopic xenografts are discriminators of the metastatic and primary human prostate carcinomas. The classification accuracy of the clinical samples is shown for clusters of 131 genes (A), 37 genes (B), 9 genes (C), and a family of 6 metastasis segregation clusters (D).  
       FIG. 50  Gene expression patterns of the selected gene clusters in highly metastatic orthotopic xenografts are discriminators of invasive ( FIG. 50A ) and recurrent ( FIG. 50B ) phenotypes of human prostate tumors.  FIG. 50A , phenotype association indices for 5 gene prostate cancer invasion predictor. Bars 1-8 tumors with positive surgical margins and prostate capsule penetration (“PSM &amp; PCP”); bars 11-16 tumors with positive surgical margins (“PSM”); bars 19-30 tumors with prostate capsule penetration (“PCP”); bars 33-58 non-invasive tumors.  FIG. 50B , phenotype association indices for 8 gene prostate cancer recurrence predictor. Bars 1-8 recurrent tumors; bars 11-23 non-recurrent tumors.  
       FIG. 51 . Gene expression profiles of selected gene clusters in highly metastatic PC3MLN4 orthotopic xenografts are concordant with the expression patterns of these genes in the recurrent (A), invasive (B), and metastatic (C) human prostate tumors. For each figure, bars show average fold change in gene expression compared to respective control for individual genes within clusters.  
       FIG. 52 . Gene expression profiles of the 25-gene recurrence predictor signature in highly metastatic PC3MLN4 orthotopic xenografts are concordant with the expression patterns of these genes in the recurrent human prostate tumors.  FIG. 52A -correlation of expression profiles in orthotopic xenografts and clinical samples for 25-gene prostate cancer recurrence predictor cluster.  FIG. 52B -Change in expression for each transcript are plotted as Log10Fold Change Average expression level in PC-3MLN40R versus Average expression level in PC-3MLN4SC and Log10Fold Change Average expression level in recurrent prostate tumors versus Average expression level in non-recurrent prostate tumors.  
       FIG. 53  is a bar graph illustrating phenotypic association indices for transcripts of the 25 genes prostate cancer recurrence predictor cluster in 8 recurrent and 13 non-recurrent human prostate tumors.  
       FIG. 54  is a bar graph illustrating expression profile of the 12 gene recurrence predictor signature in PC-3MLN4 orthotopic xenografts and recurrent human prostate tumors.  
       FIG. 55  is a scatter plot illustrating correlation of the expression profiles of the 12 genes recurrence predictor cluster in PC-3MLN4 orthotopic xenografts and recurrent human prostate tumors.  
       FIG. 56  is a bar graph illustrating phenotypic association indices for transcripts of the 12 genes prostate cancer recurrence predictor cluster in 8 recurrent and 13 non-recurrent human prostate tumors.  
       FIG. 57 . Phenotype association indices (PAIs) defined by the expression profile of the prostate cancer recurrence predictor signature 1 for 21 prostate carcinoma samples comprising a signature discovery (training) data set.  
       FIG. 58 . Kaplan-Meier analysis of the probability that patients would remain disease-free among 21 prostate cancer patients comprising a signature discovery group according to whether they had a good-prognosis or poor-prognosis signatures defined by the recurrence predictor signature 1 ( FIG. 58A ), recurrence predictor signature 2 ( FIG. 58B ), recurrence predictor signature 3 ( FIG. 58C ), and the recurrence predictor algorithm that takes into account calls from all three signatures ( FIG. 58D ).  
       FIG. 59 . Kaplan-Meier analysis of the probability that patients would remain disease-free among 79 prostate cancer patients comprising a signature validation group for all patients ( FIG. 59A ), patients with high ( FIG. 59B ) or low ( FIG. 59C ) preoperative PSA level in blood according to whether they had a good-prognosis or poor-prognosis signatures defined by the recurrence predictor algorithm or whether they had high or low preoperative PSA level in the blood ( FIG. 59D ).  
       FIG. 60 . Kaplan-Meier analysis of the probability that patients would remain disease-free among prostate cancer patients with Gleason sum 6 &amp; 7 tumors ( FIG. 60A ) and patients with Gleason sum 8 &amp; 9 tumors ( FIG. 60B ) according to whether they had a good-prognosis or poor-prognosis signatures defined by the recurrence predictor algorithm or whether they had Gleason sum 8 &amp; 9 or Gleason sum 6 &amp; 7 prostate tumors ( FIG. 60C ).  
       FIG. 61 . Kaplan-Meier analysis of the probability that patients would remain disease-free among 79 prostate cancer patients comprising a signature validation group for all patients ( FIG. 61A ), patients with poor prognosis ( FIG. 61B ) or good prognosis ( FIG. 60C ) defined by the Kattan nomogram according to whether they had a good-prognosis or poor-prognosis signatures defined by the recurrence predictor algorithm ( FIGS. 61B and 61C ) or whether they had poor or good prognosis defined by the Kattan nomogram ( FIG. 61A ).  
       FIG. 62 . Kaplan-Meier analysis of the probability that patients would remain disease-free among prostate cancer patients with stage 1C tumors ( FIG. 62A ) and patients with stage 2A tumors ( FIG. 62B ) according to whether they had a good-prognosis or poor-prognosis signatures defined by the recurrence predictor algorithm.  
       FIG. 63 . Kaplan Meier survival curves.  FIG. 63A  Survival of 151 breast cancer patients with lymph node negative disease (stratified by 14 gene signature).  FIG. 63B  Survival of 109 breast cancer patients with estrogen receptor positive tumors and lymph node negative disease (stratified by 14 gene signature);  FIG. 63C  Survival of 42 breast cancer patients with estrogen receptor negative tumors and lymph node negative disease (stratified by 4 and/or 3 gene signatures).  
       FIG. 64 . Kaplan Meier survival curves.  FIG. 64A  Survival of breast cancer patients with estrogen receptor positive and estrogen receptor negative tumors;  FIG. 64B  Survival or 69 breast cancer patients with estrogen receptor negative tumors (stratified by 5 and/or three gene signatures).  
       FIG. 65 . Metastasis-free survival of 78 breast cancer patients.  FIG. 65A  survival stratified by 4 gene signature;  FIG. 65B  survival stratified by 6 gene signature;  FIG. 65C , survival stratified by 13 gene signature;  FIG. 65D  survival stratified by 14 gene signature.  
       FIG. 66 . Survival of breast cancer patients classified into subgroups using gene signatures.  FIG. 66A  Survival of 144 breast cancer patients with lymph node positive disease stratified according to 14 gene survival predictor cluster;  FIG. 66B  Survival of 117 breast cancer patients with estrogen receptor positive tumors and lymph node positive disease stratified according to 14 gene survival predictor cluster;  FIG. 66C  Survival of 27 breast cancer patients with estrogen receptor negative tumors and lymph node positive disease stratified according to 4 and 3 gene signatures.  
       FIG. 67 . Survival of estrogen receptor positive breast cancer patients.  FIG. 67A  stratified according to positive and negative 14 gene signature;  FIG. 67B  stratified according to relative values of 14 gene signature.  
       FIG. 68 . Survival of breast cancer patients.  FIG. 68A  Survival of 295 breast cancer patients with positive and negative 14 gene signature (0.00 cut off);  FIG. 68B  Survival of 295 breast cancer patients with positive and negative 14 gene signature (−0.55 cut off);  FIG. 68C  Survival of breast cancer patients with positive and negative 14-gene signature;  FIG. 68D  Survival of breast cancer patients with positive and negative 14 gene signature;  FIG. 68E  Survival of breast cancer patients classified based on relative values of the 14 gene signature.  
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
      Definitions  
      All terms, unless specifically defined below, are intended to have their ordinary meanings as understood by those of skill in the art. Claimed masses and volumes are intended to encompass variations in the sated quantities compatible with the practice of the invention. Such variations are contemplated to be within, e.g. about +10-20 percent of the stated quantities. In case of conflict between the specific definitions contained in this section and the ordinary meanings as understood by those of skill in the art, the definitions supplied below are to control.  
      “Identifying a set of expressed genes” refers to any method now known or later developed to assess gene expression, including but not limited to measurements relating to the biological processes of nucleic acid amplification, transcription, RNA splicing, and translation. Thus, direct and indirect measures of gene copy number (e.g., as by fluorescence in situ hybridization or other type of quantitative hybridization measurement, or by quantitative PCR), transcript concentration (e.g., as by Northern blotting, expression array measurements or quantitative RT-PCR), and protein concentration (e.g., by quantitative 2-D gel electrophoresis, mass spectrometry, Western blotting, ELISA, or other method for determining protein concentration) are intended to be encompassed within the scope of the definition.  
      “Differentially expressed” refers to the existence of a difference in the expression level of a gene as compared between two sample classes. Differences in the expression levels of “differentially expressed” genes preferably are statistically significant.  
      “Tumor” is to be construed broadly to refer to any and all types of solid and diffuse malignant neoplasias including but not limited to sarcomas, carcinomas, leukaemias, lymphomas, etc., and includes by way of example, but not limitation, tumors found within prostate, breast, colon, lung, and ovarian tissues.  
      A “tumor cell line” refers to a transformed cell line derived from a tumor sample. Usually, a “tumor cell line” is capable of generating a tumor upon explant into an appropriate host. A “tumor cell line” line usually retains, in vitro, properties in common with the tumor from which it is derived, including, e.g., loss of differentiation, loss of contact inhibition, and will undergo essentially unlimited cell divisions in vitro.  
      A “control cell line” refers to a non-transformed, usually primary culture of a normally differentiated cell type. In the practice of the invention, it is preferable to use a “control cell line” and a “tumor cell line” that are related with respect to the tissue of origin, to improve the likelihood that observed gene expression differences are related to gene expression changes underlying the transformation from control cell to tumor.  
      An “unclassified sample” refers to a sample for which classification is obtained by applying the methods of the present invention. An “unclassified sample” may be one that has been classified previously using the methods of the present invention, or through the use of other molecular biological or pathohistological analyses. Alternatively, an “unclassified sample” may be one on which no classification has been carried out prior to the use of the sample for classification by the methods of the present invention.  
      “According to the sign of” a correlation coefficient refers to a determination based on the sign, i.e., positive or negative, of the referenced correlation coefficient. For example, a sample may be classified as belonging to a first set of samples if the sign of the correlation coefficient is positive, or as belonging to a second set of samples if the correlation coefficient is negative.  
      “Orthotopic” refers to the placement of cells in an organ or tissue of origin, and is intended to encompass placement within the same species or in a different species from which the cells are originally derived.  
      “Ectopic” refers to the placement of cells in an organ or tissue other than the organ or tissue of origin, and is intended to encompass placement within the same species or in a different species from which the cells are originally derived.  
      Introduction  
      Completion of the draft sequence of the human genome offers an unprecedented opportunity to study the genetic basis of human cancer progression. During malignant progression, genomic instability leads to continuously emerging phenotypic diversity, clonal evolution, and clonal selection resulting in the remarkable cellular heterogeneity of tumors. The phenotypic diversity of cancer cells is associated with significant mutation-driven changes in gene expression, although not all mutations and differences in gene expression are crucial or even relevant to the malignant phenotype. It therefore is important to identify expression changes that are highly relevant and characteristic of malignant phenotypes and progression pathways, more than one of which may exist (Hanahan, D., Weinberg, R. A. The hallmarks of cancer. Cell. 2000. 100: 57-70, incorporated herein by reference.). The methods of the present invention address this goal by providing analytical techniques to identify those expression changes highly correlated with and indeed predictive of certain clinically relevant features of malignant phenotypes and progression pathways.  
      In a broad and general sense, as applied to the analysis of tumor samples, the methods of the invention use gene expression data from a set of tumor cell lines and compare those data with gene expression data from a set of control cell lines to identify those genes that are differentially expressed in the tumor cell lines as compared to the control cell lines. In preferred embodiments, each of these sets includes more than a single member, although it is contemplated to be within the scope of the present invention to practice embodiments in which either or both of the set of tumor cell lines and the set of control cell lines includes only one member. The identified genes are referred to as a first reference set of expressed genes. Preferably, the control cell line and the tumor cell lines are related insofar as the control cell lines represent physiologically normal cells from the tissue or organ from which the tumor represented by the tumor cell lines arose. For example, if the tumor cell lines are derived from a prostate tumor, the control cell lines preferably are primary cultures of normal prostate epithelial cells. In the preferred embodiments, more than one tumor cell line and more than one control cell line is used to generate the reference set so as to reduce the number of genes in the first reference set by eliminating those genes that are not consistently differentially expressed between the tumor and control cell lines.  
      In other embodiments, the method may be practiced using only one tumor cell line and one control cell line, and identifying the set of genes differentially expressed between the tumor cell line and the control cell line. However, by carrying out a series of comparison between multiple control cell lines and multiple tumor cell lines the first reference set is more likely to contain only those genes that are consistently differentially expressed between the normal and tumor classes of cell lines (i.e., a gene is included within the first reference set if its expression level is always higher in each of the tumor cell lines examined as compared to each of the control cell lines examined, or if its expression level is always lower in each of the tumor cell lines examined as compared to each of the control cell lines examined).  
      In yet another embodiment, exemplified below as Example 6, the methods of the invention may be practiced without the use of cell lines, using instead data derived only from clinical samples. In a similar manner, the methods of the invention may be practiced using only data derived from cell lines.  
      For example, consider an embodiment in which the first reference set is derived using data obtained from three separate control cell lines and six separate tumor cell lines. For each gene considered for inclusion within the first reference set, pairwise comparisons are carried out for each of the 3×6 or 18 pairwise combinations between control cell lines and tumor cell lines. A candidate gene will be included in the first reference set if each of the 18 pairwise comparisons reveals the gene to be consistently differentially expressed (i.e., gene expression always is higher in the control cell line or always higher in the tumor cell line for each of the 18 pairwise comparisons). As one of ordinary skill readily will appreciate, it may sometimes be necessary to scale the datasets prior to carrying out the pairwise comparisons. Such scaling may be routinely implemented in the analysis software provided by commercial suppliers of expression arrays or array readers (such as, e.g., Affymetrix, Santa Clara, Calif.). For a general discussion of data scaling for and differential gene expression analysis, see, e.g., Affymetrix Microarray Suite 4.0 User Guide, Affymetrix, Santa Clara, Calif., incorporated herein by reference.  
      The first reference set therefore is a set of genes that have met a screening criterion requiring that the genes be differentially expressed between tumor and control cell lines. This criterion reflects the hypothesis that differences in the tumor and control cell phenotypes are driven, at least in part, by differences in gene expression patterns in the tumor and control cells. In the practice of the invention, generating a first reference set typically results in an order of magnitude or greater reduction in the number of genes that remain under consideration for inclusion in a cluster or for use in the sample classification methods.  
      Because the tumor and control cell lines have at some point been cultured in vitro, their gene expression patterns likely will not exactly correspond with the expression patterns of their counterparts grown in vivo. Consequently, the methods of the invention use additional steps to establish a second reference set of expressed genes that are differentially expressed in cells of biological samples that differ with respect to a classification. The classification may be an outcome predictor or cellular phenotype or any type of classification that may be used for classifying biological samples. The classification may be binary (i.e., for two mutually exclusive classes such as, e.g., invasive/non-invasive, metastatic/non-metastatic, etc.), or may be continuously or discretely variable (i.e., a classification that can assume more than two values such as, e.g., Gleason scores, survival odds, etc.) The only requirement is that the classified trait must be something that can be observed and characterized by the assignment of a variable or other type of identifier so that samples belonging to the same class may be grouped together during the analysis.  
      The second reference set of expressed genes may be obtained following essentially the same techniques described above for the first reference set, except sets of samples obtained from in vivo sources are used instead of sets of cell lines. In embodiments of the invention directed toward tumor analysis, classification or prognostication, the sample sets preferably consist of tumor samples obtained from patients that are analyzed without any intervening tissue culturing steps so that the gene expression patterns reflect as closely as possible the pattern within cells growing in their undisturbed, in vivo environment. Here again, the goal is to obtain a reference set that includes genes differentially expressed between samples belonging to different classifications. As is the case with the first reference set, it is preferable to include several independent samples within a classified set and to carry out a plurality of pairwise comparisons to identify differentially expressed genes for inclusion into the second reference set.  
      For example, assume the classification of interest is invasiveness (e.g., turning on whether tumor-free surgical margins are observed). It is preferable to use as the sample sets a number of invasive samples and a number of non-invasive samples. The number of pairwise comparisons that can be carried out is of course equal to the product of the numbers of independent samples in each category. Ideally, each of these pairwise comparisons is carried out and the same consistently differentially expressed criterion described above is used to select genes for inclusion into the second reference set.  
      It is contemplated, that in certain instances, especially, e.g., when the variance within a sample set is low, it will not be necessary to carry out all pairwise comparisons to select genes for inclusion into the first or second reference set. In practice, one of ordinary skill can readily determine whether it is advantageous to carry out all pairwise comparisons, or fewer than all pairwise comparisons by examining the convergence behavior of the reference sets as additional comparisons are carried out. If the sets apparently converge prior to completion of all possible pairwise comparisons, then the added benefit of exhaustive comparison may be small and so can be avoided.  
      Similar principles drive the selection of the numbers of cell lines and cell samples used to derive the first and second reference sets as apply to the study of other cell and molecular biological phenomena. One of ordinary skill readily will appreciate that the accuracy of the reference sets can increase as more cell lines and samples are used so that statistical noise is minimized. It currently is contemplated that preferred numbers of different cell lines and samples per set used for calculating reference sets be in the range of 2 to 50 per set, or in the range of 2 to 25, or in the range of 2 to 10, or in the range of 3 to 5 per set. While not preferred, it also is contemplated to be within the scope of the present invention to use sets consisting of a single type of cell in one or more of the four sets of input cells used to calculate the first and second reference sets (i.e., tumor cell lines, control cell lines, first sample, and second sample). Direct statistical analysis using T-test and/or Mann-Whitney test for identification of genes differentially expressed in sets of biological samples that differ with respect to a classification is also applicable to the methods of the present invention. The average expression values for genes across the first and second sets of biological samples that differ with respect to a classification are used for calculation of fold expression changes (see below).  
      After the first and second reference sets of differentially expressed genes are identified, a concordance set of expressed genes is identified. The concordance set is obtained by comparing the first and second reference sets. Two criteria preferably are used to identify genes for inclusion into the concordance set: 1) the candidate gene is present in first and second reference sets; 2) the direction of the candidate gene&#39;s differential is the same in the first and second reference sets. Again, as one of ordinary skill readily will recognize, there is a certain degree of arbitrariness to the sign of the differential, as it is determined by, e.g., the direction of the comparison between samples [sample 1/sample 2, cf. sample 2/sample 1, or alternatively, sample 1-sample 2, cf. sample 2-sample 1]. In any event, the arbitrariness does not affect the results because the direction of the comparison is the same across the entire set of expressed genes. The first criterion is, in general, required for inclusion of a gene within the concordance set, while the second criterion is preferred, but optional. In practical terms, identification of a single reference set of differentially expressed genes could serve as a starting point for identification of a concordant set of transcripts. For example, one can identify a reference set of differentially regulated genes in a panel of biological samples subject to a classification and proceed directly to identification of a concordant set of differentially regulated genes in cell lines.  
      Once the concordance set has been established, information about the rank order of expression differences is used to establish another subset of genes. This subset is referred to as the minimum segregation set. The minimum segregation set may conveniently be selected by generating a scatter plot from which may be determined correlations between the −fold expression change or difference in the cell lines and the samples. In preferred embodiments, the −fold expression change is used, and is calculated by obtaining for gene x the ratio of the average expression value obtained across all tumor cell lines and across all control cell lines, and across the first and in the second sample sets, i.e., 
          −fold change =&lt;expression&gt; 1 /&lt;expression&gt; 2  
 
 where &lt;expression&gt; 1  is the average expression for gene x across all observations in set 1, and likewise, &lt;expression&gt; 2  is the average expression for gene x across all observations in set 2. Explicitly,  
         &lt;   expression   &gt;=       1   N     ⁢       ∑     n   =   1     N     ⁢     E   n           ,       
 
 where N equals the number of observations of expression value E for gene x in the set. In the case of the cell line data, set 1 preferably correspond to the tumor cell line set, and set 2 preferably corresponds to the control cell line set. Similarly, for the sample data, set 1 preferably corresponds to the first set of samples and set 2 preferably corresponds to the second set of samples. 
       

      In another preferred embodiment, differences in expression values are used and are calculated as: 
 
difference =&lt;expression&gt; 1 −&lt;expression&gt; 2 , 
          where &lt;expression&gt; 1  and &lt;expression&gt; 2  have the same meanings as in the − fold change expression.        

      In other embodiments, preferred if the number of observations of gene x expression in each set is small, (i.e., on the order of one or two), a modified average fold change across all observations, &lt;expression&gt; m , can be used in lieu of &lt;expression&gt; 1 /&lt;expression&gt; 2  to improve the performance of the method. The modified average fold change &lt;expression&gt; m  explicitly is defined as: 
 
&lt;expression&gt; m =&lt;expression&gt; 1 /&lt;expression 1 +expression 2 &gt;
 
           &lt;   expression   ⁢     &gt;   m       =         1   N     ⁢       ∑     n   =   1     N     ⁢     E   n             1     N   +   M       ⁢       ∑     n   =   1       N   +   M       ⁢     E   n             ,       
 
 where there are observations of expression value E for gene x from set 1 and M observation of expression value E for gene x from set 2. Improvement in the method performance can be determined using samples of known classification, and assessing the overall accuracy of the method in classifying known samples using &lt;expression&gt; m  in lieu of &lt;expression&gt; 1 /&lt;expression&gt; 2 . 
 
      Consider the following observations of expression values E for gene x in which N=M=5:  
                              Expression Values, E, for gene x                             Set 1 Data   Set 2 Data                       5   1           4   2           8   1           7   4           3   2           sum = 27   sum = 10           &lt;expression&gt; 1  = 27/5 = 5.4   &lt;expression&gt; 2  = 10/5 = 2                 &lt;expression&gt; 1 /&lt;expression&gt; 2  = 5.4/2 = 2.7       &lt;expression&gt; m  = &lt;expression&gt; 1 /&lt;expression 1  + expression 2 &gt; =       5.4/3.7 = 1.5                  
 
      A scatter plot can be generated for genes within the concordance set in which each gene is assigned a point in the scatter plot. The (x,y) location of that point will be, or will be proportional to, the −fold expression change or difference in the cell line data (e.g., x), and the −fold expression change or difference in the sample data (e.g., y). Of course, the selection of the data assigned to be plotted on the abscissa and that to be plotted on the ordinate is arbitrary, so that one could have the x value correspond to the sample data and the y value correspond to the cell line data. In preferred embodiments, the −fold expression change or difference data is logarithmically transformed prior to plotting said data on the scatter plot.  
      The scatter plot potentially will be populated by data points that fall within any of the four quadrants of a graph in which the axes intersect at (0,0). Define quadrant I as negative x, positive y, quadrant II as positive x, positive y, quadrant III as positive x, negative y, and quadrant IV as negative x, negative y. The minimum segregation class is selected so as to include genes that fall within quadrants II and IV, and preferably to include only those genes within quadrants II and IV whose −fold expression changes or differences are highly positively correlated between the cell line and sample data. Alternatively, the minimum segregation class may be selected so as to include genes that fall within quadrants I and III, and preferably to include only those genes within quadrants I and III whose −fold expression changes or differences are highly negatively correlated between the cell line and sample data.  
      The scatter plots described above provide a convenient graphical representation of the data used in the clustering and classification methods of the present invention, although it is not necessary to generate such plots in the practice of the invention. Correlation coefficients can be generated for arrays of data without first plotting the data as described above. The expression data can be sorted by the values of the fold expression changes or differences and subsets of highly correlated data can be selected visually or with the aid of, e.g., regression analysis. Correlation coefficients may then be calculated on the subset of data.  
      Genes whose expression changes are highly correlated (positively or negatively) between the cell line and sample data may be identified by calculating a correlation coefficient for one or more subsets of genes that fall within quadrants II and IV (or alternatively for those that fall within quadrants I and III) of a scatter plot, and selecting as the minimum segregation set, those genes for which the correlation coefficient exceeds a predetermined value. Any one of a number of commonly used correlation coefficients may be used, including correlation coefficients generated for linear and non-linear regression lines through the data. Representative correlation coefficients include the correlation coefficient, px,y, that ranges between −1 and +1, such as is generated by Microsoft Excel&#39;s CORREL function, the Pearson product moment correlation coefficient, r, that also ranges between −1 and +1, that that reflects the extent of a linear relationship between two data sets, such as is generated by Microsoft Excel&#39;s PEARSON function, or the square of the Pearson product moment correlation coefficient, r 2 , through data points in known y&#39;s and known x&#39;s, such as is generated by Microsoft Excel&#39;s RSQ function. The r 2  value can be interpreted as the proportion of the variance in y attributable to the variance in x.  
      In a preferred embodiment, the −fold expression change or difference data are logarithmically transformed (e.g., log 10  transformed), and the minimum segregation set is selected so that the correlation coefficient, Px,y, is greater than or equal to 0.8, or is greater than or equal to 0.9, or is greater than or equal to 0.95, or is greater than or equal to 0.995. One of ordinary skill can readily work out equivalent values for other types of transformations (e.g. natural log transformations) and other types of correlation coefficients either mathematically, or empirically using samples of known classification.  
      The method can be terminated at the step of selecting the minimum segregation set. This set will consist of a collection or cluster of genes that is coordinately regulated during processes that result in phenotypic changes between the types of samples that comprise the sample sets.  
      The method may be continued, as described immediately below, to classify a sample as belonging to the first sample set or to the second sample set. The classification method uses a minimum segregation set of expressed genes to calculate a second correlation coefficient referred to as a “phenotype association index.” The method contemplates several different embodiments for calculating the second correlation coefficient. In a preferred embodiment, the second correlation coefficient is calculated by determining for an individual sample for which classification is sought, the −fold expression change for each gene x within the minimum segregation set. Preferably, the − fold expression change is determined with respect to the average value of expression for gene x across all samples used to identify the minimum segregation set. In the table above, assume set 1 data correspond to a first set of samples and that set 2 data correspond to a second set of samples. The average expression value for gene x across these samples is equal to 3.7. In this preferred embodiment, the −fold expression change is determined by computing the ratio of the expression value for gene x in the individual sample to the 3.7 average value across all the samples used to identify the minimum segregation set. For example, if the observed gene x expression value in the sample is 7, then the −fold expression change calculated according to this embodiment is 7/3.7=1.9. If the data were logarithmically transformed prior to identifying the minimum segregation set, then the same logarithmic transformation is carried out on the individual sample data prior to calculating the correlation coefficient.  
      In this preferred embodiment the classification is made according to the sign of this second correlation coefficient (phenotype association index). Given the setup outlined above, using −fold expression changes &lt;expression&gt; 1 /&lt;expression 1 +expression 2 &gt; for the sample sets to calculate the minimum segregation set, a positive correlation coefficient obtained for the classified sample indicates that the sample is a member of sample set 1, while a negative correlation coefficient indicates the sample belongs to sample set 2.  
      In a refinement of this preferred embodiment, the magnitude of the correlation coefficient can be used as a threshold for classification. The larger the magnitude of the correlation coefficient, the greater the confidence that the classification is accurate. As one of ordinary skill readily will appreciate, the appropriate threshold can be determined through the use of test data that seek to classify samples of known classification using the methods of the present invention. The threshold is adjusted so that a desired level of accuracy (e.g., greater than about 70% or greater than about 80%, or greater than about 90% or greater than about 95% or greater than about 99% accuracy is obtained). This accuracy refers to the likelihood that an assigned classification is correct. Of course, the tradeoff for the higher confidence is an increase in the fraction of samples that are unable to be classified according to the method. That is, the increase in confidence comes at the cost of a loss in sensitivity.  
      In another preferred embodiment, multiple minimum segregation sets can be identified and used to increase the sensitivity of the method. Here again, test data from samples of known classification are used to identify the minimum segregation sets and classify the individual samples. In a preferred embodiment, successive minimum segregation classes are identified using expression data from true positive and false positive samples. The expression data from these samples is again broken down into two sample sets, with the true positives assigned to, e.g., sample set 1, and the false positives assigned to sample set 2. The re-apportioned expression data are used to identify another concordance set and another minimum segregation set. This additional minimum segregation set is used to re-score the samples with particular attention paid to the ability of the set to properly classify the false positives.  
      Several such iterations can be done, and criteria developed to improve the accuracy of the method by evaluating the behavior of known samples against a number of minimum segregation sets. Such analysis can be used to show, e.g., that true positives score with the correct phenotype association index in, e.g., 3 of 3 minimum segregation sets.  
      As one of ordinary skill will recognize, a similar approach can be used with false negatives, wherein the true negatives and the false negatives are used in an iterative embodiment of the invention, with the false negatives re-assigned to sample set 1 and the true negatives assigned to sample set 2. Blended methods also may be used in which, e.g., the true positives and false negatives are assigned to sample set 1 and the true negatives and false positives assigned to sample set 2, or any other logical combination that uses mis-classified samples to iteratively obtain minimum segregation sets that are used either alone or in conjunction with other sets to improve the accuracy of the classification methods of the present invention.  
      While the clustering and classification methods have been described primarily with reference to tumor samples, they are readily applicable to any biological analysis for which appropriate cell lines and samples can be obtained. These include by way of example, but not limitation, omnipotent stem cells, pluripotent precursor cells, various terminally differentiated cells, etc. The clustering methods applied to cell differentiation analyses will identify gene clusters that are coordinately regulated in differentiation programs. These genes are useful not only from a basic research point of view (e.g., to identify novel transcription factors or response elements), but also to identify gene products specifically expressed in one but not another cell type. Such gene products are useful for, e.g., targeting of therapeutic molecules using reagents that have affinity for the specifically expressed gene products.  
      Application of the methods of the present invention to the study and classification of cancers represents an important advance made possible in large part by the ready availability of gene expression data. Recent gene expression analysis data revealed that direct comparison of expression profiles for individual tumors to identify the transcriptome of human cancer progression is extremely challenging. Continuous phenotypic changes in cancer cells during tumor progression, individual phenotypic variations, intrinsic cellular heterogeneity, and variability in cellular composition of the primary and metastatic tumors render extremely problematic the selection of the gene expression changes relevant to tumor progression and metastasis. Furthermore, the use of human tumors and metastatic material, itself, limits the direct manipulation of variables that might otherwise reveal regulatory defects that are not apparent in the ground state expression patterns of in vivo tumors.  
      A complementary experimental approach to the extensive clinical sampling was developed employing gene expression analysis of selected cancer cell lines representing divergent clinically relevant variants of cancer progression (Table 1). These cell lines were surveyed under various in vitro and in vivo conditions that model microenvironments favorable to the malignant phenotype, including differential serum withdrawal responsiveness in vitro and induction of experimental tumors in nude mice, ultimately to identify expression changes characteristic of human cancer progression. These cell lines provide a representative group of tumor cell lines that can be used in the practice of the methods of the invention (although other transformed cell lines, such as are readily available from depositories such as ATCC or commercial suppliers also can be used). The methods of the invention also may be practiced using, e.g., one or more of the 38 human breast cancer cell lines described in Forozan, F., Mahlamaki, E. H., Monni, O., Chen, Y., Veldman, R., Jiang, Y., Gooden, G. C., Ethier, S. P., Kallioniemi, A., Kallioniemi, O—P. Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data.  Cancer Res.  2000. 60: 4519-4525, incorporated herein by reference. The methods of the invention also may be practiced using one or more of the 60 human cancer cell lines representing multiple forms of human cancer and utilized in the National Cancer Institute&#39;s screen for anti-cancer drug was described in Ross, T D, Scherf, U, Eisen, M B, Perou, C M, Rees, C, Spellman, P, Iyer, V, Jeffrey, SS, Van de Rijn, M, Waltham, M, Pergamenschikov, A, Lee, J C F, Lashkari, D, Shalon, D, Myers, TG, Weinstein, J N, Botstein, D, Brown, P O. Systematic variation in gene expression patterns in human cancer cell lines. Nature Genetics, 24: 227-235, 2000, incorporated herein by reference. Classification of the human cancer cell lines based on the observed gene expression profiles revealed a correspondence to the tissue of origins of the corresponding tumors from which the cell lines were derived (Ross, D T, et al, 2000).  
      Each cell line and experimental condition provided a criterion that a gene met in order to be retained in the next step of analysis. Thus, the cancer cell lines represented in Table 1 are especially useful for the practice of the clustering and classification methods of the invention. Each step in the gene selection process (i.e., identification of a first and a second reference set, identification of a concordance set and finally, identification of a minimum segregation set) can be thought of as a cut-off criterion that allows genes to pass to the next stage in the analysis. The identified set of candidate genes that satisfies these criteria comprises genes, the differential expression of which is associated with certain features of the malignant phenotype and that is relatively insensitive to significant alterations in cell type and environmental context. Consequently, these genes represent reliable starting points for identifying genes that are commonly altered in human cancer and represent a consensus transcriptome of cancer progression. Other cell line combinations suitable for practicing the methods of the present invention are set forth in Tables 2-4. Table 2 lists representative cell line combinations for normal cells and certain cancers (e.g., breast, prostate, lung). These combinations are especially useful for identifying genetic markers that serve as diagnostics for a malignant phenotype. Such markers, in addition to providing diagnostic information, can also provide drug discovery targets. Table 2 also lists representative cell line combinations for precursor and differentiated cells, useful for identifying differentiation markers. Such markers can be used to screen for agents that activate differentiation programs to further basic research, as well as tissue engineering work. Table 3 lists additional tumor cell/control cell line combinations useful for practicing the methods of the invention to identify markers of malignant phenotype for diagnostic as well as drug discovery purposes. Table 4 provides additional primary tumor/metastatic tumor cell line combinations useful for practicing the methods of the invention to identify markers of metastatic potential for diagnostic, prognostic and therapeutic applications.  
               TABLE 1                          Model Human Cancer Cell Systems Exhibiting Graded Metastatic Potential                                     METASTATIC           CELLS   DEFINITION   POTENTIAL   REMARKS               Breast Cancer   A panel of human   Metastatic potential   This series of cells       (metastatic   breast carcinoma cell   varies from 0 (MDA-   exhibits differential       potential)   lines of graded   MB-361) to 10-90%   metastatic potential in       MDAMB-361 (0)   metastatic potential.   (MDA-MB-435 and   nude mice, differential       MDAMB-468 (5%)   High met variant   variants) incidence of   homotypic aggregation       MDAMB-231 (30%)   (lung2), low met   lung metastasis in   and clonogenic growth       MDA-MB-435 (60%)   revertant (Br), and   nude mice following   properties, differential       MB-435lung2 (90%)   blood-survival variant   orthotopic   sensitivity toward       MB-435Br (10%)   (Bl3) were derived   implantation.   apoptosis, in vivo and       MB-435Bl3 (?)   from parental MB-435       vitro sensitivity to           cells.       glycoamines, galectin-                   dependent adhesion.       PC3 System   Parental, 1 in vivo   Poorly metastatic   High metastatic       (Prostate-1)   passage   Small prostate tumors   potential is associated       PC-3M   4 in vivo serial   Metastatic   with high resistance       PC-3M-Pro4   passages in prostate   Highly metastatic   toward apoptosis.       PC-3M-LN4   4 in vivo serial       Glycoamine-sensitive           passage; LN4 &gt; Pro4       cell lines. From liver                   met. of splenic PC3                   implant.                   Exhibit rapid large                   prostate tumor growth.                   Exhibit small prostate                   tumors, large LN                   metastatic tumors.       LNCap System   parental   Poorly metastatic   Only androgen-sensitive       (Prostate-2)   5 in vivo serial   Highly metastatic   system. This panel       LNCaP   passages in prostate       exhibits differential       LNCaP-Pro5   3 in vivo serial       metastatic potential,       LNCaP-LN3   passages; LN3 &gt; Pro5       differential sensitivity                   toward apoptosis, and in                   vitro glycoamine                   sensitivity. LN3 exhibit                   decreased androgen                   dependency, increased                   PSA level, high                   frequency and load of                   regional LN metastasis.       BPE System   SV40 large T antigen   Approximately 11%   Cell line system suitable       (Prostate-3)   immortalized benign   tumorigenicity with 6   for determination of the       P69   prostate epithelial   mo. latency.   gene expression changes       2182   cells (BPE).   Lung and diaphragm   associated with       M12   3 serial passages in   metastases.   alterations within major           vivo as xenograft       tumor suppressor                   pathways.       Colon cancer   Colon carcinoma cell   Differential capability   High metastatic       KM12-C   lines selected from a   to generate liver   potential within this cell       KM12-SP   single parental cell   metastasis following   line system is associated       KM12-SM   line for differential   intrasplenic   with increased       KM12-L4   metastatic potential   implantation in nude   expression of a sialyl           through in vivo   mice.   Lewis family of           passages in nude mice.       glycoantigens and                   higher selectin-mediated                   adhesion.                  
 
      References: Pettaway, C. et al. Clin. Cancer Res., 2: 1627, 1996; Bae, V. et al. Int. J. Cancer, 58:721, 1994; Plymate, et al. J. Clin. Endocrinol., Met. 81: 3709, 1996; Morikawa et al. Cancer Res., 48: 1943, 1988; Morikawa et al. Cancer Res., 48: 6863, 1988; Schackert et al. Am. J. Pathol., 136: 95, 1990; Zhang et al. Cancer Res., 51: 2029, 1991; Zhang et al. Invasion Metastasis, 11: 204, 1991; Price et al. Cancer Res., 50: 717, 1990; Mukhopadhyay et al. Clin Exp Met., 17: 325, 1999; Glinsky et al. Clin. Exper. Metastasis, 14: 253, 1996; Glinsky et al. Cancer Res., 56: 5319, 1996; Glinsky et al. Cancer Lett., 115: 185, 1997; McConkey et al. Cancer Res., 56: 5594, 1996; Glinsky et al. Transf Med Rev 14: 326, 2000 (incorporated herein by reference).  
               TABLE 2                       Representative Cell Line Combinations                                            Tumor Cell Line   Control Cell Line   Reference/comments                         Breast Cancer                         See Table 1   Clonetics ™ human   ATCC collection,           mammary epithelial cells   incorporated herein by           (Cat. #CC2551 from   reference; Cambrex, Inc.           Cambrex, Inc., East   2002 Biotech Catalog,           Rutherford, NJ)   incorporated herein by               reference                 Prostate Cancer                         See Table 1   Clonetics ™ prostate   ATCC collection,           epithelial cells (Cat. #   incorporated herein by           CC2555 from Cambrex,   reference; Cambrex, Inc.           Inc., East Rutherford, NJ)   2002 Biotech Catalog,               incorporated herein by               reference                 Lung Cancer                         See Table 3   ATCC# CCL-256.1; NCI-   ATCC collection,           BL2126; peripheral blood;   incorporated herein by           Clonetics ™ bronchial   reference;           epithelial cells (Cat. #   Cambrex, Inc. 2002           CC2540 from Cambrex,   Biotech Catalog,           Inc., East Rutherford, NJ);   incorporated herein by           Clonetics ™ small airway   reference           epithelial cells (Cat. #           CC2547 from Cambrex,           Inc., East Rutherford, NJ);           See Table 3                 Other types of cancers                         See Table 3   See Table 3                         Differentiation Pathway                                 Reference/       Precursor/Stem Cell Line   Differentiated Cell Line   comments               CD133+ cells Cat. # 2M-   mononuclear cells Cat   ATCC collec-       102A - bone marrow   #2M-125C; CD4+ T-cells   tion, incor-       derived; Cat # 2G102 - G-   Cat. # 1C-200; human   porated herein       CSF derived; Cat. # 2L-   astrocytes Cat. # CC2565;   by reference;       102A - fetal liver derived;   human hepatocytes Cat. #   Cambrex, Inc.       CD36+ erythroid   CC2591; NHEM neonatal   2002 Biotech       progenitors Cat # 2C-250;   melanocytes Cat. #   Catalog, in-       cord blood CD19+ B cells   CC2513; SkMC - Skeletal   corporated       Cat # 1C-300; dendritic   Muscle Cells Cat. #   herein by       cell precursors Cat # 2P-   CC2561 (all from   reference       105; NHNP neural   Cambrex, Inc., East       progenitor cells Cat. #   Rutherford, NJ)       CC2599; hMSC -       mesenchymal stem cells,       human bone marrow Cat. #       PT-2501 (all from       Cambrex, Inc., East       Rutherford, NJ)                  
 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                   
               
               
                 Representative Tumor/Control Cell Line Combinations Available 
               
               
                 from American Type Culture Collection (ATCC) 
               
            
           
           
               
               
            
               
                 Tumor Cell Line 
                 Control Cell Line 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ATCC 
                   
                 Cancer 
                 Tissue 
                 ATCC 
                   
                 Tissue 
               
               
                 No. 
                 Name 
                 Type 
                 Source 
                 No. 
                 Name 
                 Source 
               
               
                   
               
               
                 CCL-256 
                 NCI-H2126 
                 carcinoma; non- 
                 lung 
                 CCL-256.1 
                 NCI-BL2126 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                   
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
               
               
                 CRL-5868 
                 NCI-H1395 
                 adenocarcinoma 
                 lung 
                 CRL-5957 
                 NCI-BL1395 
                 peripheral 
               
               
                   
                   
                   
                   
                   
                   
                 blood 
               
               
                 CRL-5882 
                 NCI-H1648 
                 adenocarcinoma 
                 lung 
                 CRL-5954 
                 NCI-BL1648 
                 peripheral 
               
               
                   
                   
                   
                   
                   
                   
                 blood 
               
               
                 CRL-5911 
                 NCI-H2009 
                 adenocarcinoma 
                 lung 
                 CRL-5961 
                 NCI-BL2009 
                 peripheral 
               
               
                   
                   
                   
                   
                   
                   
                 blood 
               
               
                 CRL-5985 
                 NCI-H2122 
                 adenocarcinoma 
                 pleural 
                 CRL-5967 
                 NCI-BL2122 
                 peripheral 
               
               
                   
                   
                   
                 effusion 
                   
                   
                 blood 
               
               
                 CRL-5922 
                 NCI-H2087 
                 adenocarcinoma 
                 lymph node 
                 CRL-5965 
                 NCI-BL2087 
                 peripheral 
               
               
                   
                   
                   
                 (metastasis) 
                   
                   
                 blood 
               
               
                 CRL-5886 
                 NCI-H1672 
                 carcinoma; 
                 lung 
                 CRL-5959 
                 NCI-BL1672 
                 peripheral 
               
               
                   
                   
                 classic small 
                   
                   
                   
                 blood 
               
               
                   
                   
                 cell lung cancer 
               
               
                 CRL-5929 
                 NCI-H2171 
                 carcinoma; 
                 lung 
                 CRL-5969 
                 NCI-BL2171 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                   
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
               
               
                 CRL-5931 
                 NCI-H2195 
                 carcinoma; 
                 lung 
                 CRL-5956 
                 NCI-BL2195 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                   
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
               
               
                 CRL-5858 
                 NCI-H1184 
                 carcinoma; 
                 lymph node 
                 CRL-5949 
                 NCI-BL1184 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                 (metastasis) 
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
               
               
                 HTB-172 
                 NCI-H209 
                 carcinoma; 
                 bone 
                 CRL-5948 
                 NCI-BL209 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                 marrow 
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
                 (metastasis) 
               
               
                 CRL-5983 
                 NCI-H2107 
                 carcinoma; 
                 bone 
                 CRL-5966 
                 NCI-BL2107 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                 marrow 
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
                 (metastasis) 
               
               
                 HTB-120 
                 NCI-H128 
                 carcinoma; 
                 pleural 
                 CRL-5947 
                 NCI-BL128 
                 peripheral 
               
               
                   
                   
                 small cell lung 
                 effusion 
                   
                   
                 blood 
               
               
                   
                   
                 cancer 
               
               
                 CRL-5915 
                 NCI-H2052 
                 mesothelioma 
                 pleural 
                 CRL-5963 
                 NCI-BL2052 
                 peripheral 
               
               
                   
                   
                   
                 effusion 
                   
                   
                 blood 
               
               
                 CRL-5893 
                 NCI-H1770 
                 neuroendocrine 
                 lymph node 
                 CRL-5960 
                 NCI-BL1770 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 (metastasis) 
                   
                   
                 blood 
               
               
                 HTB-126 
                 Hs 578T 
                 ductal 
                 mammary 
                 HTB-125 
                 Hs 578Bst 
                 mammary 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 gland; 
               
               
                   
                   
                   
                 breast 
                   
                   
                 breast 
               
               
                 CRL-2320 
                 HCC1008 
                 ductal 
                 mammary 
                 CRL-2319 
                 HCC1007 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2338 
                 HCC1954 
                 ductal 
                 mammary 
                 CRL-2339 
                 HCC1954 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2314 
                 HCC38 
                 primary ductal 
                 mammary 
                 CRL-2346 
                 HCC38 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2321 
                 HCC1143 
                 primary ductal 
                 mammary 
                 CRL-2362 
                 HCC1143 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2322 
                 HCC1187 
                 primary ductal 
                 mammary 
                 CRL-2323 
                 HCC1187 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2324 
                 HCC1395 
                 primary ductal 
                 mammary 
                 CRL-2325 
                 HCC1395 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2331 
                 HCC1599 
                 primary ductal 
                 mammary 
                 CRL-2332 
                 HCC1599 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2336 
                 HCC1937 
                 primary ductal 
                 mammary 
                 CRL-2337 
                 HCC1937 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2340 
                 HCC2157 
                 primary ductal 
                 mammary 
                 CRL-2341 
                 HCC2157 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-2343 
                 HCC2218 
                 primary ductal 
                 mammary 
                 CRL-2363 
                 HCC2218 BL 
                 peripheral 
               
               
                   
                   
                 carcinoma 
                 gland; 
                   
                   
                 blood 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-7345 
                 Hs 574.T 
                 ductal 
                 mammary 
                 CRL-7346 
                 Hs 574.Sk 
                 skin 
               
               
                   
                   
                 carcinoma 
                 gland; 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-7482 
                 Hs 742.T 
                 scirrhous 
                 mammary 
                 CRL-7481 
                 Hs 742.Sk 
                 skin 
               
               
                   
                   
                 adenocarcinoma 
                 gland; 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-7365 
                 Hs 605.T 
                 carcinoma 
                 mammary 
                 CRL-7364 
                 Hs 605.Sk 
                 skin 
               
               
                   
                   
                   
                 gland; 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-7368 
                 Hs 606 
                 carcinoma 
                 mammary 
                 CRL-7367 
                 Hs 606.Sk 
                 skin 
               
               
                   
                   
                   
                 gland; 
               
               
                   
                   
                   
                 breast 
               
               
                 CRL-1974 
                 COLO 829 
                 malignant 
                 skin 
                 CRL-1980 
                 COLO 829BL 
                 peripheral 
               
               
                   
                   
                 melanoma 
                   
                   
                   
                 blood 
               
               
                 CRL-7762 
                 TE 354.T 
                 basal cell 
                 skin 
                 CRL-7761 
                 TE 353.Sk 
                 skin 
               
               
                   
                   
                 carcinoma 
               
               
                 CRL-7677 
                 Hs 925.T 
                 pagetoid 
                 skin 
                 CRL-7676 
                 Hs 925.Sk 
                 skin 
               
               
                   
                   
                 sarcoma 
               
               
                 CRL-7672 
                 Hs 919.T 
                 benign osteoid 
                 bone 
                 CRL-7671 
                 Hs 919.Sk 
                 skin 
               
               
                   
                   
                 osteoma 
               
               
                 CRL-7554 
                 Hs 821.T 
                 giant cell 
                 bone 
                 CRL-7553 
                 Hs 821.Sk 
                 skin 
               
               
                   
                   
                 sarcoma 
               
               
                 CRL-7552 
                 Hs 820.T 
                 heterophilic 
                 bone 
                 CRL-7551 
                 Hs 820.Sk 
                 skin 
               
               
                   
                   
                 osteofication 
               
               
                 CRL-7444 
                 Hs 704.T 
                 osteosarcoma 
                 bone 
                 CRL-7443 
                 Hs 704.Sk 
                 skin 
               
               
                 CRL-7448 
                 Hs 707(A).T 
                 osteosarcoma 
                 bone 
                 CRL-7449 
                 Hs 707(B).Ep 
                 skin 
               
               
                 CRL-7471 
                 Hs 735.T 
                 osteosarcoma 
                 bone 
                 CRL-7865 
                 Hs 735.Sk 
                 skin 
               
               
                 CRL-7595 
                 Hs 860.T 
                 osteosarcoma 
                 bone 
                 CRL-7519 
                 Hs 791.Sk 
                 skin 
               
               
                 CRL-7622 
                 Hs 888.T 
                 osteosarcoma 
                 bone 
                 CCL-211 
                 Hs888Lu 
                 lung 
               
               
                 CRL-7626 
                 Hs 889.T 
                 osteosarcoma 
                 bone 
                 CRL-7625 
                 Hs 889.Sk 
                 skin 
               
               
                 CRL-7628 
                 Hs 890.T 
                 osteosarcoma 
                 bone 
                 CRL-7627 
                 Hs 890.Sk 
                 skin 
               
               
                 CRL-7453 
                 Hs 709.T 
                 periostitis; 
                 bone 
                 CRL-7452 
                 Hs 709.Sk 
                 skin 
               
               
                   
                   
                 granuloma 
               
               
                 CRL-7886 
                 Hs 789.T 
                 transitional cell 
                 ureter 
                 CRL-7518 
                 Hs 789.Sk 
                 skin 
               
               
                   
                   
                 carcinoma 
               
               
                 CRL-7547 
                 Hs 814.T 
                 giant cell 
                 vertebral 
                 CRL-7546 
                 Hs 814.Sk 
                 skin 
               
               
                   
                   
                 sarcoma 
                 column 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
               
               
                 Representative Primary Tumor/Metastatic Tumor Cell Line Combinations 
               
               
                 Available from American Type Culture Collection (ATCC) 
               
            
           
           
               
               
            
               
                 Primary Cell Line 
                 Metastatic Cell Line 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ATCC 
                   
                   
                   
                 ATCC 
                   
                   
               
               
                 No. 
                 Name 
                 Disease 
                 Tissue 
                 No. 
                 Name 
                 Tissue 
               
               
                   
               
               
                 CCL-228 
                 SW480 
                 colorectal 
                 colon 
                 CCL-227 
                 SW620 
                 lymph 
               
               
                   
                   
                 adenocarcinoma 
                   
                   
                   
                 node 
               
               
                 CRL-1864 
                 RF-1 
                 gastric 
                 stomach 
                 CRL-1863 
                 RF-48 
                 ascites 
               
               
                   
                   
                 adenocarcinoma 
               
               
                 CRL-1675 
                 WM-115 
                 melanoma 
                 skin 
                 CRL-1676 
                 WM-266-4 
                 n/a 
               
               
                 CRL-7425 
                 Hs 
                 melanoma 
                 skin 
                 CRL-7426 
                 Hs 688(B).T 
                 lymph 
               
               
                   
                 688(A).T 
                   
                   
                   
                   
                 node 
               
               
                   
               
            
           
         
       
     
      Application of the methods of the invention to the study of particular cancers is described generally below, and is followed by specific working examples demonstrating aspects of the invention.  
      Prostate Cancer  
      As many as 50% of men, aged 70 years and over have microscopic foci of prostate cancer without clinical evidence of disease (Trump, D. L., Robertson, C. N., Holland, J. F., Frei, E., Bast, R. C., Kufe, D. W., Morton, D. L., and Weishselbaum, R. R. Neoplasms of the prostate. In: D. L. Trump, C. N. Robertson, J. F. Holland, E. Frei, R. C. Bast, D. W. Kufe, D. L. Morton, and R. R. Weishselbaum (eds.), Cancer Med, Vol. 3, pp. 1562-86. Philadelphia: Lea &amp; Febiger, 1993.). Although some prostate cancers remain indolent and confined to the gland, other prostate cancers behave more aggressively and metastasize if not adequately treated. Prostate cancer is the second most lethal neoplasia in males after lung cancer. Because of widespread screening programs utilizing serum PSA values, many more cases of early stage disease are being diagnosed. In 1988 approximately 50% of patients were diagnosed with early stage disease (stage I and II). Today, about 75% of patients have early stage disease that is potentially curable.  
      Unfortunately, the only potentially curative therapy for prostate cancer consists of radical prostatectomy or other local therapies such as external irradiation, implanted irradiation seeds, or cryotherapy. The use of prostatectomy has increased in step with the amount of diagnosed early stage prostate cancer. SEER data indicates an increase in prostatectomies from 17.4 per 100,000 in 1988 to 54.6 per 100,000 in 1992. Insufficient treatment leads to local disease extension and metastasis. Current methods, such as Gleason scores are not perfectly reliably correlated with whether a tumor is aggressive or indolent. Thus, developing a treatment strategy appropriate for any individual is difficult. The recognition of those genetic changes that portend metastatic prostate cancer would, therefore, be a breakthrough. The methods of the present invention readily identify such genetic changes.  
      Breast Cancer  
      Breast cancer is the most common cancer among women in North America and Western Europe and is the second leading cause of female cancer death in the United States. In the United States, age-adjusted breast cancer incidence rates have considerably increased during last century. Approximately 40% of patients diagnosed with breast cancer have disease that has regional or distant metastases and, at present, there is no efficient curative therapy for breast cancer patients with advanced metastatic disease. Thus, developing a treatment strategy appropriate for any individual with early stage disease is difficult and insufficient treatment leads to local disease extension and metastasis. Therefore, there is an urgent clinical need for novel diagnostic methods that would allow early identification of those breast cancer patients who are likely to develop metastatic disease and would require the most aggressive and advanced forms of therapy for increased chance of survival. The identification of those genetic changes that distinguish aggressive metastatic disease and predict metastatic behavior would, therefore, be a breakthrough. The methods of the present invention provide information that allows prognostication of aggressive metastatic disease.  
      Recent gene expression analysis of human tumor samples employing cDNA microarray technology underscores the difficulties in identification of the cellular origin of differentially expressed transcripts in clinical samples due to the remarkable cellular heterogeneity and variability in cellular compositions of human tumors (Golub, T. R., Slonim, D. K., Tamayo, P., Huard, C., Gaasenbeek, M., Mesirov, J. P., Coller, H., Loh, M. L., Downing, J. R., Caliguri, M. A., Bloomfield, C. D., Lander, E. S. 1999. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science, 286: 531-537; Perou C M, Jeffrey S S, van de Rijn M, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci USA. 1999. 96:9212-9217; Perou C M, Sorlie T, Eisen M B, et al. Molecular portrait of human breast tumors. Nature. 2000. 406:747-752, incorporated herein by reference). However, a cDNA microarray analysis of gene expression in melanoma cell lines of distinct metastatic potential, was successfully employed for identification of RhoC as an essential gene for the acquisition of metastatic phenotype by melanoma cells (Clark, E A, Golub T R, Lander E S, Hynes R O. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000. 406:532-535, incorporated herein by reference). Established human cancer cell lines were utilized for parallel comparisons of the alterations in DNA copy number and gene expression associated with human breast cancer (Pollack, J. R., Perou, C. M., Alizadeh, A. A., Eisen, M. B., Pergamenschikov, A., Williams, C. F., Jeffrey, S. S., Botstein, D., Brown, P. O. Genome-wide analysis of DNA-copy number changes using cDNA microarrays. Nature Genetics. 1999. 23: 41-46; Forozan, F., Mahlamaki, E. H., Monni, O., Chen, Y., Veldman, R., Jiang, Y., Gooden, G. C., Ethier, S. P., Kallioniemi, A., Kallioniemi, O-P. Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data. Cancer Res. 2000. 60: 4519-4525, incorporated herein by reference). Thus, model systems are a reasonable source of gene candidates to be studied in the much more heterogeneous environment of real human tumors.  
      Analysis of gene expression in normal and neoplastic ovarian human tissues using methods of the present invention revealed that high malignant potential ovarian cancers exhibited gene expression profile somewhat similar to the ovarian cancer cell lines (Welsh, J. B., Zarrinkar, P. P., Sapinoso, L. M., Kern, S. G., Behling, C. A., Monk, B. J., Lockhart, D. J., Burger, R. A., Hampton, G. M. Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc Natl Acad Sci USA. 2001. 98:1176-1181, incorporated herein by reference), further validating the complementary gene expression analysis approach utilizing selected established cancer cell lines and clinical samples.  
      Metastasis  
      Cancer cells have exceedingly low survival rates in the circulation (reviewed in [Glinsky, G. V. 1993. Cell adhesion and metastasis: is the site specificity of cancer metastasis determined by leukocyte-endothelial cell recognition and adhesion? Crit. Rev. Onc./Hemat., 14: 229-278, incorporated herein by reference). Even if the bloodstream contains many cancer cells, there may be no clinical or pathohistological evidence of metastatic dissemination into the target organs (Williams, W. R. The theory of Metastasis. In The Natural History of Cancer. 1908; 442-448; Goldmann, E. 1907. The growth of malignant disease in man and the lower animals, with special reference to the vascular system. Proc. R. Soc. Med., 1:1-13; Schmidt, M. B. In Die Verbreitungswege der Karzinome und die bezienhung generalisiertes sarkome su den leukamischen neubildungen. Fischer, Jena, 1903, incorporated herein by reference). The levels of metastatic efficiency at the intramicrovascular (postintravasation) phase of metastatic dissemination were shown to be only 0.2% and 0.003% in high and low metastatic variants of B16 melanoma cells, respectively, injected at a concentration of 10 5  cells into the tail veins of laboratory mice (Weiss, L. 1990. Metastatic inefficiency. Adv. Cancer Res., 54: 159-211; Weiss, L., Mayhew, E., Glaves-Rapp, D., Holmes, J. C. 1982. Metastatic inefficiency in mice bearing B16 melanomas. Br. J. Cancer, 45: 44-53, incorporated herein by reference). The fate of cancer cells in the circulation is a rapid phase of intramicrovascular cancer cell death, which is completed in &lt;5 minutes and accounts for 85% of arrested cancer cells. This is followed by a slow phase of cell death, which accounts for the vast majority of the remainder (Weiss, L. 1988. Biomechanical destruction of cancer cells in the hart: a rate regulator of hematogenous metastasis. Invas. Metastasis, 8: 228-237; Weiss, L., Orr, F. W., Honn, K. V. 1988. Interactions of cancer cells with the microvasculature during metastasis. FSEB J., 2: 12-21; Weiss, L., Harlos, J. P., Elkin, G. 1989. Mechanism of mechanical trauma to Ehrlich ascites tumor cells in vitro and its relationship to rapid intravascular death during metastasis. Int. J. Cancer, 44: 143-148, incorporated herein by reference).  
      For example, the number of tumor cells in the lungs declined very rapidly after intravenous injection i.e., 90-99% had disappeared after 24 hours (Hewitt, H. B., Blake, A. 1975. Quantitative studies of translymphonodal passage of tumor cells naturally disseminating from a nonimmunogenic murine squamous carcinoma. Br. J. Cancer, 31: 25-35; Fidler, I. J. 1970. Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5 iodo-2′-deoxyuridine. J. Natl. Cancer Inst., 45: 773-782; Proctor, J. W. 1976. Rat sarcoma model supports both soil seed and mechanical theories of metastatic spread. Br. J. Cancer, 34: 651-654; Proctor, J. W., Auclair, B. G., Rudenstam, C. M. 1976. The distribution and fate of blood-born 125IudR-labeled tumor cells in immune syngeneic rats. Int. J. Cancer, 18: 255-262; Weston, B. J., Carter, R. L., Eastry, G. C., Connell, D. I., Davies, A. J. C. 1974. The growth and metastasis of an allografted lymphoma in normal, deprived and reconstituted mice. Int. J. Cancer, 14: 176-185; Kodama, M., Kodama, T. 1975. Enhancing effect of hydrocortisone on hematogenous metastasis of Ehrlich ascites tumor in mice. Cancer Res., 35: 1015-1021, incorporated herein by reference) and after 3 days generally less than 1% remained (Fidler, I. J. 1970. Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5 iodo-2′-deoxyuridine. J. Natl. Cancer Inst., 45: 773-782; Weston, B. J., Carter, R. L., Eastry, G. C., Connell, D. I., Davies, A. J. C. 1974. The growth and metastasis of an allografted lymphoma in normal, deprived and reconstituted mice. Int. J. Cancer, 14: 176-185; Kodama, M., Kodama, T. 1975. Enhancing effect of hydrocortisone on hematogenous metastasis of Ehrlich ascites tumor in mice. Cancer Res., 35: 1015-1021, incorporated herein by reference). This decline is due to a rapid degeneration of cancer cells (Fidler, I. J. 1970. Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5 iodo-2′-deoxyuridine. J. Natl Cancer Inst., 45: 773-782; Roos, E., Dingemans, K. P. 1979. Mechanisms of metastasis. Biochim. Biophys. Acta, 560: 135-166, incorporated herein by reference). Therefore, the individual ‘average’ cancer cell survives only a short time in the circulation. The successful metastatic cancer cells are able to find a largely unknown survival and escape route. Patients at high risk for metastatic disease could be better managed if gene expression patterns correlated with a clinical metastatic phenotype are identified. The methods of the present invention identify such gene expression patterns. Patients&#39; tumor samples can be tested to see whether the gene expression pattern is associated with an increased risk of metastasis, and if so, the patients can be treated with more aggressive therapies to lower the risk of metastasis. As explained in greater detail below, the present invention provides for methods that allow identification of such gene expression patterns, and sample classification based on those patterns.  
      Models of Human Cancer Metastasis of Graded Metastatic Potential  
      We have acquired several well-established and characterized model human cancer cell systems of graded metastatic potential (Table 1). The collection of these human cancer cell line panels provides different backgrounds upon which increased metastatic potential is superimposed. We have studied these cell line systems extensively for many years both in vitro and in vivo (Glinsky, G. V. 1998. Failure of Apoptosis and Cancer Metastasis. Berlin/Heidelberg: Springer-Verlag, pp. 178 et seq.; Glinsky, G. V., Mossine, V. V., Price, J. E., Bielenberg, D., Glinsky, V. V., Ananthaswamy, H. N., Feather, M. S. 1996. Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamines. Clin. Exp. Metastasis, 14: 253-267; Glinsky, G. V., Price, J. E., Glinsky, V. V., Mossine, V. V., Kiriakova, G., Metcalf, J. B. 1996. Inhibition of human breast cancer metastasis in nude mice by synthetic glycoamines. Cancer Res., 56: 5319-5324; Glinsky, G. V., Glinsky, V. V. 1996. Apoptosis and metastasis: a superior resistance of metastatic cancer cells to the programmed cell death. Cancer Lett., 101: 43-51; Glinsky, G. V., Glinsky, V. V., Ivanova, A. B., Hueser, C. N. 1997. Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms. Cancer Lett., 115: 185-193, incorporated herein by reference) and, therefore, have considerable experience in the maintenance of cell lines preserving graded metastatic potentials. These models provide an excellent opportunity to test whether concordant changes in gene expression underlie the metastasis process and to test the efficacy of drugs designed to block one or more crucial targets.  
      Four important features of the selected models have been documented (Glinsky, G. V. 1997. Apoptosis in metastatic cancer cells. Crit. Rev. Onc/Hemat., 25:175-186; Glinsky, G. V. 1998. Anti-adhesion cancer therapy. Cancer and Metastasis Reviews, 17: 171-185. Glinsky, G. V. 1998. Failure of Apoptosis and Cancer Metastasis. Berlin/Heidelberg: Springer-Verlag, pp 178 et seq.; Glinsky, G. V., Mossine, V. V., Price, J. E., Bielenberg, D., Glinsky, V. V., Ananthaswamy, H. N., Feather, M. S. 1996. Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamines. Clin. Exp. Metastasis, 14: 253-267; Glinsky, G. V., Price, J. E., Glinsky, V. V., Mossine, V. V., Kiriakova, G., Metcalf, J. B. 1996. Inhibition of human breast cancer metastasis in nude mice by synthetic glycoamines. Cancer Res., 56: 5319-5324; Glinsky, G. V., Glinsky, V. V. 1996. Apoptosis and metastasis: a superior resistance of metastatic cancer cells to the programmed cell death. Cancer Lett., 101: 43-51; Glinsky, G. V., Glinsky, V. V., Ivanova, A. B., Hueser, C. N. 1997. Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms. Cancer Lett., 115: 185-193, incorporated herein by reference): a) highly metastatic cell variants possess an increased survival ability, high clonogenic growth potential, and enhanced resistance to apoptosis compared to parental or poorly metastatic counterparts; b) treatment of highly metastatic cell variants with certain synthetic glycoamine analogues caused inhibition of clonogenic growth and survival and reversal of apoptosis resistance in vitro, as well as significant reduction of metastatic potential in vivo; c) these cell lines maintain their distinct in vivo metastatic potentials during in vitro passage for at least several months, indicating that metastatic ability is preserved in vitro; d) differential transcription profiles of four metastasis-associated genes between high and low metastatic cell variants was shown to be similar in vitro and in vivo (Greene, G. F., Kitadai, Y., Pettaway, C. A., von Eschenbach, A. C., Bucana, C. D., Fidler, I. J. 1997. Correlation of metastasis-related gene expression with metastatic potential in human prostate carcinoma cells implanted in nude mice using an in situ messenger RNA hybridization technique. American J. Pathology, 150: 1571-1582, incorporated herein by reference) indicating the potential relevance of in vitro gene expression patterns to the metastatic phenotype. Thus, in accordance with the methods of the present invention, these cellular systems can be used to identify relevant gene expression patterns associated with phenotypes of interest (such as, e.g., metastasis, invasiveness, etc.) by comparing patterns of differential gene expression in one or more independently selected cell line variants with those in different types of clinical human cancer samples.  
      Orthotopic Model of Human Cancer Metastasis in Nude Mice  
      When human tumor cells are injected into ectopic sites in nude mice most do not metastasize (Fidler, I. J. The nude mouse model for studies of human cancer metastasis. In: V. Schirrmacher and R. Schwartz-Abliez (eds.). pp. 11-17. Berlin: Springer-Verlag, 1989; Fidler, I. J. Critical factors in the biology of human cancer metastasis. 1990. Cancer Res., 50, 6130-6138, incorporated herein by reference). The normal host tissue environment influences metastatic ability of cancer cells in such a way that many human and animal tumors transplanted into nude mice metastasize only if placed in the orthotopic organ (Fidler, I. J. The nude mouse model for studies of human cancer metastasis. In: V. Schirrmacher and R. Schwartz-Abliez (eds.). pp. 11-17. Berlin: Springer-Verlag, 1989; Fidler, I. J. Critical factors in the biology of human cancer metastasis. 1990. Cancer Res., 50, 6130-6138; Fidler, I. J., Naito, S., Pathak, S. 1990. Orthotopic implantation is essential for the selection, growth and metastasis of human renal cell cancer in nude mice. Cancer Metastasis Rev., 9, 149-165; Giavazzi, R., Campbell, D. E., Jessup, J. M., Cleary, K., and Fidler, I. J. 1986. Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res., 46: 1928-1948; Naito, S., von Eschenbach, A. C., Giavazzi, R., and Fidler, I. J. 1986. Growth and metastasis of tumor cells isolated from a renal cell carcinoma implanted into different organs of nude mice. Cancer Res., 46: 4109-4115; McLemore, T. L., et al. 1987. Novel intrapulmonary model for orthotopic propagation of human lung cancer in athymic nude mice. Cancer Res., 47: 5132-5140, incorporated herein by reference). These observations pointed out the unique opportunity to study gene expression changes associated with aggressive metastatic phenotype. A comparison of gene expression patterns using the same high metastatic variant implanted at orthotopic (metastasis promoting model) and ectopic (metastasis suppressing model) sites should provide unique information regarding differential gene expression profiles associated with metastatic behavior in vivo.  
      Several orthotopic models of human cancer metastasis have been developed (Fu, X., Herrera, H., and Hoffman, R. M. 1992. Orthotopic growth and metastasis of human prostate carcinoma in nude mice after transplantation of histologically intact tissue. Int. J. Cancer, 52: 987-990; Stephenson, R. A., Dinney, C. P. N., Gohji, K., Ordonez, N. G., Killion, J. J., and Fidler, I. J. 1992. Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J. Natl. Cancer Inst., 84: 951-957; Pettaway, C. A., Stephenson, R. A., and Fidler, I. J. 1993. Development of orthotopic models of metastatic human prostate cancer. Cancer Bull. (Houst.), 45: 424-429; An, Z., Wang, X., Geller, J., Moossa, A. R., and Hoffman, R. M. 1998. Surgical orthotopic implantation allows high lung and lymph node metastasis expression of human prostate carcinoma cell line PC-3 in nude mice. The Prostate, 34: 169-174; Wang, X., An, Z., Geller, J., and Hoffman, R. M. 1999. High-malignancy orthotopic mouse model of human prostate cancer LNCaP. The Prostate, 39: 182-186; Yang, M., Jiang, P., Sun, F.-X., Hasegawa, S., Baranov, E., Chishima, T., Shimada, H., Moosa, A. R., and Hofman, R. M. 1999. A fluorescent orthotopic bone metastasis model of human prostate cancer. Cancer Res., 59: 781-786, incorporated herein by reference). The orthotopic model of human cancer metastasis in nude mice was used for in vivo selection of highly and poorly metastatic cell variants, employing either established panels of human cancer cell lines or cell variants derived from the same parental cell lines (Giavazzi, R., Campbell, D. E., Jessup, J. M., Cleary, K., and Fidler, I. J. 1986. Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res., 46: 1928-1948; Morikawa, K., Walker, S. M., Jessup, J. M., Cleary, K., and Fidler, I. J. 1988. In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinoma implanted in nude mice. Cancer Res., 48: 1943-1948; Dinney, C. P. N. et al. 1995. Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice. J. Urol., 154: 1532-1538, incorporated herein by reference).  
      This approach was successfully applied to develop a human breast cancer model of graded metastatic potential (see Glinsky, G. V., Mossine, V. V., Price, J. E., Bielenberg, D., Glinsky, V. V., Ananthaswamy, H. N., Feather, M. S. 1996. Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamines. Clin. Exp. Metastasis, 14: 253-267; Glinsky, G. V., Price, J. E., Glinsky, V. V., Mossine, V. V., Kiriakova, G., Metcalf, J. B. 1996. Inhibition of human breast cancer metastasis in nude mice by synthetic glycoamines. Cancer Res., 56: 5319-5324, incorporated herein by reference) as well as three independent panels of human prostate cancer cell lines with distinct metastatic potential (Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R., Killion, J. J., and Fidler, I. J. 1996. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clinical Cancer Res., 2: 1627-1636; Bae, V. L., Jackson-Cook, C. K., Brothman, A. R., Maygarden, S. J., and Ware, J. Tumorugenicity of SV40 T antigen immortalized human prostate epithelial cells: association with decreased epidermal growth factor receptor (EGFR) expression. Int. J. Cancer 1994;58:721-29; Plymate, et al., The effect of the IGF system in human prostate epithelial cells of immortalization and transformation by SV-40 T antigen. J. Clin. Endocrinol. Met. 1996:81;3709-16; Jackson-Cook, C., Bae, V., Edelman W., Brothman, A., and Ware, J. Cytogenetic characterization of the human prostate cancer cell line P69SV40T and its novel tumorigenic sublines M2182 and M15. Cancer Genet. &amp; Cytogenet 1996;87:14-23; Bae, V. L., Jackson-Cook, C. K., Maygarden, S. J., Plymate, S. R., Chen, J., and Ware, J. L. Metastatic subline of an SV40 large T antigen immortalized human prostate epithelial cell line. Prostate 1998;34:275-82, incorporated herein by reference). Recent experimental evidence indicates that enhancement of metastatic capability of human cancer cells transplanted orthotopically is associated with differential expression of several metastasis-associated genes that have been implicated earlier in certain key features of the metastatic phenotype (Greene, G. F., Kitadai, Y., Pettaway, C. A., von Eschenbach, A. C., Bucana, C. D., Fidler, I. J. 1997. Correlation of metastasis-related gene expression with metastatic potential in human prostate carcinoma cells implanted in nude mice using an in situ messenger RNA hybridization technique. American J. Pathology, 150: 1571-1582, incorporated herein by reference). These data support the rationale for the methods of the present invention to identify gene expression profiles associated with the phenotypes of clinical tumor samples based on a combination of in vitro gene expression analysis in one or more cell lines having a phenotype of interest (e.g., metastatic potential, invasiveness, etc.) and gene expression analysis of clinical samples.  
      A similar rationale supports the use of the methods of the present invention to identify gene expression patterns correlated with specific differentiation pathways associated with defined cell types (e.g., liver, skin, bone, muscle, blood, etc.), although in this instance, the preferred relevant comparisons are the gene expression profiles of one or more stem cell lines with that of the terminally differentiated cell type. (See, e.g., Table 2, supra.) In a related method of the present invention, expression analysis may be carried out on one or more different cell types using sets of genes (i.e., gene clusters) previously identified in, e.g., a biological sample analysis experiment such as the described tumor classification methods, to identify concordantly regulated genes that can be used as tissue-specific markers, or to screen for agents that may affect cellular differentiation or other aspects of cellular phenotype. Phenotype association indices can be calculated for normally differentiated tissue samples by calculating a correlation coefficient for a particular normally differentiated tissue sample against, e.g., −fold expression changes or expression differences for a minimum segregation set identified in a cancer analysis, as described above. The −fold expression changes or expression differences for the normally differentiated tissue sample can be calculated with reference to average values of gene x expression across a collection of different normal tissue samples. Expression data derived from the large collections of normal human and mouse tissue samples are available as supplemental data reported by Su, A. I. et al. Large-scale analysis of the human and mouse transcriptomes. PNAS 99: 4465-4470, 2002, incorporated herein by reference, and are available from the publicly accessible website http://expression.gnf.org, incorporated herein by reference.  
      Three possible outcomes are observed. In the first, no correlation is observed between the minimum segregation set and the normal tissue sample expression data implying that the regulatory pathway represented by the transcript abundance rank order within the minimum segregation set is not active. In the second, a positive correlation is seen between the −fold expression changes or differences in the minimum segregation set and the normal tissue sample implying that the regulatory pathway represented by the transcript abundance rank order within the minimum segregation set is active. In this outcome, the minimum segregation set represents a cluster of genes involved in a differentiation program and/or regulatory pathway that operates in the normal tissue sample and in the tumor cell lines. In the third outcome, a negative correlation is seen between the −fold expression changes or differences in the minimum segregation set and the normal tissue sample implying that the alternative regulatory pathway to one represented by the transcript abundance rank order within the minimum segregation set is active. In this outcome, the minimum segregation set represents a cluster of genes co-regulated in a differentiation program and/or regulatory pathway that operates in the normal tissue samples but that has failed in the tumor cell lines. Because the expression rank order of the genes within the minimum segregation class was derived from a comparison of the fold expression changes in tumor cell lines versus normal epithelial cells of the organ of cancer origin, this scenario may serve as an indicator of an active tumor suppression pathway. Gene expression profiles of human normal prostate epithelial cells and prostate cancer cell lines in culture  
      To identify genes expression of which is consistently altered in human prostate cancer cell lines, we searched for genes whose differential expression is retained as cells diverge through mutation, genomic instability, and possibly epigenetic mechanisms during repeated cycles of in vivo prostate cancer growth and progression in nude mice. To model this behavior, cell lines established from LNCap- and PC3-derived human prostate carcinoma xenografts were studied. Parental LNCap and PC3 cell lines represent divergent clinically relevant prostate cancer progression variants. LNCap is a relatively less aggressive, androgen-dependent cell line with wild-type p53, and PC3 is an aggressive, p53 mutated (21), and androgen independent cell line. The five cell lines, LNCapLN3, LNCapPro5, PC3M, PC3MLN4, PC3 MPro4 (Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R., Killion, J. J. and Fidler, I. J. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res. 1996;2:1627-36, incorporated herein by reference) represent lineages that have been derived from xenografts passaged repeatedly in the mouse to model prostate cancer growth and metastatic progression (see Table 1 and accompanying legend). The number of successive in vivo progression and in vitro expansion cycles varied from 1 to 5 in different lineages (Table 1).  
      The model design was based on the following considerations. Genes regulated similarly in five lineages would be expected to biased towards those genes that are relatively insensitive to the individual genetic differences in the cell&#39;s in vitro regulatory program. Furthermore, genes that are sensitive to environmental perturbations may be a source of changes that are stress-induced or are handling artifacts. This consideration also is relevant for changes associated with surgically-derived samples isolated from patients. We chose the early response to serum starvation (two hours) as a convenient method to identify and remove genes that are sensitive to environmental perturbations. Following these criteria, we identified 214 transcripts that are differentially expressed in the same direction in all five prostate cancer cell lines, relative to normal prostate epithelium (NPE), regardless of the presence or absence of serum (vs. 292 observed using data from high serum alone). 43 of these genes were consistently up-regulated and 171 were consistently down-regulated at least two-fold in all five cancer cell lines relative to NPE.  
      Of the 78 genes excluded by this experimental condition, only the Id3 protein and two alternatively spliced transcripts from the Id1 gene showed a common differential response to serum withdrawal within all five PC3— and LNCap-derived cell lines. Id1 and Id3 gene products are dominant negative regulators of the HLH transcription factors (Lyden, D., Young, A. Z., Zagzag, D., Yan, W., Gerald, W., O&#39;Reilly, R., Bader, B. L., Hynes, R. O., Zhuang, Y., Manova, K., Benezra, R. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumor xenografts. Nature 1999;401:670-77, incorporated herein by reference). The remaining 75 genes were differentially regulated with respect to serum withdrawal in ways that depended on the cell type. This is consistent with the view that the serum withdrawal criterion removes genes that are sensitive to both external environmental variables and internal cell line-specific context.  
      Gene Expression Profiles of PC3-Derived Orthotopic Tumors  
      To test whether the altered gene expression pattern of 214 genes identified in vitro is maintained in vivo, the common set of differentially expressed genes identified in the five cell lines relative to NPE were compared with genes that were differentially expressed in orthotopic tumors induced in nude mice using donor tumors for the PC3 lineage.  
      We identified a concordant gene expression profile for two tumors each independently derived from the three cell lines PC3 parental, PC3M, and PC3MLN4.79% (170 of 214 genes) of the transcripts differentially expressed in five prostate cancer cell lines in vitro were also differentially regulated in the same direction in vivo in all six orthotopic tumors. This gene set is exhaustively authenticated in thirty separate comparisons, which should, theoretically, put their regulation in these systems beyond doubt. Nevertheless, a sample of twelve up- and two down-regulated genes was tested using Q-PCR on an ABI7900 using the vendor&#39;s recommended protocols available at http://www.appliedbiosystems.con/support/tutorials/ (incorporated herein by reference). This PCR experiment used a further new batch of RNA from normal human prostate epithelial cell line and PC3M cells and human transcript-specific pairs of PCR primers. For several genes two separate sets of primers were designed and tested. Regulation was confirmed in the correct direction for these 14 genes, although the arrays tended to underestimate the magnitude of the change.  
      Therefore, the differential expression pattern of many of the prostate cancer-associated transcripts of PC3/LNCap consensus class identified in vitro using cell line concordance and media shift refractivity is retained in vivo in orthotopic human prostate tumors in mice. In the context of present invention, these data suggest that human prostate carcinoma xenografts may serve as a useful source of samples for identification of the reference standard data sets.  
      In Vivo Versus In Vitro Selection of Human Prostate Cancer-Associated Genes  
      To determine whether the consensus set of 214 differentially expressed genes identified here is retained in the parental cell lines, the PC3 and LNCaP cell lines that have not been serially passaged through mice were examined by microarray analysis, both in high and low serum. When concordance analysis was performed comparing the consensus list of 214 genes and genes that were differentially regulated relative to NPE in parental PC3 and LNCap cell lines, the majority of the down-regulated transcripts (133 genes; 78%) were similarly down-regulated in all 7 cell lines. However, only a small fraction (10 genes; 23%) of up-regulated transcripts was similarly differentially regulated in both parental cell lines. Thus, when compared with the five tumor-derived cell lines, PC3 and LNCaP parental cell lines have substantially smaller similarity with respect to the up-regulated transcripts, indicating that the transcripts with increased mRNA abundance levels in a set of 214 genes do not reflect in vitro selection. The significant degree of conservation of the consensus set of 214 genes in both xenograft-derived and plastic-maintained series of cancer cell lines supports the notion that plastic maintained cancer cell lines may serve as a useful source of samples for identification of the reference standard data sets.  
      Comparison with Clinical Human Prostate Tumors  
      While the genes described here are of undoubted interest as their expression is consistently altered in the multiple mouse model systems of human prostate cancer, it is not possible to say, as yet, whether they are of relevance to human disease. However, the expression levels of the genes in our stable set were analyzed published data from a group of clinical samples (Welsh, J. B., Sapinoso, L. M., Su, A. I., Kern, S. G., Wang-Rodriguez, J., Moskaluk, C. A., Frierson, H. F., Jr., Hampton, G. M. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res., 61: 5974-5978, 2001, (supplemental data obtained from http://www.gnf.org/cancer/prostate), incorporated herein by reference).  
      These data must be treated with caution because the human clinical samples are highly heterogeneous, consisting of different amounts of cells of epithelial, stromal, and other origins. Nevertheless, of the genes that could be cross-referenced, 31 out of 41 up-regulated genes (76%) were more highly expressed in the majority of 24 human tumors than in a normal epithelial cell line. 32 of these genes were more highly expressed in the majority of tumors than the average expression found in nine adjacent normal prostate tissue samples. Similarly, 141 of 166 down-regulated genes (88%) were down regulated in tumors relative to normal epithelial cells, and 122 were down-regulated in tumors relative to adjacent normal prostate tissue. The similarity in the altered regulation of many of these genes in clinical tumors is an indication that these genes are relevant to the human disease.  
      Materials and Methods  
      Cell culture. Cell lines used in this study are described in Table 1. The PC3— and LNCap-derived cell lines were developed by consecutive serial orthotopic implantation, either from metastases to the lymph node (for the LN series), or reimplanted from the prostate (Pro series). This procedure generated cell variants with differing tumorigenicity, frequency and latency of regional lymph node metastasis (Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R., Killion, J. J. and Fidler, I. J. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res. 1996;2:1627-36, incorporated herein by reference). The LNCaP and PC-3 panels of human prostate carcinoma cell lines of graded metastatic potential were provided by Dr. C. Pettaway (M. D. Anderson Cancer Center, Houston, Tex.) and described earlier (Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R., Killion, J. J. and Fidler, I. J. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res. 1996;2:1627-36, incorporated herein by reference). A third progression model is represented by the P69 cell line, an SV40 large T-antigen-immortalized prostate epithelial line, and M12, a metastatic derivative of P69 (Bae, V. L., Jackson-Cook, C. K., Brothman, A. R., Maygarden, S. J., and Ware, J. Tumorugenicity of SV40 T antigen immortalized human prostate epithelial cells: association with decreased epidermal growth factor receptor (EGFR) expression. Int. J. Cancer 1994;58:721-29; Jackson-Cook, C., Bae, V., Edelman W., Brothman, A., and Ware, J. Cytogenetic characterization of the human prostate cancer cell line P69SV40T and its novel tumorigenic sublines M2182 and M15. Cancer Genet. &amp; Cytogenet 1996;87:14-23; Bae, V. L., Jackson-Cook, C. K., Maygarden, S. J., Plymate, S. R., Chen, J., and Ware, J. L. Metastatic subline of an SV40 large T antigen immortalized human prostate epithelial cell line. Prostate 1998;34:275-82, incorporated herein by reference). The P69 cell line and M12 cell line were obtained from Dr. S. Plymate and Dr. J. Ware. Two primary human prostate epithelial and one primary human prostate stromal cell line were obtained from Clonetics/BioWhittaker (San Diego, Calif.) and grown in complete prostate epithelial and stromal growth medium provided by the supplier. Except where noted, other cell lines were grown in RPM11640 supplemented with 10% fetal bovine serum and gentamycin (Gibco BRL) to 70-80% confluence and subjected to serum starvation as described (14-16), or maintained in fresh complete media, supplemented with 10% FBS.  
      RNA extraction. For gene expression analysis, cells were harvested in lysis buffer 2 hrs after the last media change at 70-80% confluence and total RNA or mRNA was extracted using the RNeasy (Qiagen, Chatsworth, Calif.) or FastTract kits (Invitrogen, Carlsbad, Calif.). Cell lines were not split more than 5 times, except where noted.  
      Orthotopic xenografts. Orthotopic xenografts of human prostate PC3 cells and sublines (Table 1) were developed by surgical orthotopic implantation as previously described (An, Z., Wang, X., Geller, J., Moossa, A. R., Hoffman, R. M. Surgical orthotopic implantation allows high lung and lymph node metastatic expression of human prostate carcinoma cell line PC-3 in nude mice. Prostate 1998;34:169-74, incorporated herein by reference). Briefly, 2×10 6  cultured PC3 cells, PC3M cells, or PC3M sublines were injected subcutaneously into male athymic mice, and allowed to develop into firm palpable and visible tumors over the course of 2-4 weeks. Intact tissue was harvested from a single subcutaneous tumor and surgically implanted in the ventral lateral lobes of the prostate gland in a series of six athymic mice per cell line subtype. The mice were examined periodically for suprapubic masses, which appeared for all subline cell types, in the order PC3MLN4&gt;PC3M&gt;&gt;PC3. Tumor-bearing mice were sacrificed by CO 2  inhalation over dry ice and necropsy was carried out in a 2-4° C. cold room. Typically, bilaterally symmetric prostate gland tumors in the shape of greatly distended prostate glands were apparent. Prostate tumor tissue was excised and snap frozen in liquid nitrogen. The elapsed time from sacrifice to snap freezing was &lt;20 min. A systematic gross and microscopic post mortem examination was carried out.  
      Tissue processing for mRNA isolation. Fresh frozen orthotopic tumor was examined by use of hematoxylin and eosin stained frozen sections. Orthotopic tumors of all sublines exhibited similar morphology consisting of sheets of monotonous closely packed tumor cells with little evidence of differentiation interrupted by only occasional zones of largely stromal components, vascular lakes, or lymphocytic infiltrates. Fragments of tumor judged free of these non-epithelial clusters were used for mRNA preparation. Frozen tissue (1-3 mm×1-3 mm) was submerged in liquid nitrogen in a ceramic mortar and ground to powder. The frozen tissue powder was dissolved and immediately processed for mRNA isolation using a Fast Tract kit for mRNA extraction (Invitrogen, Carlsbad, Calif., see above) according to the manufacturers instructions.  
      Affymetrix arrays. The protocol for mRNA quality control and gene expression analysis was that recommended by the array manufacturer, Affymetrix, Inc. (Santa Clara, Calif. http://www.affymetrix.com). In brief, approximately one microgram of mRNA was reverse transcribed with an oligo(dT) primer that has a T7 RNA polymerase promoter at the 5′ end. Second strand synthesis was followed by cRNA production incorporating a biotinylated base. Hybridization to Affymetrix Hu6800 arrays representing 7,129 transcripts or Affymetrix U95Av2 array representing 12,626 transcripts overnight for 16 h was followed by washing and labeling using a fluorescently labeled antibody. The arrays were read and data processed using Affymetrix equipment and software (Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H. and Brown, E. L. Expression monitoring by hybridization to high-density oligonucleotide arrays [see comments]. Nat. Biotechnol. 1996;14:1675-80, incorporated herein by reference). Detailed protocols for data analysis and documentation of the sensitivity, reproducibility and other aspects of the quantitative microarray analysis using Affymetrix technology have been reported (Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H. and Brown, E. L. Expression monitoring by hybridization to high-density oligonucleotide arrays [see comments]. Nat. Biotechnol. 1996;14: 1675-80, incorporated herein by reference).  
      To determine the quantitative difference in the mRNA abundance levels between two samples, in each individual sample for each gene the average expression differences were calculated from intensity measurements of perfect match (PM) probes minus corresponding control probes representing a single nucleotide mismatch (MM) oligonucleotides for each gene-specific set of 20 PM/MM pairs of oligonucleotides, after discarding the maximum, the minimum, and any outliers beyond 3 standard deviations (SD) from the average. The averages of pairwise comparisons for each individual gene were made between the samples, and the corresponding expression difference calls (see below) were made with Affymetrix software. Microsoft Access was used for other aspects of data management and storage. For each gene, a matrix-based decision concerning the difference in the mRNA abundance level between two samples was made by the software and reported as a “Difference call” (No change (NC), Increase (1), Decrease (D), Marginal increase (MI), and Marginal decrease (MD)) and the corresponding fold change ratio was calculated. 40-50% of the surveyed genes were called present by the Affymetrix software in these experiments. The concordance analysis of differential gene expression across the data set was performed using Microsoft Access and Affymetrix MicroDB software. For experiments involving study of prostate cancer, three of the normal prostate epithelial (NPE) microarrays are used as controls, and referred to as the NPE expression profile. Thus, when a gene is required to show a 2-fold or greater change relative to NPE, this must occur in all three microarrays, for either positive or negative changes. These stringent criteria exclude genes for which one of the three microarrays is in error. The strategy in this study is based on the idea that expression differences will not be called by chance in the same direction in multiple arrays (see below for statistical justification). Each gene in the final list of the 214 differentially expressed genes was required to be called exclusively as either concordantly up- or down-regulated in 30 separate comparisons (5 prostate cancer cell lines×2 experimental serum conditions×3 NPE controls) or 15 separate comparisons (5 prostate cancer cell lines×1 experimental serum condition×3 NPE controls).  
      Statistical analysis and quality performance criteria. We used a stringent analytical approach to test the hypothesis that there are common genes with altered mRNA abundance levels whish appear to be significantly associated with the studied phenotypes. The Affymetrix MicroDB and Affymetrix DMT software was used to identify in any given comparison of two chips only genes that are determined to be expressed at statistically significantly different (p&lt;0.05) levels. These transcripts are called as differentially expressed. To be included in our final differentially regulated gene class the given transcript was required to be determined as differentially regulated in the same direction (up or down) at the statistically significant levels (p&lt;0.05) e.g., in 30 independent comparisons (5 experimental cell lines×2 experimental conditions×3 control cell lines). To be recognized as differentially regulated in the orthotopic tumors any given gene of the PC3/LNCap consensus class was required to be determined differentially regulated in the same direction at the statistically significant level (p&lt;0.05) in 18 additional independent comparisons (6 orthotopic tumors×3 controls). Despite that identified set of 214 genes is differentially expressed in described experimental systems with the extremely high level of confidence, we carried out Q-PCR confirmation analysis for a sub-set of identified genes and confirmed their differential expression in all instances using an additional independent normal human prostate epithelial cell line as a control.  
      Quality performance criteria adopted for the Affymetrix GeneChip system and applied in this study. 40-50% of the surveyed genes were called present by the Affymetrix software in these experiments. This is at the high end of the required standard adopted in many peer-reviewed publications using the same experimental system. Transcripts that are called present by the Affymetrix software in any given experiment were determined to have the signal intensities higher in the perfect match probe sets compared to single-nucleotide mismatch probe sets and background at the statistically significant level. This analysis was performed for each individual transcript using unique set of 20 perfect matches versus 20 single nucleotide mismatch probes. In our final list of 214 genes all transcripts were called present in at least one experimental setting. The inclusion error associated with two mRNA samples from identical cell lines was 2.7% for a difference called by the Affymetrix software. Thus, two independently obtained mRNA from the same cell lines will have 2.7% false positives. When a third independently derived epithelial cell line was included, only 4 genes (0.06%) out of 7,129 were called differentially expressed. The expression profiles of the normal prostate epithelial cell lines used in our experiments were determined to be indistinguishable. Therefore, controls are not likely source of errors in gene expression analysis performed in this study. This is particularly important, since the strategy adopted in this study is based on the idea that expression differences will not be called statistically significant by chance in the same direction in multiple arrays and during multiple independent comparisons of different phenotypes and variable experimental conditions. To impose additional stringent restrictions on possibility of a gene to be detected as concordantly differentially regulated by chance, we apply the use of multiple experimental models and vastly variable experimental settings such as in vitro and in vivo growth and varying growth conditions. Similar strategy for identification of consistent gene expression changes based on a concordant behavior of the differentially regulated genes using Affymetrix GeneChip system and software was applied and validated in several peer-reviewed published papers (see for example, Lee C K, Klopp, R G, Weindruch, R, Prolla, T A. Gene expression profile of aging and its retardation by caloric restriction. Science 1999; 285: 1390-1393; Ishida, S, Huang, E, Zuzan, H, Spang, R, Leone, G, West, M, Nevins, JR. Role for E2F in control of both DNA replication and mitotic function as revealed from DNA microarray analysis. Mol Cell Biol 2001; 21: 4684-4699, incorporated herein by reference). We applied more stringent criteria in our study requiring a concordance in at least 30 of 30 experiments compared to 6 of 6 comparisons in (Lee C K, Klopp, R G, Weindruch, R, Prolla, T A. Gene expression profile of aging and its retardation by caloric restriction. Science 1999; 285: 1390-1393, incorporated herein by reference); and 4 of 6 comparisons in (Ishida, S, Huang, E, Zuzan, H, Spang, R, Leone, G, West, M, Nevins, JR. Role for E2F in control of both DNA replication and mitotic function as revealed from DNA microarray analysis. Mol Cell Biol 2001; 21: 4684-4699, incorporated herein by reference). Ishida, et al. (Ishida, S, Huang, E, Zuzan, H, Spang, R, Leone, G, West, M, Nevins, JR. Role for E2F in control of both DNA replication and mitotic function as revealed from DNA microarray analysis. Mol Cell Biol 2001; 21: 4684-4699, incorporated herein by reference) provided a formal statistical justification that four or more concordant calls out of six comparisons cannot be explained by chance, with the probability in the range of 1 in 10 4 .  
      Q-PCR confirmation analysis of the differentially regulated genes. To confirm differential regulation of the transcripts comprising a PC3/LNCap-consensus class using an independent method a sample of 14 genes (12 up-regulated and 2 down-regulated) was tested using Q-PCR on an ABI7900 according to the vendor&#39;s recommended protocols (available at http://www.appliedbiosystems.com/support/tutorials/). This PCR experiment used a further new batch of RNA from a third normal human prostate epithelial cell line and human transcript-specific pairs of PCR primers.  
     EXAMPLE 1  
     Classification of Human Prostate Tumors  
      A. General  
      A first reference set for human prostate tumors was obtained by obtaining gene expression data from five prostate cancer cell lines (cell lines used were LNCapLN3; LNCapPro5; PC3M; PC3MLN4; PC3 Mpro4; see Table 1) and two different normal human prostate epithelial cell lines were obtained from Clonetics/BioWhittaker (San Diego, Calif.) and grown in complete prostate epithelial growth medium provided by the supplier. An original and a replicate data set was obtained for the first normal cell line, and the second cell line represented an independent data set from an independent epithelial cell line. Each of the tumor cell lines was derived from aggressively metastatic human prostate tumors. Consequently, we expected that these tumor cell lines should have an “invasive” phenotype because had they not been “invasive,” they would not have penetrated the prostate capsule, a step pre-requisite to metastasis.  
      The expression data were obtained using an Affymetrix Human Genome-U95Av2 (“HG-U95Av2”) expression array chip (Affymetrix, Santa Clara, Calif.). The HG-U95Av2 Array represents approximately 10,000 full-length genes. Data were obtained from the HG-U95Av2 according to the manufacturer&#39;s suggested protocols, as outlined in the Materials &amp; Methods Section above  
      The original data set thus comprised a total of eight separate sets of gene expression data, five from the set of tumor cell lines and three from the set of epithelial cell lines. Fifteen separate pairwise comparisons were carried out to identify a first reference set of genes that were differentially expressed in the tumor cell lines and the epithelial cell lines. Differential expression was determined using Affymetrix&#39;s Microarray Suite software (versions 4.0 and 5.0). To be included in the first reference set, a candidate gene needed to meet two criteria: 1) the candidate gene was shown to be differentially expressed in each of the 15 pairwise comparisons; and 2) the direction of the differential (i.e. greater expression in the tumor cell lines cf. the epithelial cell lines or vice-versa) was consistent in each of the 15 pairwise comparisons. The first reference set comprised of 629 genes.  
      B. Recurrence Predictor Cluster and Sample Classification  
      The methods of the invention were used to identify gene clusters associated with increased likelihood of tumor recurrence. A second reference set was obtained using expression data obtained from clinical human prostate tumor samples. These data were the supplemental data reported in Singh, D., Febbo, P. G., et al., “Gene Expression Correlates of Clinical Prostate Cancer Behavior,” Cancer Cell March 20021:203-209, incorporated herein by reference. The clinical human prostate tumor samples were divided into two groups, recurrent and non-recurrent, as reported in Singh, et al. (2002). Data from twenty-one patients were evaluable with respect to recurrence following surgery. Recurrence was defined as two successive PSA values&gt;0.2 ng/ml. Of the twenty-one patients, eight had recurrences, and thirteen patients remained relapse-free for at least four years.  
      Affymetrix MicroDB (version 3.0) and Affymetrix Data Mining Tools (DMT) (version 3.0) data analysis software were used to identify genes that were differentially regulated in recurrence group compared to relapse-free group of patients at the statistically significant level (p&lt;0.05; Student T-test). Candidate genes were included in the second reference set if they were identified by the DMT software as having p values of 0.05 or less both for up-regulated and down-regulated genes. 316 genes were identified as being members of the second reference set.  
      A concordance set of genes was identified from the first and second reference sets. Genes were included in the concordance set if they met the following criteria: 1) the gene was identified as a member of both the first and the second reference sets; and 2) the direction of the differential was consistent in the first and the second reference sets (i.e., the gene transcript was more abundant in the tumor cell lines cf. the control cell lines and more abundant in the recurrent cf. the non-recurrent samples, or the gene transcript was less abundant in the tumor cell lines cf. the control cell lines and less abundant in the recurrent cf. the non-recurrent samples). The first criterion provides a way of minimizing the number of genes for which the pairwise comparisons are carried out for the sample data. Only those genes that are members of the first reference set need to be compared for generating the second reference set because the first criterion requires that the candidate gene be a member of both the first and second reference sets. The concordance set comprises of 19 genes.  
      The minimum segregation set was obtained as follows. For each gene in the concordance set, the −fold expression changes (as determined by the ratio of the relative transcript abundance levels) was determined. This was done for the cell line data by computing for each gene in the concordance set the ratio of the average expression in the tumor cell lines to the average expression in the control cell lines, and similarly the ratio of the average expression in the samples obtained from patients who relapsed (recurrent population) from those who did not relapse (non-recurrent population). Using the notation described above, this corresponds to calculating &lt;expression&gt; 1 /&lt;expression&gt; 2  for the cell line and clinical samples data. For the cell line data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all tumor cell lines and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all control cell lines. For the clinical sample data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who relapsed and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who did not relapse.  
      The −fold expression change data were log 10  transformed and the transformed data were entered as two arrays in a Microsoft Excel spreadsheet. The Excel CORREL function was used to generate a correlation coefficient that characterizes the degree to which the concordance set −fold expression changes were correlated between the cell line and clinical sample data. Typically, we observe correlation coefficients at this stage of the analysis in the range of about 0.7 to about 0.9. A scatter plot showing the relationship between the log-transformed −fold expression changes in the cell line and clinical sample data is shown in  FIG. 1 . In the scatter plot, each point represents an individual gene belonging to the concordance set. The correlation coefficient for this concordance set was 0.777.  
      A minimum segregation set was selected from the concordance set. This set was chosen by looking at the scatter plot ( FIG. 1 ) and manually selecting sub-sets of genes within the concordance set whose representative points fell closest to an imaginary regression line drawn through the data. Of course, this procedure can be automated. A second correlation coefficient was calculated using the Microsoft Excel CORREL function for several sub-sets of genes within the concordance set to arrive at a highly-correlated sub-set. These genes are members of the minimum segregation set, and represent genes whose −fold expression changes are most highly correlated between the cell line and clinical sample data. Typically, we identified minimum segregation sets that comprised on the order of from about 3 to about 20 genes and that produced correlation coefficients on the order of ≧0.98.  
      Using this method, a total of nine genes was selected for the recurrence predictor minimum segregation set. This recurrence predictor minimum segregation set had a correlation coefficient of 0.995 for the cell line and sample −fold expression change differences. See  FIG. 2 . Members of this recurrence predictor minimum segregation set are shown in Table 5.  
               TABLE 5                          Prostate Tumor Recurrence Predictor Minimum Segregation Set.                                 Affymetrix   LocusLink               Probe Set ID   Identifier 1     Description 2                         41435_at   8541   PPFIA3: protein tyrosine                   phosphatase, receptor type,                   f polypeptide (PTPRF),                   interacting protein (liprin),                   alpha 3           33228_g_at   3588   IL10RB: interleukin 10                   receptor, beta           40522_at   2752   GLUL: glutamate-                   ammonia ligase (glutamine                   synthase)           37026_at   1316   COPEB: core promoter                   element binding protein           33436_at   6662   SOX9: SRY (sex                   determining region Y)-box                   9 (campomelic dysplasia,                   autosomal sex-reversal)           39631_at   2013   EMP2: epithelial                   membrane protein 2           1915_s_at   2353   FOS: v-fos FBJ murine                   osteosarcoma viral                   oncogene homolog           37286_at   3726   JUNB: jun B proto-                   oncogene           40448_at   7538   ZFP36: zinc finger protein                   36, C3H type, homolog                   (mouse)                           1 LocusLink provides a single query interface to curated sequence and descriptive information about genetic loci. It presents information on official nomenclature, aliases, sequence accessions, phenotypes, EC numbers, MIM numbers, UniGene clusters, homology, map locations, and related web sites. It may be accessed through the National Center for Biotechnology Information (NCBI) website at http://www.ncbi.nlm.nih.gov/LocusLink/.                  2 The first entry in each cell of this column corresponds to the HUGO Gene Nomenclature Committee (“HGNC”) Approved Symbol for the gene corresponding to the Affymetrix Probe Set and LocusLink Identifiers within the same row. Information for the subject gene, associated cDNA, mRNA, and protein sequences may be obtained using the LocusLink identifier or the HGNC Approved Symbol by querying the search page at http://www.ncbi.nlm.nih.gov/LocusLink.                Note, the footnotes associated with Table 5 apply to every table in this specification that follows the same or similar format as Table 3 (i.e., column 1 contains information on the Affymetrix Probe Set ID, column 2 contains the LocusLink Identifier, and column 3 contains the gene description.             
 
      The recurrence predictor minimum segregation set was used to calculate a phenotype association indices for each of the twenty-one tumors removed from the patients described in Singh, et al. (2002) that were evaluated for recurrence. The phenotype association index was obtained by calculating for each individual tumor sample, the −fold expression change for each of the nine genes in the recurrence predictor minimum segregation set. The −fold expression change was calculated as: 
 
expression/&lt;expression 1 +expression 2 &gt;
 
 where “expression” is the observed expression level for gene x for the individual tumor, and “&lt;expression 1 +expression 2 &gt;” is the average gene expression level for gene x across the set of 21 tumors used to generate the recurrence predictor minimum segregation set. The −fold expression changes for these nine genes were log 10  transformed, the transformed data entered as an array in a Microsoft Excel spreadsheet, and the Excel CORREL function was used to generate a correlation coefficient between the individual tumor data array and the corresponding log 10  transformed data for the average −fold expression changes in the cell lines for the same nine genes (i.e., log 10 (&lt;expression&gt; 1 /&lt;expression&gt; 2 ). This second correlation coefficient is the phenotype association index. The phenotype association index has the surprising and unexpected property of allowing the samples to be classified according to the sign of the index.  FIG. 3  shows the phenotype association index for each of the twenty-one tumors classified using the recurrence predictor minimum segregation class described above. 7 out of 8 tumors associated with recurrences had positive association indices, while 11 out of 13 tumors associated with no recurrence had negative association indices. Thus, the method correctly classified 18/21 or 86% of the tumors. 
 
      B-1. Prostate Cancer Predictor Clusters and Sample Classification  
      The methods of the invention were used to identify gene clusters associated with the presence of prostate carcinoma cells in a tissue sample compared to the adjacent normal tissue samples that were determined to be cancer cell free. The first reference data set was derived as described above in A. A second reference set was obtained using expression data obtained from clinical human prostate tumor samples. These data were two independent sets of the supplemental data reported in Welsh, J. B., et al., “Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer,” Cancer Research, 2001, 61: 5974-5978; and Singh, D., Febbo, P. G., et al., “Gene Expression Correlates of Clinical Prostate Cancer Behavior,” Cancer Cell March 2002 1:203-209, incorporated herein by reference. The clinical human prostate tumor samples were divided into two groups, cancer samples and adjacent normal tissue samples, as reported in Welsh, et al. (2001). Data from twenty-five cancer samples (analysis of one tumor samples was carried out in duplicate) and nine adjacent normal tissue samples were used to identify the concordance gene set with high correlation coefficient and significant sample segregation power thus comprising genes with the properties of the minimum segregation class.  
      Genes were included in the concordance set if the direction of the differential was consistent in the first reference set and in the clinical samples (i.e., the gene transcript was more abundant in the tumor cell lines cf. the control cell lines and more abundant in the cancer samples cf. the adjacent normal tissue (ANT) samples, or the gene transcript was less abundant in the tumor cell lines cf. the control cell lines and less abundant in the cancer samples cf. the ANT samples. The concordance set comprising 54 genes was identified with correlation coefficient 0.823. Members of this concordance set are shown in Table 6. When applied to individual clinical samples, this gene set yielded sample segregation power of 91%. 30 of 33 clinical samples were classified correctly; 9 of 9 ANT samples displayed negative phenotype association indices while 21 of 24 cancer samples had positive phenotype association indices ( FIG. 4 ).  
               TABLE 6                          54 genes of the prostate cancer/normal tissue concordant set.                                     Affymetrix                   Affymetrix Probe   Probe Set ID   UniGene   LocusLink       Set ID (HuFL6800)   (U95Av2)   Identifier   Identifier   Description               U03735_f_at   34575_f_at   Hs.36978   MAGEA3   MAGE-3 antigen                       (MAGE-3) gene       L77701_at   40427_at   Hs.16297   COX17   COX17 mRNA       X70940_s_at   35175_f_at   Hs.2642   EEF1A2   mRNA for elongation                       factor 1 alpha-2       U33053_at   175_s_at   Hs.2499   PRKCL1   lipid-activated protein                       kinase PRK1 mRNA       L18920_f_at   34575_f_at   Hs.36980   MAGEA2   MAGE-2 gene exons 1-4       M77140_at   35879_at   Hs.1907   GAL   pro-galanin mRNA       X92896_at   40891_f_at   Hs.18212   DXS9879E   mRNA for ITBA2                       protein       L18877_f_at   34575_f_at   Hs.169246   MAGEA12   MAGE-12 protein gene       M77481_rna1_f_at   36302_f_at   Hs.72879   MAGEA12   antigen (MAGE-1)                       gene       U77413_at   38614_s_at   Hs.100293   OGT   O-linked GlcNAc                       transferase mRNA       U73514_at   40778_at   Hs.171280   HADH2   short-chain alcohol                       dehydrogenase                       (XH98G2) mRNA       U39840_at   37141_at   Hs.299867   HNF3A   hepatocyte nuclear                       factor-3 alpha (HNF-3                       alpha) mRNA       L41559_at   34352_at   Hs.3192   PCBD   pterin-4a-                       carbinolamine                       dehydratase (PCBD)                       mRNA       U90907_at   37961_at   Hs.88051   PIK3R3   clone 23907 mRNA                       sequence       D00860_at   36489_at   Hs.56   PRPS1   mRNA for                       phosphoribosyl                       pyrophosphate                       synthetase (EC 2.7.6.1)                       subunit I       U81599_at   40327_at   Hs.66731   HOXB13   homeodomain protein                       HOXB13 mRNA       M80254_at   40840_at   Hs.173125   PPIF   cyclophilin isoform                       (hCyP3) mRNA       HG1612-HT1612_at   36174_at   Hs.75061   MACMARCKS   Macmarcks       D85131_s_at   1764_s_at   Hs.7647   MAZ   mRNA for Myc-                       associated zinc-finger                       protein ofislet       U79274_at   31838_at   Hs.150555   HSU79274   clone 23733 mRNA       Z22548_at   39729_at   Hs.146354   PRDX2   thiol-specific                       antioxidant protein                       mRNA       HG4312-   36188_at   Hs.75113   GTF3A   Transcription Factor       HT4582_s_at               IIIa       J04444_at   1160_at   Hs.289271   CYC1   cytochrome c-1 gene       X79865_at   39812_at   Hs.109059   MRPL12   Mrp17 mRNA       U37022_rna1_at   1942_s_at   Hs.95577   CDK4   cyclin-dependent                       kinase 4 (CDK4) gene       U07424_at   34291_at   Hs.23111   FARSL   putative tRNA                       synthetase-like protein                       mRNA       U79287_at   40955_at   Hs.19555   PTOV1   clone 23867 mRNA                       sequence       M34338_s_at   241_g_at   Hs.76244   SRM   spermidine synthase                       mRNA       L37936_at   39659_at   Hs.340959   TSFM   nuclear-encoded                       mitochondrial                       elongation factor Ts                       (EF-Ts) mRNA       X07979_at   32808_at   Hs.287797   ITGB1   mRNA for fibronectin                       receptor beta subunit       X54232_at   33929_at   Hs.2699   GPC1   mRNA for heparan                       sulfate proteaglycan                       (glypican)       M55210_at   232_at   Hs.214982   LAMC1   laminin B2 chain                       (LAMB2) gene       S74017_at   853_at   Hs.155396   NFE2L2   Nrf2 = NF-E2-like basic                       leucine zipper                       transcriptional activator                       [human       U90913_at   39416_at   Hs.12956   TIP-1   clone 23665 mRNA                       sequence       X52425_at   404_at   Hs.75545   IL4R   IL-4-R mRNA for the                       interleukin 4 receptor       U90878_at   36937_s_at   Hs.75807   PDLIM1   LIM domain protein                       CLP-36 mRNA       X86163_at   39310_at   Hs.250882   BDKRB2   mRNA for B2-                       bradykinin receptor       U73377_at   38118_at   Hs.81972   SHC1   p66shc (SHC) mRNA       Z29083_at   368_at   Hs.82128   TPBG   5T4 gene for 5T4                       Oncofetal antigen       M31013_at   39738_at   Hs.146550   MYH9   nonmuscle myosin                       heavy chain (NMHC)                       mRNA       M77349_at   1385_at   Hs.118787   TGFBI   transforming growth                       factor-beta induced                       gene product (BIGH3)                       mRNA       U04636_rna1_at   1069_at   Hs.196384   PTGS2   cyclooxygenase-2                       (hCox-2) gene       X15414_at   36589_at   Hs.75313   AKR1B1   mRNA for aldose                       reductase(EC 1.1.1.2)       M65292_s_at   32249_at   Hs.278568   HFL1   factor H homologue                       mRNA       X07438_s_at   38634_at   Hs.101850   RBP1   DNA for cellular                       retinol binding protein                       (CRBP) exons 3 and                       4 /gb = X07438 /ntype =                       DNA /annot = exon       X79882_at   38064_at   Hs.80680   MVP   lrp mRNA       M11433_at   38634_at   Hs.101850   RBP1   cellular retinol-binding                       protein mRNA       U60060_at   37743_at   Hs.79226   FEZ1   FEZ1 mRNA       X04412_at   32612_at   Hs.290070   GSN   mRNA for plasma                       gelsolin       X93510_at   32610_at   Hs.79691   RIL   mRNA for 37 kDa LIM                       domain protein       M12125_at   32313_at   Hs.300772   TPM2   fibroblast muscle-type                       tropomyosin mRNA       L13210_at   37754_at   Hs.79339   LGALS3BP   Mac-2 binding protein                       mRNA       M21186_at   35807_at   Hs.68877   CYBA   neutrophil cytochrome                       b light chain p22                       phagocyte b-                       cytochrome mRNA       L13720_at   1598_g_at   Hs.78501   GAS6   growth-arrest-specific                       protein (gas) mRNA                  
 
      The minimum segregation set was obtained as follows. For each gene in the concordance set, the −fold expression changes (as determined by the ratio of the relative transcript abundance levels) was determined. This was done for the cell line data by computing for each gene in the concordance set the ratio of the average expression in the tumor cell lines to the average expression in the control cell lines, and similarly the ratio of the average expression values in the samples obtained from cancer samples (malignant population) from those from ANT samples (non-malignant population). Using the notation described above, this corresponds to calculating &lt;expression&gt; 1 /&lt;expression&gt; 2  for the cell line and clinical samples data. For the cell line data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all tumor cell lines and &lt;expression&gt;2 corresponds to the average expression value for gene x over all control cell lines. For the clinical sample data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all cancer samples and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all ANT samples.  
      The −fold expression change data were log 10  transformed and the transformed data were entered as two arrays in a Microsoft Excel spreadsheet. The Excel CORREL function was used to generate a correlation coefficient that characterizes the degree to which the concordance set −fold expression changes were correlated between the cell line and clinical sample data. Typically, we observe correlation coefficients at this stage of the analysis in the range of about 0.7 to about 0.9. A scatter plot showing the relationship between the log-transformed −fold expression changes in the cell line and clinical samples data for the 54 genes of a concordance set is shown in  FIG. 5 . In the scatter plot, each point represents an individual gene belonging to the concordance set. The correlation coefficient for this concordance set was 0.823.  
      A minimum segregation set was selected from the concordance set. This set was chosen by looking at the scatter plot ( FIG. 5 ) and manually selecting sub-sets of genes within the concordance set whose representative points fell closest to an imaginary regression line drawn through the data. Of course, this procedure can be automated. A second correlation coefficient was calculated using the Microsoft Excel CORREL function for several sub-sets of genes within the concordance set to arrive at a highly-correlated sub-set. These genes are members of the minimum segregation cluster, and represent genes whose −fold expression changes are most highly correlated between the cell line and clinical sample data. Typically, we identified minimum segregation clusters that comprised on the order of from about 3 to about 20 genes and that produced correlation coefficients on the order of ≧0.98.  
      Using this method, a total of ten genes were selected for the prostate cancer/normal tissue predictor minimum segregation set 1 (i.e. cluster 1) and a total of five genes was selected for the prostate cancer/normal tissue minimum segregation set 2 (i.e., cluster 2). These prostate cancer predictor minimum segregation clusters had a correlation coefficient of 0.995 (cluster 1) and 0.997 (cluster 2) for the cell line and sample −fold expression change differences. Members of these two prostate cancer minimum segregation clusters are shown in Table 7.  
               TABLE 7                          The genes comprising prostate cancer minimum segregation set 1 (cluster       1) (ten genes) and minimum segregation set 2 (cluster 2) (five genes).                             Affymetrix                   Probe Set   Affymetrix       ID   Probe Set ID       Short       (U95Av2)   (HuFL6800)   Description   Description                         10 genes (r = 0.995)                             1160_at   J04444_at   J04444 /FEATURE = cds /DEF-   cytochrome c-1               INITION = HUMCYC1A Human               cytochrome c-1 gene, complete cds       38614_s_at   U77413_at   Cluster Incl. U77413: Human O-linked   O-linked GlcNAc               GlcNAc transferase mRNA, complete   transferase               cds /cds = (265, 3027) /gb =               U77413 /gi = 2266993 /ug =               Hs.100293 /len = 3084       37141_at   U39840_at   Cluster Incl. U39840: Human hepatocyte   hepatocyte               nuclear factor-3 alpha (HNF-3 alpha)   nuclear factor-3               mRNA, complete cds /cds = (87,   alpha (HNF-3               1508) /gb = U39840 /gi = 1066121 /ug =   alpha)               Hs.105440 /len = 2872       34352_at   L41559_at   Cluster Incl. AA631698: np79a08.s1   dimerization                 Homo sapiens  cDNA /clone = IMAGE-   cofactor of               1132502 /gb = AA631698 /gi =   hepatocyte               2554309 /ug = Hs.3192 /len = 640   nuclear factor 1                   alpha (TCF1)       40327_at   U81599_at   Cluster Incl. U57052: Human Hoxb-13   homeodomain               mRNA, complete cds /cds = (54,   protein HOXB13               908) /gb = U57052 /gi = 1519039 /ug =               Hs.66731 /len = 1026       39729_at   Z22548_at   Cluster Incl. L19185: Human natural   peroxiredoxin 2               killer cell enhancing factor (NKEFB)               mRNA, complete cds /cds = (124,               720) /gb = L19185 /gi = 440307 /ug =               Hs.146354 /len = 980       34291_at   U07424_at   Cluster Incl. U07424: Human putative   phenylalanine-               tRNA synthetase-like protein mRNA,   tRNA synthetase-               complete cds /cds = (12, 1538) /gb =   like               U07424 /gi = 2098578 /ug =               Hs.23111 /len = 1807       36937_s_at   U90878_at   Cluster Incl. U90878:  Homo sapiens     carboxy terminal               carboxyl terminal LIM domain protein   LIM domain               (CLIM1) mRNA, complete cds /cds =   protein 1               (142, 1131) /gb = U90878 /gi =               2957144 /ug = Hs.75807 /len = 1480       38634_at   X07438_s_at   Cluster Incl. M11433: Human cellular   cellular retinol               retinol-binding protein mRNA,   binding protein               complete cds /cds = (125, 532) /gb =   (CRBP)               M11433 /gi = 190947 /ug =               Hs.101850 /len = 716       32313_at   M12125_at   Cluster Incl. M12125: Human fibroblast   tropomyosin 2               muscle-type tropomyosin mRNA,   (beta)               complete cds /cds = (118, 972) /gb =               M12125 /gi = 339951 /ug =               Hs.180266 /len = 1044                 5 genes (r = 0.998)                             36174_at   HG1612-   Cluster Incl. X70326:  H. sapiens     Macmarcks           HT1612_at   MacMarcks mRNA /cds = (13,               600) /gb = X70326 /gi = 38434 /ug =               Hs.75061 /len = 1334       39812_at   X79865_at   Cluster Incl. X79865:  H. sapiens  Mrp17   ribosomal protein,               mRNA /cds = (137, 733) /gb =   mitochondrial,               X79865 /gi = 1313961 /ug =   L12               Hs.109059 /len = 1008       39310_at   X86163_at   Cluster Incl. X86163:  H. sapiens  mRNA   bradykinin               for B2-bradykinin receptor, 3 /cds =   receptor B2               (0, 41) /gb = X86163 /gi =               1220163 /ug = Hs.239809 /len = 2582       38634_at   M11433_at   Cluster Incl. M11433: Human cellular   retinol-binding               retinol-binding protein mRNA,   protein 1, cellular               complete cds /cds = (125, 532) /gb =               M11433 /gi = 190947 /ug =               Hs.101850 /len = 716       37743_at   U60060_at   Cluster Incl. U60060: Human FEZ1   fasciculation and               mRNA, complete cds /cds = (99,   elongation protein               1277) /gb = U60060 /gi =   zeta 1 (zygin I)               1927201 /ug = Hs.79226 /len =               1619                  
 
      The prostate cancer/normal tissue minimum segregation clusters were used to calculate phenotype association indices for each of the thirty-three samples from the patients described in Welsh, et al. (2001). The phenotype association index was obtained by calculating for each individual clinical sample, the −fold expression change for each of the ten and five genes in the prostate cancer predictor minimum segregation set 1 and 2. The −fold expression change was calculated as: 
 
expression/&lt;expression 1 +expression 2 &gt;
          where “expression” is the observed expression level for gene x for the individual tumor, and “&lt;expression 1 +expression 2 &gt;” is the average gene expression level for gene x across the set of 33 samples used to generate the prostate cancer predictor minimum segregation sets. The −fold expression changes for these ten and five genes were log 10  transformed, the transformed data entered as an array in a Microsoft Excel spreadsheet, and the Excel CORREL function was used to generate a correlation coefficient between the individual tumor data array and the corresponding log 10  transformed data for the average −fold expression changes in the cell lines for the same ten and five genes (i.e., log 10 (&lt;expression&gt; 1 /&lt;expression&gt; 2 ). This second correlation coefficient is the phenotype association index. The phenotype association indices had the surprising and unexpected property of allowing the samples to be classified according to the sign of the index.  FIG. 6  and  FIG. 7  show the phenotype association index for each of the thirty-three samples classified using the prostate cancer/normal tissue minimum segregation sets described above. In both instances, using either cluster 1 (ten genes) or cluster 2 (five genes), 9 out of 9 ANT samples had negative association indices, while 21 out of 24 cancer samples had positive association indices. Thus, the method correctly classified 30/33 or 91% of the samples.        

      To test the performance of prostate cancer/normal tissue minimum segregation sets or clusters on independent data sets, we applied the method to classify 94 ANT and cancer samples described in Singh, D., Febbo, P. G., et al., “Gene Expression Correlates of Clinical Prostate Cancer Behavior,”  Cancer Cell  March 2002 1:203-209, incorporated herein by reference. This set of samples comprises of 47 cancer samples and 47 adjacent normal tissue samples obtained in each instances from the same patients. The phenotype association index was obtained by calculating for each individual clinical sample, the −fold expression change for each of the ten and five genes in the prostate cancer predictor minimum segregation set 1 and 2. The −fold expression change was calculated as: 
 
expression/&lt;expression 1 +expression 2 &gt;
          where “expression” is the observed expression level for gene x for the individual tumor, and “&lt;expression 1 +expression 2 &gt;” is the average gene expression level for gene x across the set of 94 samples. The −fold expression changes for these ten and five genes were log 10  transformed, the transformed data entered as an array in a Microsoft Excel spreadsheet, and the Excel CORREL function was used to generate a correlation coefficient between the individual tumor data array and the corresponding log 10  transformed data for the average −fold expression changes in the cell lines for the same ten and five genes (i.e., log 10 (&lt;expression&gt; 1 /&lt;expression&gt; 2 ).        

       FIG. 8  and  FIG. 9  show the phenotype association index for each of the ninety-four samples classified using the prostate cancer predictor minimum segregation clusters described above. Using cluster 1 (ten genes), 34 of 47 ANT samples had negative association indices, while 40 of 47 cancer samples had positive association indices. Thus, the method correctly classified 74/94 or 79% of the samples in independent data set. Using cluster 2 (five genes), 34 of 47 ANT samples had negative association indices, while 42 of 47 cancer samples had positive association indices. Thus, the method correctly classified 76/94 or 81% of the samples in an independent data set.  
      C. Invasion Clusters and Sample Classification  
      The methods of the invention were used along with the data reported by Singh, et al. (2002) to identify gene clusters associated with an invasive phenotype. Invasive phenotype was assessed by determining the presence or absence of positive surgical margins. The same first reference set described above in part A was used to generate the concordance and minimum segregation sets for invasiveness. The second reference set was obtained following the procedures described above in part B, using the supplemental data reported in Singh, et al. (2002) for fourteen invasive and 38 non-invasive human prostate tumors. Thus, the second reference set was obtained by using the Affymetrix MicroDB (version 3.0) and Affymetrix Data Mining Tools (DMT) (version 3.0) data analysis software to identify genes that were differentially regulated in invasion group compared to non-invasive group of patients at the statistically significant level (p&lt;0.05; Student T-test). Candidate genes were included in the second reference set if they were identified by the DMT software as having p values of 0.05 or less both for up-regulated and down-regulated genes. 3869 genes were identified as being members of the second reference set.  
      The concordance set was obtained by selecting only those genes having a consistent direction of the differential in both the first and the second reference sets (i.e., greater gene expression in the tumor lines cf. the control lines and greater gene expression in the invasive tumor samples cf. the non-invasive tumor samples or vice-versa). The concordance set comprised 104 genes with an overall correlation coefficient of 0.755 ( FIG. 10 ).  
      A minimum segregation set was selected following the procedures described above in section B. A scatter plot was generated of the log 10  transformed average −fold expression change in the cell line and average −fold expression change in the sample data. For the clinical sample data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who had invasive tumors and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who had non-invasive tumors. The overall correlation coefficient for the invasiveness concordance set was 0.755. The invasiveness concordance set is shown in  FIG. 10 .  
      A minimum segregation set was identified by selecting a subset of the highly correlated genes from the invasiveness concordance set. This minimum segregation set (invasion minimum segregation set 1 or invasion cluster 1) included 20 genes listed below in Table 8. The overall correlation coefficient between the cell lines and clinical samples for invasion cluster 1 was 0.980.  FIG. 11  shows the scatter plot for invasion cluster 1.  
               TABLE 8                          Prostate Cancer Invasion Minimum Segregation Set 1.                         Affymetrix               Probe Set   LocusLink       ID (U95Av2)   Identifier   Description                                 33904_at   1365   CLDN3: claudin 3       1842_at   2521   FUS: fusion, derived from               t(12; 16) malignant               liposarcoma       37741_at   5831   PYCR1: pyrroline-5-               carboxylate reductase 1       36174_at   65108   MACMARCKS:               macrophage myristoylated               alanine-rich C kinase               substrate       1287_at   142   ADPRT: ADP-               ribosyltransferase (NAD+;               poly (ADP-ribose)               polymerase)       39729_at   7001   PRDX2: peroxiredoxin 2       39020_at   10572   SIVA: CD27-binding               (Siva) protein       40074_at   10797   MTHFD2: methylene               tetrahydrofolate               dehydrogenase (NAD+               dependent),               methenyltetrahydrofolate               cyclohydrolase       502_s_at   2709   GJB5: gap junction               protein, beta 5 (connexin               31.1)       41817_g_at   355   TNFRSF6: tumor necrosis               factor receptor               superfamily, member 6       40847_at   3675   ITGA3: integrin, alpha 3               (antigen CD49C, alpha 3               subunit of VLA-3               receptor)       41641_at   578   BAK1: BCL2-               antagonist/killer 1       40031_at   8626   TP63: tumor protein p63       38608_at   5099   PCDH7: BH-               protocadherin (brain-heart)       38288_at   N/A [Genbank Accession   KRT6E: keratin 6E           No. L42611]       34853_at   2263   FGFR2: fibroblast growth               factor receptor 2 (bacteria-               expressed kinase,               keratinocyte growth factor               receptor, craniofacial               dysostosis 1, Crouzon               syndrome, Pfeiffer               syndrome, Jackson-Weiss               syndrome)       209_at   2263   FGFR2 fibroblast growth               factor receptor 2 (bacteria-               expressed kinase,               keratinocyte growth factor               receptor, craniofacial               dysostosis 1, Crouzon               syndrome, Pfeiffer               syndrome, Jackson-Weiss               syndrome) 10q26       32719_at   27350   APOBEC3C:               apolipoprotein B mRNA               editing enzyme, catalytic               polypeptide-like 3C       1898_at   3084   NRG1: neuregulin 1       115_at   2263   FGFR2: fibroblast growth               factor receptor 2 (bacteria-               expressed kinase,               keratinocyte growth factor               receptor, craniofacial               dysostosis 1, Crouzon               syndrome, Pfeiffer               syndrome, Jackson-Weiss               syndrome)                  
 
      Note that three entries in the table correspond to the same genes, i.e., 34853_at, 209_at, and 115_at. They most likely represent the splice variants of the same gene (Hs.31989). According to Affymetrix annotation, the 34853_at is an alternative splice 3 variant of the FGFR2.  
      Individual phenotype association indices were calculated for each of the 14 invasive and each of the 38 non-invasive human prostate tumors according to the methods described in section B, above, using data for the 20 genes that make up invasion cluster 1. The phenotype association index for each tumor sample was calculated using the average −fold expression change data for the tumor cell line data and the individual −fold expression change data for the tumor sample. The data were log 10  transformed and a correlation coefficient (phenotype association index) was calculated. The results are shown in  FIG. 12 . Application of the classification method using invasion cluster 1 resulted in 12/14 invasive tumors having positively signed association indices, and so were correctly classified, while 21/38 of the non-invasive tumors had negative association indices and so were correctly classified. Thus, invasion cluster 1 accurately classified 33/52=63% of the tumors in this sample set.  
      The greatest percentage of misclassifications obtained using invasion cluster 1 involved false positives, i.e., 17/38=44% of the non-invasive tumors were mis-classified as having an expression profile associated with the invasive phenotype. To improve the overall accuracy of the method, the sample set was re-structured so as to include data only from the twelve invasive tumors correctly classified using invasion cluster 1, and from the seventeen tumors mis-classified as false positives. (The false positives were considered to be non-invasive tumors (as, in fact they were) in carrying out the method steps to generate the second reference set, the concordance set, and the minimum segregation set.) Using this set of twenty-nine samples, another second reference set was generated by using the Affymetrix MicroDB (version 3.0) and Affymetrix Data Mining Tools (DMT) (version 3.0) data analysis software to identify genes that were differentially regulated in invasion group compared to non-invasive group of patients at the statistically significant level (p&lt;0.05; Student T-test). Candidate genes were included in the second reference set if they were identified by the DMT software as having p values of 0.05 or less both for up-regulated and down-regulated genes. 458 genes were identified as being members of the second reference set.  
      Once the second reference set was generated, it was used to generate a concordance set by applying the criterion that the direction of the differential was consistent in the cell line and the clinical sample data. That is, the concordance set included only those genes present in the first and second reference sets whose expression was always greater in the tumor cell line cf. the control cell line and always greater in the invasive tumor sample cf. the non-invasive tumor sample, or vice-versa. The concordance set comprised 23 genes (r=0.809).  
      Once the concordance set was obtained using the data from the 29-member set of clinical samples, average expression values for genes within the concordance set were generated for the tumor cell lines, the control cell lines, the invasive tumors, and the non-invasive tumors. Average −fold expression changes were obtained, log 10  transformed, and used to generate scatter plots and first correlation coefficients, as described above. A second minimum segregation set (invasion cluster 2) was identified by selecting a subset of genes from the concordance set whose −fold expression changes were highly correlated in the cell line and clinical samples. Invasion cluster 2 included 12 genes, and had an overall correlation coefficient of 0.983. See  FIG. 13 . The genes that were selected as invasion cluster 2 (invasion minimum segregation set 2) are listed in Table 9.  
               TABLE 9                          Prostate Cancer Invasion Minimum Segregation Set 2.       12 genes (r = 0.983)                     Affymetrix Probe           Set ID (U95Av2)   Description               1018_at   U81787 /FEATURE = /DEFINITION = HSU81787 Human Wnt10B           mRNA, complete cds       38336_at   Cluster Incl. AB023230:  Homo sapiens  mRNA for KIAA1013           protein, partial cds /cds = (0, 3188) /gb = AB023230 /gi = 4589675 /ug =           Hs.96427 /len = 4783       41619_at   Cluster Incl. AL022398: dJ434O14.4 (Interferon Regulatory Factor 6) /cds =           (68, 1471) /gb = AL022398 /gi = 3355547 /ug = Hs.11H801 /len =           4077       33369_at   Cluster Incl. AI535653: P9-C4.T3.P9.D4  Homo sapiens  cDNA, 3 end /clone_end =           3 /gb = AI535653 /gi = 4449788 /ug = Hs.223018 /len = 590       37978_at   Cluster Incl. D78177:  Homo sapiens  mRNA for quinolinate           phosphoribosyl transferase, complete cds /cds = (0, 893) /gb =           D78177 /gi = 1060906 /ug = Hs.8935 /len = 894       377_g_at   AB000220 /FEATURE = /DEFINITION = AB000220  Homo sapiens             mRNA for semaphorin E, complete cds       39411_at   Cluster Incl. AL080156:  Homo sapiens  mRNA; cDNA           DKFZp434J214 (from clone DKFZp434J214) /cds = (0, 1081) /gb =           AL080156 /gi = 5262614 /ug = Hs.12813 /len = 2697       38772_at   Cluster Incl. Y11307:  H. sapiens  CYR61 mRNA /cds = (223,           1368) /gb = Y11307/ gi = 2791897 /ug = Hs.8867 /len = 2052       39248_at   Cluster Incl. N74607: za55a01.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-296424 /clone_end = 3 /gb =N74607 /gi =           1231892 /ug = Hs.234642 /len = 487       41193_at   Cluster Incl. AB013382:  Homo sapiens  mRNA for DUSP6, complete           cds /cds = (351, 1496) /gb = AB013382 /gi = 3869139 /ug =           Hs.180383 /len = 2390       672_at   J03764 /FEATURE = cds /DEFINITION = HUMPAIA Human,           plasminogen activator inhibitor-1 gene, exons 2 to 9       39052_at   Cluster Incl. J00124:  Homo sapiens  50 kDa type I epidermal keratin           gene, complete cds /cds = (61, 1479) /gb = J00124 /gi = 186704 /ug =           Hs.117729 /len = 1634                  
 
      Individual phenotype association indices were calculated for each of the 12 invasive and each of the 17 non-invasive human prostate tumors used to generate invasion cluster 2 according to the methods described in section B, above, using data for the 12 genes that make up invasion cluster 2. The phenotype association index for each tumor sample was calculated using the average −fold expression change data for the tumor cell line data and the individual −fold expression change data for the tumor sample. The data were log 10  transformed and a correlation coefficient (phenotype association index) was calculated. The results are shown in  FIG. 14 . Application of the classification method using invasion cluster 2 resulted in 11/12 invasive tumors having positively signed association indices, and so were correctly classified, while 10/17 of the non-invasive tumors had negative association indices and so were correctly classified. There thus were 7 false positives identified using invasion cluster 2. Overall, invasion cluster 2 accurately classified 21/29=72% of the tumors in this sample set.  
      The method was iterated using the 11 properly classified invasive tumors and the 7 non-invasive tumors mis-classified as false positives using invasion cluster 2. Using the expression data from these 18 tumors (11 invasive and 7 non-invasive) and following the identical procedures as outlined above, a new second reference set of 449 genes, concordance set of 16 genes (r=0.908), and minimum segregation set (minimum segregation set 3 or invasion cluster 3) were generated. Invasion cluster 3 includes the 10 genes listed in Table 10, and had an overall correlation coefficient of 0.998, as shown in  FIG. 15 .  
               TABLE 10                          Prostate Cancer Invasion Minimum Segregation Set 3.       10 genes (r = 0.998)                     Affymetrix Probe           Set ID (U95Av2)   Description               35704_at   Cluster Incl. X92814:  H. sapiens  mRNA for rat HREV107-like           protein /cds = (407, 895) /gb = X92814 /gi = 1054751 /ug =           Hs.37189 /len = 1070       41850_s_at   Cluster Incl. U63825: Human hepatitis delta antigen interacting           protein A (dipA) mRNA, complete cds /cds = (28, 636) /gb =           U63825 /gi = 1488313 /ug = Hs.66713 /len = 879       39072_at   Cluster Incl. L07648: Human MXI1 mRNA, complete cds /cds =           (208, 894) /gb = L07648 /gi = 506626 /ug = Hs.118630 /len = 2400       38771_at   Cluster Incl. D50405: Human mRNA for RPD3 protein, complete           cds /cds = (63, 1511) /gb = D50405 /gi = 1665722 /ug = Hs.88556 /len = 2091       34987_s_at   Cluster Incl. X79536:  H. sapiens  mRNA for hnRNPcore protein           A1 /cds = (26, 988) /gb = X79536 /gi = 496897 /ug = Hs.151604 /len = 1198       37040_at   Cluster Incl. D42041: Human mRNA for KIAA0088 gene, partial           cds /cds = (0, 2832) /gb = D42041 /gi = 577294 /ug = Hs.76847 /len = 3820       851_s_at   S62539 /FEATURE = /DEFINITION = S62539 insulin receptor           substrate-1 [human, skeletal muscle, mRNA, 5828 nt]       209_at   M94167 /FEATURE = /DEFINITION = HUMHERGC Human           heregulin-beta2 gene, complete cds       936_s_at   Protein Phosphatase Inhibitor Homolog       115_at   X14787 /FEATURE = cds /DEFINITION = HSTS Human mRNA for           thrombospondin                  
 
      As was done with the previous invasion clusters, individual phenotype association indices were calculated for each of the 11 invasive and each of the 7 non-invasive human prostate tumors used to generate invasion cluster 3 according to the methods described in section B, above, using data for the 10 genes that make up invasion cluster 3. The results are shown in  FIG. 16 . Application of the classification method using invasion cluster 3 resulted in 10/11 invasive tumors having positively signed association indices, and so were correctly classified, while 7/7 of the non-invasive tumors had negative association indices and so were correctly classified. There thus were 0 false positives identified using invasion cluster 3. Overall, invasion cluster 3 accurately classified 17/18=94% of the tumors in this sample set.  
      Of the fourteen invasive tumors comprising the original data set, 10/14=71% scored positive phenotype association indices in all three invasion clusters, 3/14=21% scored positive phenotype association indices in two of the three invasion clusters, and 1/14=7% scored a positive phenotype association index in only a single of the three invasion clusters. These data are summarized in Table 11.  
               TABLE 11                          Classification of Invasive Prostate Tumors       using Invasion Clusters 1-3.                                     Invasion   Invasion   Invasion   No. of Correct       Tumor   Cluster 1   Cluster 2   Cluster 3   Classifications                                         T33   0   1   0   1       T46   0   1   1   2       T54   1   1   0   2       T58   1   0   1   2       T01   1   1   1   3       T10   1   1   1   3       T24   1   1   1   3       T29   1   1   1   3       T30   1   1   1   3       T32   1   1   1   3       T47   1   1   1   3       T57   1   1   1   3       T59   1   1   1   3       T62   1   1   1   3       No. Genes in   20   12   10       Cluster       Correlation   0.98   0.983   0.998       Coefficient of       Cluster                 Note:            1 = Positive phenotype association index;            0 = negative phenotype association index.             
 
      A similar analysis can be carried out for the 38 non-invasive tumors that comprised the original sample set. Of these thirty eight non-invasive tumors, 17/38=45% scored a positive phenotype association index in one of the three invasion clusters (one non-invasive tumor (T5) scored negatively in all three invasion clusters and included in this group), and 21/38=55% scored a positive phenotype association index in two of the three invasion clusters. These data are summarized in Table 12.  
               TABLE 12                          Classification of Non-Invasive Prostate       Tumors using Invasion Clusters 1-3.                                     Invasion   Invasion   Invasion   No. of Correct       Tumor   Cluster 1   Cluster 2   Cluster 3   Classifications                                         T5   0   0   0   3       T3   0   1   0   2       T6   0   1   0   2       T11   0   1   0   2       T15   0   1   0   2       T17   0   1   0   2       T18   0   1   0   2       T19   0   1   0   2       T20   0   1   0   2       T21   0   1   0   2       T22   0   1   0   2       T23   0   1   0   2       T26   0   1   0   2       T34   0   1   0   2       T41   0   1   0   2       T49   0   1   0   2       T55   1   0   0   2       T2   0   1   1   1       T4   1   1   0   1       T13   0   1   1   1       T14   0   1   1   1       T16   1   0   1   1       T25   0   1   1   1       T27   1   1   0   1       T28   1   0   1   1       T31   1   0   1   1       T36   1   0   1   1       T37   1   1   0   1       T38   1   1   0   1       T39   0   1   1   1       T40   1   1   0   1       T42   1   1   0   1       T43   1   1   0   1       T45   1   0   1   1       T50   1   0   1   1       T53   1   0   1   1       T56   1   0   1   1       T60   1   0   1   1       No. Genes in   20   12   10       Cluster       Correlation   0.98   0.983   0.998       Coefficient of       Cluster                 Note:            1 = Positive phenotype association index;            0 = negative phenotype association index.             
 
      Three of the invasive tumors scored positively in two of the three invasion clusters, and twenty-one of the non-invasive tumors also scored positively in two of the three invasion clusters. We iterated the method, as described above, using this group of three invasive and twenty-one non-invasive tumors to generate another second reference set, concordance set and minimum segregation set (minimum segregation set 4 or invasion cluster 4). The purpose of this experiment was to determine how well invasion cluster 4 could differentiate this set of three invasive and twenty-one non-invasive prostate tumors.  
      Invasion cluster 4 includes the 13 genes listed in Table 13, and had an overall correlation coefficient of 0.986, as shown in  FIG. 17 .  
               TABLE 13                          Prostate Cancer Invasion Minimum Segregation Set 4.       13 genes (r = 0.986)                     Affymetrix Probe           Set ID (U95Av2)   Description               1375_s_at   M32304 /FEATURE = /DEFINITION = HUMMET Human           metalloproteinase inhibitor mRNA, complete cds       41393_at   Cluster Incl. AF003540:  Homo sapiens  Krueppel family zinc finger           protein (znfp104) mRNA, complete cds /cds = (45, 1934) /gb =           AF003540 /gi = 2384652 /ug = Hs.104382 /len = 2394       870_f_at   M93311 /FEATURE = cds /DEFINITION = HUMMETIII Human           metallothionein-III gene, complete cds       39594_f_at   J04152 /FEATURE = mRNA /DEFINITION = HUMGA733A Human           gastrointestinal tumor-associated antigen GA733-1 protein gene,           complete cds, clone 05516       609_f_at   S62539 /FEATURE = /DEFINITION = S62539 insulin receptor           substrate-1 [human, skeletal muscle, mRNA, 5828 nt]       40031_at   L33930 /FEATURE = /DEFINITION = HUMCD24B  Homo sapiens             CD24 signal transducer mRNA, complete cds and 3 region       38608_at   Cluster Incl. M10943: Human metallothionein-If gene           (hMT-If) /cds = (0, 185) /gb = M10943 /gi =           187540 /ug = Hs.203936 /len = 186       38288_at   AB000220 /FEATURE = /DEFINITION = AB000220  Homo sapiens             mRNA for semaphorin E, complete cds       36883_at   Cluster Incl. L41827:  Homo sapiens  sensory and motor neuron derived           factor (SMDF) mRNA, complete cds /cds = (500, 1390) /gb =           L41827 /gi = 862422 /ug = Hs.172816 /len = 1860       36130_f_at   Cluster Incl. M74542: Human aldehyde dehydrogenase type III           (ALDHIII) mRNA, complete cds /cds = (42, 1403) /gb =           M74542 /gi = 178401 /ug = Hs.575 /len = 1636       35577_at   Cluster Incl. AF027866:  Homo sapiens  megsin mRNA, complete           cds /cds = (364, 1506) /gb = AF027866 /gi = 3769372 /ug =           Hs.138202 /len = 2249       32719_at   L20852 /FEATURE = /DEFINITION = HUMGLVR2X Human           leukemia virus receptor 2 (GLVR2) mRNA, complete cds       291_s_at   Cluster Incl. U40038: Human GTP-binding protein alpha q subunit           (GNAQ) mRNA, complete cds /cds = (42, 1121) /gb = U40038 /gi =           1181670 /ug = Hs.180950 /len = 1450                  
 
      As shown in  FIG. 18 , when phenotype association indices were calculated for this set of samples applying genes of the invasion cluster 4, 3/3 invasive and 16/21 non-invasive tumors were correctly classified. Overall, 19 of 24 (79%) samples in this data set were correctly classified. As one skilled in art may determine from the  FIG. 18 , adjustment of the discrimination threshold (requiring, e.g., a positive association index of at least about 0.4) would yield a more accurate classification close to 100% accuracy.  
      D. Gleason Score Clusters and Sample Classifications  
      The methods of the invention were used along with the data reported by Singh, et al. (2002) to identify gene clusters capable of distinguishing tumor samples having a Gleason score of 6 or 7 (low grade tumors) from those having a Gleason score of 8 or 9 (high grade tumors). The same first reference set described above in part A was used to generate concordance and minimum segregation sets for Gleason score stratification. The second reference set was obtained following the procedures described above in part B, using the supplemental data reported in Singh, et al. (2002) for 46 low grade tumors and six high-grade tumors. Thus, the second reference set was generated by using the Affymetrix MicroDB (version 3.0) and Affymetrix Data Mining Tools (DMT) (version 3.0) data analysis software to identify genes that were differentially regulated in high grade group compared to low grade group of patients at the statistically significant level (p&lt;0.05; Student T-test). Candidate genes were included in the second reference set if they were identified by the DMT software as having p values of 0.05 or less both for up-regulated and down-regulated genes. 2144 genes were identified as being members of the second reference set.  
      The concordance set was obtained by selecting only those genes having a consistent direction of the differential in both the first and the second reference sets (i.e., greater gene expression in the tumor lines cf. the control lines and greater gene expression in the high grade cf. the low-grade tumor samples or vice-versa). The concordance set comprised 58 genes with an overall correlation coefficient equal to 0.823 (see  FIG. 19 ).  
      A minimum segregation set was selected following the procedures described above in section B. A scatter plot was generated of the log 10  transformed average −fold expression change in the cell line and average −fold expression change in the sample data. For the clinical sample data, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who had tumors with Gleason scores of 8 or 9 (high grade) and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who had tumors with Gleason scores of 6 or 7 (low grade). The overall correlation coefficient for the high grade concordance set was 0.823. The high grade concordance set is shown in  FIG. 19 .  
      A minimum segregation set was identified by selecting a subset of the highly correlated genes from the high grade concordance set. This minimum segregation set (Gleason Score 8/9 minimum segregation set 1 or high grade cluster 1) included 17 genes listed below in Table 14. The overall correlation coefficient between the cell lines and clinical samples for high grade cluster 1 was 0.986.  FIG. 20  shows the scatter plot for high grade cluster 1.  
               TABLE 14                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 1.       17 genes (r = 0.986)                     Affymetrix Probe           Set ID (U95Av2)   Description               34801_at   Cluster Incl. AB014610:  Homo sapiens  mRNA for KIAA0710           protein, complete cds /cds = (203, 3550) /gb =           AB014610 /gi = 3327233 /ug = Hs.4198 /len = 4607       35627_at   Cluster Incl. U40571: Human alpha1-syntrophin (SNT A1) mRNA,           complete cds /cds = (37, 1554) /gb = U40571 /gi =           1145727 /ug = Hs.31121 /len = 2110       33132_at   Cluster Incl. U37012: Human cleavage and polyadenylation           specificity factor mRNA, complete cds /cds = (51,           4379) /gb = U37012 /gi = 1045573 /ug = Hs.83727 /len =           4463       39812_at   Cluster Incl. X79865:  H. sapiens  Mrp17 mRNA /cds = (137,           733) /gb = X79865 /gi = 1313961 /ug = Hs.109059 /len =           1008       34366_g_at   Cluster Incl. AF042386:  Homo sapiens  cyclophilin-33B (CYP-33)           mRNA, complete cds /cds = (60, 950) /gb = AF042386 /gi =           2828150 /ug = Hs.33251 /len = 1099       33436_at   Cluster Incl. Z46629:  Homo sapiens  SOX9 mRNA /cds = (359,           1888) /gb = Z46629 /gi = 758102 /ug = Hs.2316 /len = 3923       1143_s_at   Fibroblast Growth Factor Receptor K-Sam, Alt. Splice 3, K-Sam III       39407_at   Cluster Incl. M22488: Human bone morphogenetic protein 1 (BMP-1)           mRNA /cds = (29, 2221) /gb = M22488 /gi = 179499 /ug =           Hs.1274 /len = 2487       1343_s_at   S66896 /FEATURE = /DEFINITION = S66896 squamous cell           carcinoma antigen = serine protease inhibitor [human, mRNA, 1711           nt]       2073_s_at   L34058 /FEATURE = /DEFINITION = HUMCA13A  Homo sapiens             cadherin-13 mRNA, complete cds       33272_at   Cluster Incl. AA829286: of08a01.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-1420488 /clone_end = 3 /gb =           AA829286 /gi = 2902385 /ug = Hs.181062 /len = 559       1440_s_at   X83490 /FEATURE = exon /DEFINITION = HSFAS34  H. sapiens             mRNA for Fas/Apo-1 (clone pCRTM11-Fasdelta(3, 4))       32382_at   Cluster Incl. AB015234:  Homo sapiens  mRNA for uroplakin 1b,           complete cds /cds = (0, 782) /gb = AB015234 /gi =           3721857 /ug = Hs.198650 /len = 783       988_at   X16354 /FEATURE = /DEFINITION = HSTM1CEA Human mRNA           for transmembrane carcinoembryonic antigen BGPa (formerly TM1-           CEA)       779_at   D21337 /FEATURE = /DEFINITION = HUMCO Human mRNA for           collagen       39721_at   Cluster Incl. U09303: Human T cell leukemia LERK-2 (EPLG2)           mRNA, complete cds /cds = (701, 1741) /gb = U09303 /gi =           1783360 /ug = Hs.144700 /len = 2895       37989_at   Cluster Incl. J03802: Human renal carcinoma parathgrad hormone-           like peptide mRNA, complete cds /cds = (303, 830) /gb =           J03802 /gi = 190717 /ug = Hs.89626 /len = 1595                  
 
      Individual phenotype association indices were calculated for each of the six high grade and each of the 46 low grade human prostate tumors used to generate high grade cluster 1 according to the methods described in section B, above, using data for the 17 genes that make up high grade cluster 1 (data not shown). Application of the classification method using high grade cluster 1 resulted in 6/6 high grade tumors having positively signed association indices, and so were correctly classified, while 26/46 of the low grade tumors had negative association indices and so were correctly classified. There thus were 20 false positives (i.e., low grade tumors improperly classified as high grade tumors) identified using high grade cluster 1. Overall, high grade cluster 1 accurately classified 32/52=62% of the tumors in this sample set.  
      To improve the accuracy of the method, we selected from the concordance set of 58 genes additional minimum segregation sets and tested their ability to classify tumor samples. A second minimum segregation set was identified by selecting a smaller subset of the highly correlated genes from the high grade minimum segregation cluster 1. This minimum segregation set (Gleason Score 8/9 minimum segregation set 2 or high grade cluster 2) included 12 genes listed below in Table 15. The overall correlation coefficient between the cell lines and clinical samples for high grade cluster 2 was 0.994.  FIG. 21  shows the scatter plot for high grade cluster 2.  
               TABLE 15                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 2.       12 genes (r = 0.994)                     Affymetrix Probe           Set ID (U95Av2)   Description               34801_at   Cluster Incl. AB014610:  Homo sapiens  mRNA for KIAA0710           protein, complete cds /cds = (203, 3550) /gb = AB014610 /gi =           3327233 /ug = Hs.4198 /len = 4607       35627_at   Cluster Incl. U40571: Human alpha1-syntrophin (SNT A1) mRNA,           complete cds /cds = (37, 1554) /gb = U40571 /gi =           1145727 /ug = Hs.31121 /len = 2110       33132_at   Cluster Incl. U37012: Human cleavage and polyadenylation           specificity factor mRNA, complete cds /cds = (51, 4379) /gb =           U37012 /gi = 1045573 /ug = Hs.83727 /len = 4463       39812_at   Cluster Incl. X79865:  H. sapiens  Mrp17 mRNA /cds = (137,           733) /gb = X79865 /gi = 1313961 /ug = Hs.109059 /len =           1008       34366_g_at   Cluster Incl. AF042386:  Homo sapiens  cyclophilin-33B (CYP-33)           mRNA, complete cds /cds = (60, 950) /gb = AF042386 /gi =           2828150 /ug = Hs.33251 /len = 1099       40712_at   Cluster Incl. D26579:  Homo sapiens  mRNA for transmembrane           protein, complete cds /cds = (9, 2483) /gb = D26579 /gi =           1864004 /ug = Hs.86947 /len = 3236       38903_at   Cluster Incl. AF099731:  Homo sapiens  connexin 31.1 (GJB5) gene,           complete cds /cds = (27, 848) /gb = AF099731 /gi = 4009521 /ug =           Hs.198249 /len = 1370       1687_s_at   X84213 /FEATURE = cds /DEFINITION = HSCEBP1  H. sapiens  BAK           mRNA for BCl-2 homologue       40448_at   Cluster Incl. M92843:  H. sapiens  zinc finger transcriptional regulator           mRNA, complete cds /cds = (59, 1039) /gb = M92843 /gi = 183442 /ug =           Hs.1665 /len = 1746       39721_at   Cluster Incl. U09303: Human T cell leukemia LERK-2 (EPLG2)           mRNA, complete cds /cds = (701, 1741) /gb = U09303 /gi =           1783360 /ug = Hs.144700 /len = 2895       36543_at   Cluster Incl. J02931: Human placental tissue factor (two forms)           mRNA, complete cds /cds = (111, 998) /gb = J02931 /gi =           339501 /ug = Hs.62192 /len = 2141       37989_at   Cluster Incl. J03802: Human renal carcinoma parathgrad hormone-           like peptide mRNA, complete cds /cds = (303, 830) /gb =           J03802 /gi = 190717 /ug = Hs.89626 /len = 1595                  
 
      Individual phenotype association indices were calculated for each of the six high grade and each of the 46 low grade human prostate tumors according to the methods described in section B, above, using data for the 12 genes that make up high grade cluster 2 (data not shown). Application of the classification method using high grade cluster 2 resulted in 6/6 high grade tumors having positively signed association indices, and so were correctly classified, while 30/46 of the low grade tumors had negative association indices and so were correctly classified. There thus were 16 false positives (i.e., low grade tumors improperly classified as high grade tumors) identified using high grade cluster 2. Overall, high grade cluster 2 accurately classified 36/52=69% of the tumors in this sample set.  
      A third minimum segregation set was identified by selecting a smaller subset of the highly correlated genes from the high grade minimum segregation cluster 2. This minimum segregation set (Gleason Score 8/9 minimum segregation set 3 or high grade cluster 3) included the 7 genes listed below in Table 16. The overall correlation coefficient between the cell lines and clinical samples for high grade cluster 3 was 0.970 ( FIG. 22 ).  
               TABLE 16                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 3.       7 genes (r = 0.97)                     Affymetrix Probe           Set ID (U95Av2)   Description               40712_at   Cluster Incl. D26579:  Homo sapiens  mRNA for transmembrane           protein, complete cds /cds = (9, 2483) /gb = D26579 /gi =           1864004 /ug = Hs.86947 /len = 3236       38903_at   Cluster Incl. AF099731:  Homo sapiens  connexin 31.1 (GJB5) gene,           complete cds /cds = (27, 848) /gb = AF099731 /gi = 4009521 /ug =           Hs.198249 /len = 1370       1687_s_at   X84213 /FEATURE = cds /DEFINITION = HSCEBP1  H. sapiens  BAK           mRNA for BCl-2 homologue       40448_at   Cluster Incl. M92843:  H. sapiens  zinc finger transcriptional regulator           mRNA, complete cds /cds = (59, 1039) /gb = M92843 /gi = 183442 /ug =           Hs.1665 /len = 1746       39721_at   Cluster Incl. U09303: Human T cell leukemia LERK-2 (EPLG2)           mRNA, complete cds /cds = (701, 1741) /gb = U09303 /gi =           1783360 /ug = Hs.144700 /len = 2895       36543_at   Cluster Incl. J02931: Human placental tissue factor (two forms)           mRNA, complete cds /cds = (111, 998) /gb = J02931 /gi =           339501 /ug = Hs.62192 /len = 2141       37989_at   Cluster Incl. J03802: Human renal carcinoma parathgrad hormone-           like peptide mRNA, complete cds /cds = (303, 830) /gb =           J03802 /gi = 190717 /ug = Hs.89626 /len = 1595                  
 
      Individual phenotype association indices were calculated for each of the six high grade and each of the 46 low grade human prostate tumors according to the methods described in section B, above, using data for the 7 genes that make up high grade cluster 3 (data not shown). Application of the classification method using high grade cluster 3 again resulted in 6/6 high grade tumors having positively signed association indices, and so were correctly classified, while 17/46 of the low grade tumors had negative association indices and so were correctly classified. There thus were 29 false positives (i.e., low grade tumors improperly classified as high grade tumors) identified using high grade cluster 3. Overall, high grade cluster 3 accurately classified 23/52=44% of the tumors in this sample set.  
      A summary of the accuracy with which the first three high grade clusters distinguished high grade (Gleason score 8 or 9) from low grade (Gleason score 6 or 7) tumors is provided in Table 17.  
               TABLE 17                          Classification of High Grade &amp; Low Grade Prostate       Tumors using High Grade Clusters 1-3.                                                 No. of Cor-           High Grade   High Grade   High Grade   rect Classi-       Tumor   Cluster 1   Cluster 2   Cluster 3   fications                         Gleason Score 8 or 9 (high grade) Tumors                                 T26   1   1   1   3       T31   1   1   1   3       T45   1   1   1   3       T57   1   1   1   3       T58   1   1   1   3       T59   1   1   1   3                 Gleason Score 6 or 7 (low grade) Tumors                                 T01   1   1   1   0       T02   0   0   1   2       T03   0   0   0   3       T04   0   0   0   3       T05   0   0   1   2       T06   0   0   0   3       T10   1   1   0   1       T11   0   0   1   2       T13   0   0   1   2       T14   0   0   1   2       T15   0   0   0   3       T16   1   0   0   2       T17   0   0   1   2       T18   0   0   1   2       T19   0   0   0   3       T20   0   0   0   3       T21   0   0   1   2       T22   0   0   0   3       T23   0   0   1   2       T24   0   0   1   2       T25   1   0   1   1       T27   0   0   0   3       T28   1   1   1   0       T29   1   1   1   0       T30   1   0   0   2       T32   0   0   0   3       T33   0   0   1   2       T34   0   0   1   2       T36   1   1   1   0       T37   0   0   1   2       T38   0   0   1   2       T39   0   0   1   2       T40   1   1   1   0       T41   1   0   0   2       T42   1   1   1   0       T43   1   1   1   0       T46   1   1   0   1       T47   1   1   1   0       T49   0   0   1   2       T50   0   0   0   3       T53   1   1   1   0       T54   1   1   1   0       T55   1   1   1   0       T56   1   1   0   1       T60   1   1   1   0       T62   1   1   0   1       No. Genes in   17   12   7       Cluster       Correlation   0.986   0.994   0.97       Coefficient of       Cluster                 Note:            1 = Positive phenotype association index;            0 = negative phenotype association index.             
 
      Since the overall classification accuracy of high grade cluster 3 was lower than that of high grade cluster 1 and 2, additional high grade clusters were generated from a high grade concordance set of 58 genes. The resulting alternative minimum segregation set (ALT high grade cluster) included a total of 38 genes listed below in Table 18. The overall correlation coefficient between the cell line and clinical samples for this high grade cluster (Gleason Score 8/9 ALT high grade cluster) was 0.929 ( FIG. 23 ). Phenotype association indices were calculated for each of the 6 high grade and each of the 46 low grade tumors to determine how well this high grade cluster would classify the samples. All six of the high grade tumors were correctly classified, while 26/46 of the low grade tumors were correctly classified. Thus overall, this minimum segregation set correctly classified 32/52=62% of the samples.  
               TABLE 18                          Prostate Cancer Gleason Score 8/9 ALT High Grade Minimum       Segregation Set (38 genes).       38 genes (r = 0.929)                     Affymetrix           Probe Set ID       (U95Av2)   Description               34801_at   Cluster Incl. AB014610:  Homo sapiens  mRNA for KIAA0710 protein,           complete cds /cds = (203, 3550) /gb = AB014610 /gi =           3327233 /ug = Hs.4198 /len = 4607       35627_at   Cluster Incl. U40571: Human alpha1-syntrophin (SNT A1) mRNA,           complete cds /cds = (37, 1554) /gb = U40571 /gi = 1145727 /ug =           Hs.31121 /len = 2110       33132_at   Cluster Incl. U37012: Human cleavage and polyadenylation specificity           factor mRNA, complete cds /cds = (51, 4379) /gb = U37012 /gi =           1045573 /ug = Hs.83727 /len = 4463       39812_at   Cluster Incl. X79865:  H. sapiens  Mrp17 mRNA /cds = (137,           733) /gb = X79865 /gi = 1313961 /ug = Hs.109059 /len = 1008       34366_g_at   Cluster Incl. AF042386:  Homo sapiens  cyclophilin-33B (CYP-33)           mRNA, complete cds /cds = (60, 950) /gb = AF042386 /gi =           2828150 /ug = Hs.33251 /len = 1099       32545_r_at   Cluster Incl. L12535: Human RSU-1/RSP-1 mRNA, complete           cds /cds = (827, 1660) /gb = L12535 /gi = 434050 /ug =           Hs.75551 /len = 2194       35899_at   Cluster Incl. AF109401:  Homo sapiens  neurotrophic factor artemin           precursor (ARTN) mRNA, complete cds /cds = (298, 960) /gb =           AF109401 /gi = 4071352 /ug = Hs.194689 /len = 1003       32855_at   Cluster Incl. L00352: Human low density lipoprotein receptor           gene /cds = (93, 2675) /gb = L00352 /gi = 460289 /ug =           Hs.213289 /len = 5175       41817_g_at   Cluster Incl. AL049851: Human DNA sequence from clone 889J22B           on chromosome 22q13.1 /cds = (0, 1000) /gb = AL049851 /gi =           4826526 /ug = Hs.57973 /len = 1798       33436_at   Cluster Incl. Z46629:  Homo sapiens  SOX9 mRNA /cds = (359,           1888) /gb = Z46629 /gi = 758102 /ug = Hs.2316 /len = 3923       41663_at   Cluster Incl. AF038202:  Homo sapiens  clone 23570 mRNA           sequence /cds = UNKNOWN /gb = AF038202 /gi = 2795923 /ug =           Hs.12311 /len = 1742       188_at   U09303 /FEATURE = /DEFINITION = HSU09303 Human T cell           leukemia LERK-2 (EPLG2) mRNA, complete cds       38822_at   Cluster Incl. AB011420:  Homo sapiens  mRNA for DRAK1, complete           cds /cds = (117, 1361) /gb = AB011420 /gi = 3834353 /ug =           Hs.9075 /len = 2641       38913_at   Cluster Incl. U60319:  Homo sapiens  haemochromatosis protein (HLA-           H) mRNA, complete cds /cds = (221, 1267) /gb = U60319 /gi =           1469789 /ug = Hs.20019 /len = 2716       1143_s_at   Fibroblast Growth Factor Receptor K-Sam, Alt. Splice 3, K-Sam III       40712_at   Cluster Incl. D26579:  Homo sapiens  mRNA for transmembrane           protein, complete cds /cds = (9, 2483) /gb = D26579 /gi =           1864004 /ug = Hs.86947 /len = 3236       39407_at   Cluster Incl. M22488: Human bone morphogenetic protein 1 (BMP-1)           mRNA /cds = (29, 2221) /gb = M22488 /gi = 179499 /ug =           Hs.1274 /len = 2487       34044_at   Cluster Incl. AB007131:  Homo sapiens  mRNA for HSF2BP, complete           cds /cds = (332, 1336) /gb = AB007131 /gi = 3345673 /ug =           Hs.97624 /len = 1898       39320_at   Cluster Incl. U13697: Human interleukin 1-beta converting enzyme           isoform beta (IL1BCE) mRNA, complete cds /cds = (0, 1151) /gb =           U13697 /gi = 717039 /ug = Hs.2490 /len = 1185       38608_at   Cluster Incl. AA010777: ze22f06.r1  Homo sapiens  cDNA, 5           end /clone = IMAGE-359747 /clone_end = 5 /gb =           AA010777 /gi = 1471804 /ug = Hs.99923 /len = 521       35194_at   Cluster Incl. X53463: Human mRNA for glutathione peroxidase-like           protein /cds = (51, 623) /gb = X53463 /gi = 31894 /ug =           Hs.2704 /len = 951       1343_s_at   S66896 /FEATURE = /DEFINITION = S66896 squamous cell           carcinoma antigen = serine protease inhibitor [human, mRNA, 1711 nt]       2073_s_at   L34058 /FEATURE = /DEFINITION = HUMCA13A  Homo sapiens             cadherin-13 mRNA, complete cds       38903_at   Cluster Incl. AF099731:  Homo sapiens  connexin 31.1 (GJB5) gene,           complete cds /cds = (27, 848) /gb = AF099731 /gi = 4009521 /ug =           Hs.198249 /len = 1370       33272_at   Cluster Incl. AA829286: of08a01.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-1420488 /clone_end = 3 /gb =           AA829286 /gi = 2902385 /ug = Hs.181062 /len = 559       1687_s_at   X84213 /FEATURE = cds /DEFINITION = HSCEBP1  H. sapiens  BAK           mRNA for BCl-2 homologue       1440_s_at   X83490 /FEATURE = exon /DEFINITION = HSFAS34  H. sapiens             mRNA for Fas/Apo-1 (clone pCRTM11-Fasdelta(3, 4))       32382_at   Cluster Incl. AB015234:  Homo sapiens  mRNA for uroplakin 1b,           complete cds /cds = (0, 782) /gb = AB015234 /gi =           3721857 /ug = Hs.198650 /len = 783       40448_at   Cluster Incl. M92843:  H. sapiens  zinc finger transcriptional regulator           mRNA, complete cds /cds = (59, 1039) /gb = M92843 /gi = 183442 /ug =           Hs.1665 /len = 1746       988_at   X16354 /FEATURE = /DEFINITION = HSTM1CEA Human mRNA           for transmembrane carcinoembryonic antigen BGPa (formerly TM1-           CEA)       41481_at   Cluster Incl. X17033: Human mRNA for integrin alpha-2           subunit /cds = (48, 3593) /gb = X17033 /gi = 33906 /ug =           Hs.1142 /len = 5373       35444_at   Cluster Incl. AC004030:  Homo sapiens  DNA from chromosome 19,           cosmid F21856 /cds = (0, 2039) /gb = AC004030 /gi =           2804590 /ug = Hs.169508 /len = 2040       779_at   D21337 /FEATURE = /DEFINITION = HUMCO Human mRNA for           collagen       38746_at   Cluster Incl. AF011375:  Homo sapiens  integrin variant beta4E           (ITGB4) mRNA, complete cds /cds = (0, 2894) /gb =           AF011375 /gi = 2293520 /ug = Hs.85266 /len = 2895       32821_at   Cluster Incl. AI762213: wi54d04.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2394055 /clone_end = 3 /gb =           AI762213 /gi = 5177880 /ug = Hs.204238 /len = 677       39721_at   Cluster Incl. U09303: Human T cell leukemia LERK-2 (EPLG2)           mRNA, complete cds /cds = (701, 1741) /gb = U09303 /gi =           1783360 /ug = Hs.144700 /len = 2895       36543_at   Cluster Incl. J02931: Human placental tissue factor (two forms)           mRNA, complete cds /cds = (111, 998) /gb = J02931 /gi =           339501 /ug = Hs.62192 /len = 2141       37989_at   Cluster Incl. J03802: Human renal carcinoma parathgrad hormone-like           peptide mRNA, complete cds /cds = (303, 830) /gb = J03802 /gi =           190717 /ug = Hs.89626 /len = 1595                  
 
      To further improve the overall classification accuracy, additional high grade clusters were generated by culling a subset of sample data made up of all the true positives (ie., the 6 high grade tumors correctly classified using each of the first three high grade clusters) and the set of 12 low grade tumors that scored as false positives in 3/3 of the first 3 high grade clusters (i.e., all the Gleason score 6&amp;7 tumors that had a “0” in the “No. of Correct Classifications” column in Table 15). This subset was used to generate another second reference set, and concordance set using the same procedures outlined above. From this concordance set of 33 genes (r=0.731), a fourth minimum segregation set was identified by selecting a subset of the highly correlated genes from the new high grade concordance set. This minimum segregation set (Gleason Score 8/9 minimum segregation set 4 or high grade cluster 4) included 5 genes listed below in Table 19. The overall correlation coefficient between the cell lines and clinical samples for high grade cluster 4 was 0.995.  FIG. 24  shows the scatter plot for high grade cluster 4.  
               TABLE 19                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 4.       5 genes (r = 0.995)                     Affymetrix Probe           Set ID (U95Av2)   Description               1733_at   M60315 /FEATURE = /DEFINITION = HUMTGFBC Human           transforming growth factor-beta (tgf-beta) mRNA, complete cds       41850_s_at   Cluster Incl. U63825: Human hepatitis delta antigen interacting           protein A (dipA) mRNA, complete cds /cds = (28, 636) /gb =           U63825 /gi = 1488313 /ug = Hs.66713 /len = 879       39020_at   Cluster Incl. U82938: Human CD27BP (Siva) mRNA, complete           cds /cds = (252, 821) /gb = U82938 /gi = 2228596 /ug =           Hs.112058 /len = 1034       33436_at   Cluster Incl. Z46629:  Homo sapiens  SOX9 mRNA /cds = (359,           1888) /gb = Z46629 /gi = 758102 /ug = Hs.2316 /len = 3923       988_at   X16354 /FEATURE = /DEFINITION = HSTM1CEA Human mRNA           for transmembrane carcinoembryonic antigen BGPa (formerly TM1-           CEA)                  
 
      Phenotype association indices were calculated using the average cell line and individual sample −fold change expression data for the genes in high grade cluster 4. The sample included the 6 high grade tumors and the set of 17 low grade tumors that scored as false positives in 2/3 or 3/3 of the first three high grade clusters (i.e., all the Gleason score 6&amp;7 tumors that had a “0” or “1” in the “No. of Correct Classifications” column in Table 17).  
      High grade cluster 4 correctly classified 6/6 high grade tumors, and 12/17 low grade tumors. Overall, high grade cluster 4 accurately characterized 18/23=78% of the tumors in this set.  
      To improve the accuracy of the classification, several additional minimum segregation sets of highly correlated genes were selected. Gleason Score 8/9 minimum segregation set 5, or high grade cluster 5, was used to generate phenotype association indices for the 6 high grade tumors (true positives) and the set of 17 low grade tumors that scored as false positives in 2/3 or 3/3 of the first three high grade clusters (i.e., all the Gleason score 6&amp;7 tumors that had a “0” or “1” in the “No. of Correct Classifications” column in Table 17). High grade cluster 5 correctly classified 6/6 high grade tumors and 9/17 low grade tumors. Overall, high grade cluster 5 correctly classified 15/23=65% of the samples in this set.  
      High grade cluster 5 included 4 genes listed below in Table 20. The overall correlation coefficient between the cell lines and clinical samples for high grade cluster 5 was 0.998.  FIG. 25  shows the scatter plot for high grade cluster 5.  
               TABLE 20                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 5.       4 genes (r = 0.998)                     Affymetrix Probe           Set ID (U95Av2)   Description               41850_s_at   Cluster Incl. U63825: Human hepatitis delta antigen interacting           protein A (dipA) mRNA, complete cds /cds = (28, 636) /gb =           U63825 /gi = 1488313 /ug = Hs.66713 /len = 879       39020_at   Cluster Incl. U82938: Human CD27BP (Siva) mRNA, complete           cds /cds = (252, 821) /gb = U82938 /gi = 2228596 /ug =           Hs.112058 /len = 1034       33436_at   Cluster Incl. Z46629:  Homo sapiens  SOX9 mRNA /cds = (359,           1888) /gb = Z46629 /gi = 758102 /ug = Hs.2316 /len = 3923       988_at   X16354 /FEATURE = /DEFINITION = HSTM1CEA Human mRNA           for transmembrane carcinoembryonic antigen BGPa (formerly TM1-           CEA)                  
 
      High grade cluster 6 included 7 genes and had an overall correlation coefficient of 0.995 ( FIG. 26 ). High grade cluster 7 included 13 genes and had an overall correlation coefficient of 0.992 ( FIG. 27 ). High grade cluster 6 correctly classified 6/6 of the high grade tumors, and 13/17 of the low grade tumors. Overall, high grade cluster 6 correctly classified 19/23=83% of the samples in this set. High grade cluster 7 correctly classified 6/6 of the high grade tumors and 14/17 of the low grade tumors. Overall, high grade cluster 7 correctly classified 20/23=87% of the samples in this set. Tables 21 and 22 list the genes that make up high grade cluster 6 and high grade cluster 7. A summary of the accuracy with which high grade clusters 4-7 distinguished high grade (Gleason score 8 or 9) from the “false positive” subset of seventeen low grade (Gleason score 6 or 7) tumors is provided in Table 23.  
               TABLE 21                          Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 6.       7 genes (r = 0.995)                     Affymetrix Probe           Set ID (U95Av2)   Description               1733_at   M60315 /FEATURE = /DEFINITION = HUMTGFBC Human transforming growth factor-           beta (tgf-beta) mRNA, complete cds       41850_s_at   Cluster Incl. U63825: Human hepatitis delta antigen interacting protein A (dipA)           mRNA, complete cds /cds = (28, 636) /gb = U63825 /gi = 1488313 /ug =           Hs.66713 /len = 879       39020_at   Cluster Incl. U82938: Human CD27BP (Siva) mRNA, complete cds /cds = (252,           821) /gb = U82938 /gi = 2228596 /ug = Hs.112058 /len = 1034       37026_at   Cluster Incl. AF001461:  Homo sapiens  Kruppel-like zinc finger protein Zf9 mRNA,           complete cds /cds = (30, 881) /gb = AF001461 /gi = 3378030 /ug =           Hs.76526 /len = 1354       32587_at   Cluster Incl. U07802: Human Tis11d gene, complete cds /cds = (291, 1739) /gb =           U07802 /gi = 984508 /ug = Hs.78909 /len = 3655       40448_at   Cluster Incl. M92843:  H. sapiens  zinc finger transcriptional regulator mRNA, complete           cds /cds = (59, 1039) /gb = M92843 /gi = 183442 /ug = Hs.1665 /len = 1746       779_at   D21337 /FEATURE = /DEFINITION = HUMCO Human mRNA for collagen                  
 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                   
               
               
                 Prostate Cancer Gleason Score 8/9 Minimum Segregation Set 7. 
               
               
                 13 genes (r = 0.992) 
               
            
           
           
               
               
            
               
                 Affymetrix Probe 
                   
               
               
                 Set ID (U95Av2) 
                 Description 
               
               
                   
               
               
                 1733_at 
                 M60315 /FEATURE = /DEFINITION = HUMTGFBC Human transforming growth factor- 
               
               
                   
                 beta (tgf-beta) mRNA, complete cds 
               
               
                 41850_s_at 
                 Cluster Incl. U63825: Human hepatitis delta antigen interacting protein A (dipA) 
               
               
                   
                 mRNA, complete cds /cds = (28, 636) /gb = U63825 /gi = 1488313 /ug = 
               
               
                   
                 Hs.66713 /len = 879 
               
               
                 39020_at 
                 Cluster Incl. U82938: Human CD27BP (Siva) mRNA, complete cds /cds = (252, 
               
               
                   
                 821) /gb = U82938 /gi = 2228596 /ug = Hs.112058 /len = 1034 
               
               
                 33936_at 
                 Cluster Incl. D86181:  Homo sapiens  DNA for galactocerebrosidase /cds = (146, 
               
               
                   
                 2155) /gb = D86181 /gi = 2897770 /ug = Hs.273 /len = 3869 
               
               
                 39631_at 
                 Cluster Incl. U52100: Human XMP mRNA, complete cds /cds = (63, 566) /gb = 
               
               
                   
                 U52100 /gi = 2474095 /ug = Hs.29191 /len = 690 
               
               
                 38617_at 
                 Cluster Incl. D45906:  Homo sapiens  mRNA for LIMK-2, complete cds /cds = (114, 
               
               
                   
                 2030) /gb = D45906 /gi = 1805593 /ug = Hs.100623 /len = 3668 
               
               
                 35703_at 
                 Cluster Incl. X06374: Human mRNA for platelet-derived growth factor PDGF-A /cds = 
               
               
                   
                 (403, 993) /gb = X06374 /gi = 35363 /ug = Hs.37040 /len = 2305 
               
               
                 41257_at 
                 Cluster Incl. D16217: Human mRNA for calpastatin, complete cds /cds = (162, 
               
               
                   
                 2288) /gb = D16217 /gi = 303598 /ug = Hs.226067 /len = 2493 
               
               
                 32786_at 
                 Cluster Incl. X51345: Human jun-B mRNA for JUN-B protein /cds = (253, 
               
               
                   
                 1296) /gb = X51345 /gi = 34014 /ug = Hs.198951 /len = 1797 
               
               
                 1052_s_at 
                 M83667 /FEATURE = mRNA /DEFINITION = HUMNFIL6BA Human NF-IL6-beta protein 
               
               
                   
                 mRNA, complete cds 
               
               
                 231_at 
                 M55153 /FEATURE = /DEFINITION = HUMTGASE Human transglutaminase (TGase) mRNA, 
               
               
                   
                 complete cds 
               
               
                 31792_at 
                 Cluster Incl. M20560: Human lipocortin-III mRNA, complete cds /cds = (46, 
               
               
                   
                 1017) /gb = M20560 /gi = 186967 /ug = Hs.1378 /len = 1339 
               
               
                 36543_at 
                 Cluster Incl. J02931: Human placental tissue factor (two forms) mRNA, complete 
               
               
                   
                 cds /cds = (111, 998) /gb = J02931 /gi = 339501 /ug = Hs.62192 /len = 2141 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                   
               
               
                 Classification of High Grade &amp; “False Positive” Low 
               
               
                 Grade Prostate Tumors using High Grade Clusters 4-7. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 No. of 
               
               
                   
                 High 
                 High 
                 High 
                 High 
                 Correct 
               
               
                   
                 Grade 
                 Grade 
                 Grade 
                 Grade 
                 Classifi- 
               
               
                 Tumor 
                 Cluster 4 
                 Cluster 5 
                 Cluster 6 
                 Cluster 7 
                 cations 
               
               
                   
               
            
           
           
               
            
               
                 Gleason Score 8 or 9 (high grade) Tumors 
               
            
           
           
               
               
               
               
               
               
            
               
                 T26 
                 1 
                 1 
                 1 
                 1 
                 4 
               
               
                 T31 
                 1 
                 1 
                 1 
                 1 
                 4 
               
               
                 T45 
                 1 
                 1 
                 1 
                 1 
                 4 
               
               
                 T57 
                 1 
                 1 
                 1 
                 1 
                 4 
               
               
                 T58 
                 1 
                 1 
                 1 
                 1 
                 4 
               
               
                 T59 
                 1 
                 1 
                 1 
                 1 
                 4 
               
            
           
           
               
            
               
                 Gleason Score 6 or 7 (low grade) Tumors 
               
            
           
           
               
               
               
               
               
               
            
               
                 T01 
                 1 
                 0 
                 0 
                 0 
                 3 
               
               
                 T10 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 T25 
                 0 
                 0 
                 0 
                 1 
                 3 
               
               
                 T28 
                 0 
                 0 
                 0 
                 0 
                 4 
               
               
                 T29 
                 0 
                 0 
                 1 
                 1 
                 2 
               
               
                 T36 
                 0 
                 0 
                 0 
                 0 
                 4 
               
               
                 T40 
                 0 
                 0 
                 1 
                 0 
                 3 
               
               
                 T42 
                 0 
                 1 
                 0 
                 0 
                 3 
               
               
                 T43 
                 1 
                 0 
                 0 
                 1 
                 2 
               
               
                 T46 
                 1 
                 1 
                 0 
                 0 
                 2 
               
               
                 T47 
                 0 
                 1 
                 0 
                 0 
                 3 
               
               
                 T53 
                 1 
                 1 
                 0 
                 0 
                 2 
               
               
                 T54 
                 0 
                 0 
                 0 
                 0 
                 4 
               
               
                 T55 
                 0 
                 0 
                 0 
                 0 
                 4 
               
               
                 T56 
                 0 
                 1 
                 0 
                 0 
                 3 
               
               
                 T60 
                 0 
                 1 
                 0 
                 0 
                 3 
               
               
                 T62 
                 0 
                 1 
                 0 
                 0 
                 3 
               
               
                 No. Genes in 
                 5 
                 4 
                 7 
                 13 
               
               
                 Cluster 
               
               
                 Correlation 
                 0.995 
                 0.998 
                 0.995 
                 0.992 
               
               
                 Coefficient of 
               
               
                 Cluster 
               
               
                   
               
               
                   Note:    
               
               
                   1 = Positive phenotype association index;    
               
               
                   0 = negative phenotype association index.    
               
            
           
         
       
     
      Application of the methods of present invention to classification of human prostate tumors according to Gleason grade revealed that high grade tumors can be readily distinguished from the majority of low grade prostate cancers based on gene expression analysis of small discrete clusters of genes. However, there is a significant fraction of low grade tumors that closely resemble transcriptional profiles of more advanced and aggressive high grade tumors suggesting that these low grade tumors may represent a precursor of aggressive metastatic disease.  
      D. Benign Prostatic Hyperplasia (BPH) Sample Classification  
      Applying method of present invention we identified a BPH vs. prostate cancer discrimination cluster comprising 14 genes listed in Table 22. In this example we utilized human prostate carcinoma cell line gene expression data to develop a first reference set and clinical sample data set presented in Stamey T A, Warrington J A, Caldwell M C, Chen Z, Fan Z, Mahadevappa M, McNeal J E, Nolley R, Zhang Z. Molecular genetic profiling of Gleason grade 4/5 prostate cancers compared to benign prostatic hyperplasia. J Urol 2001 166(6):2171-2177, 2001; incorporate herein by reference. The clinical data set consists of 17 samples obtained from 8 patients with BPH and 9 patients with prostate cancer (Stamey, T. A., et al., 2001).  
      We identified a concordance set of 54 genes (r=0.842) exhibiting concordant gene expression changes between prostate cancer cell lines vs. normal prostate epithelial cells and clinical samples of prostate cancer vs. BPH. As shown in  FIG. 28 , 7 of 8 samples from the BPH group had negative phenotype association indices, whereas 9 of 9 samples from the prostate cancer group had positive phenotype association indices yielding overall accuracy of 94% in sample classification.  
      Applying the methods of the present invention, we next identified a minimum segregation set of genes (BPH minimum segregation set 1 or BPH cluster 1 (MAGE-1 cluster)—Table 24) that is able accurately discriminates between BPH and prostate cancer in clinical tissue samples derived from human prostate. This BPH vs. prostate cancer discrimination cluster comprises 14 genes displaying a high correlation coefficient of −fold expression changes in prostate cancer cell lines vs. normal prostate epithelial cells and clinical samples of prostate cancer vs. BPH (r=0.990) and high accuracy of sample classification. As shown in  FIG. 29 , of 8 samples from the BPH group had negative phenotype association indices, whereas 9 of 9 samples from the prostate cancer group had positive phenotype association indices yielding overall accuracy of 100% in sample classification.  
               TABLE 24                          BPH Minimum Segregation Set 1.       14 genes (r = 0.990) [BPH segregation cluster (MAGE-1 cluster)]                     Affymetrix           Probe Set       ID (U95Av2)   Description               M77481_rna1_f_at   MAGE-1       U73514_at   hydroxyacyl-Coenzyme A dehydrogenase,           type II       U39840_at   hepatocyte nuclear factor-3 alpha (HNF-3           alpha)       L41559_at   dimerization cofactor of hepatocyte           nuclear factor 1 alpha (TCF1)       U90907_at   clone 23907       D00860_at   phosphoribosyl pyrophosphate synthetase           subunit I       U81599_at   homeodomain protein HOXB13       X91247_at   thioredoxin reductase 1       U79274_at   clone 23733       J03473_at   poly(ADP-ribose) synthetase       HG4312-HT4582_s_at   Transcription Factor IIIa       M55593_at   matrix metalloproteinase 2 (gelatinase           A, 72 kD gelatinase, 72 kD type IV           collagenase)       M11433_at   retinol-binding protein 1, cellular       X93510_at   LIM domain protein                  
 
      E. Metastatic Prostate Cancer Sample Classification  
      Applying method of present invention we identified two gene clusters comprising 17 and 19 genes useful for classifying prostate cancer metastases. In this example we utilized human prostate carcinoma cell line gene expression data and clinical sample data set presented in Dhanasekaran, S. M., Barrette, T. R., Ghosh, D., Shah, R., Varambally, S., Kurachi, K., Pienta, K. J., Rubin, M. A., Chinnalyan, A. M. Delineation of prognostic biomarkers in prostate cancer. Nature, 412:822-826, 2001, incorporated herein by reference. As a starting gene set we utilized a set of 242 genes that was identified using a combination of statistical and clustering analyses approach in Dhanasekaran, S. M., et al., 2001 and was found to be useful in classification of various clinical samples using hierarchical clustering algorithm. Our initial analysis applying the methods of the present invention was performed on a small training data set comprising three human prostate cancer cell lines (LNCap; PC3; DU145), three samples of adjacent to cancer normal prostate, one sample of prostatitis, five samples of BPH, ten samples of hormone dependent localized prostate cancer, and seven samples of hormone refractory metastatic prostate cancer.  
      The original gene expression data were presented as log transformed −fold expression changes of a gene in a sample compared to normal human prostate. For the set of 242 genes we calculated average gene expression values for three prostate cancer cell lines (first reference set) and average expression values for group of metastatic prostate tumors vs. localized prostate tumors (second reference set). The initial set of 242 genes displayed only a weak correlation coefficient of the −fold expression changes in prostate cancer cell lines and clinical samples of metastatic prostate cancer vs. localized prostate cancer (r=0.323).  
      Applying the methods of the present invention, we identified a concordance set of 72 genes (r=0.866) exhibiting concordant gene expression changes between prostate cancer cell lines and clinical samples of metastatic prostate cancer vs. localized prostate cancer. When we utilized genes of this concordance set to calculate the phenotype association indices in individual clinical samples, 3 of 3 samples from ANP group, 5 of 5 samples from the BPH group, one sample of prostatitis, and five of ten samples of localized prostate cancer had negative phenotype association indices, whereas 7 of 7 samples from the metastatic prostate cancer group had positive phenotype association indices yielding overall accuracy of 84% in sample classification.  
      Applying the methods of the present invention, we next identified two minimum segregation sets of genes capable of accurately discriminating between metastatic prostate cancer and localized prostate cancer in clinical tissue samples derived from human prostate. The first metastatic prostate cancer (MPC) vs. localized prostate cancer (LPC) minimum segregation set or cluster (metastasis minimum segregation set 1) comprises 17 genes displaying a high correlation coefficient of fold expression changes in prostate cancer cell lines and clinical samples of metastatic prostate cancer prostate cancer vs. localized prostate cancer (r=0.988) and is highly accurate in discriminating among these different types of samples. As shown in  FIG. 30 , 3 of 3 samples from ANP group, 5 of 5 samples from the BPH group, one sample of prostatitis, and nine of ten samples of localized prostate cancer had negative phenotype association indices, whereas 7 of 7 samples from the metastatic prostate cancer group had positive phenotype association indices yielding overall accuracy of 96% in sample classification.  
      The second metastatic prostate cancer vs. localized prostate cancer discrimination cluster (metastasis minimum segregation set 2) comprises 19 genes displaying a high correlation coefficient of −fold expression changes in prostate cancer cell lines and clinical samples of metastatic prostate cancer prostate cancer vs. localized prostate cancer (r=0.988) and also is highly accurate in discriminating among these different types of samples. As shown in  FIG. 31 , 3 of 3 samples from ANP group, 5 of 5 samples from the BPH group, one sample of prostatitis, and nine of ten samples of localized prostate cancer had negative phenotype association indices, whereas 7 of 7 samples from the metastatic prostate cancer group had positive phenotype association indices yielding overall accuracy of 96% in sample classification.  
      To further validate the sample classification accuracy using an independent data set, we tested the performance of the two metastatic prostate cancer discrimination clusters on a larger set of clinical samples consisting of four samples of adjacent to cancer normal prostate (ANP), one sample of prostatitis, fourteen samples of BPH, fourteen samples of hormone dependent localized prostate cancer (LPC), and twenty samples of hormone refractory metastatic prostate cancer. As shown in  FIG. 32 , when metastasis minimum segregation set 1 (i.e., the cluster of 17 genes) was utilized, 4 of 4 samples from ANP group, 14 of 14 samples from the BPH group, one sample of prostatitis, and 10 of 14 samples of localized prostate cancer had negative phenotype association indices, whereas 20 of 20 samples from the metastatic prostate cancer group had positive phenotype association indices yielding overall accuracy of 92% in sample classification.  
      As shown in  FIG. 33 , when metastasis minimum segregation set 2 (i.e., the cluster of 19 genes) was utilized, 4 of 4 samples from ANP group, 13 of 14 samples from the BPH group, one sample of prostatitis, and 12 of 14 samples of localized prostate cancer had negative phenotype association indices, whereas 20 of 20 samples from the metastatic prostate cancer group had positive phenotype association indices yielding overall accuracy of 94% in sample classification. The genes comprising prostate cancer metastasis minimum segregation sets 1 and 2 are set forth in Tables 25 and 26.  
               TABLE 25                          Prostate Cancer Metastasis Minimum Segregation Set 1.       17 genes (r = 0.988)                                     Clone   UniGene           Gene           ID   Cluster   Accession   NID   Symbol   NAME                                             469954   Hs.169449   AA030029   g1496255   PRKCA   protein kinase C, alpha       308041   Hs.3847   W24429   g1301379   PNUTL1   peanut ( Drosophila )-like 1       83605   Hs.50966   T61078   g664115   CPS1   carbamoyl-phosphate synthetase 1, mitochondrial       123755   Hs.45514   R01304   g751040   ERG   v-ets avian erythroblastosis virus E26 oncogene related       810512   Hs.87409   AA464630   g2189514   THBS1   thrombospondin 1       811028   Hs.9946   AA485373   g2214592       ESTs       767828   Hs.83951   AA418773   g2080583   HPS   Hermansky-Pudlak syndrome       417711   Hs.180255   W88967   g1404003   HLA-   major histocompatibility complex, class II, DR beta 1                       DRB1       727251   Hs.1244   AA412053   g2070642   CD9   CD9 antigen (p24)       214990   Hs.80562   H72027   g1043843   GSN   gelsolin (amyloidosis, Finnish type)       788566   Hs.80296   AA452966   g2166635   PCP4   Purkinje cell protein 4       205049   Hs.111676   H57494   g1010326       ESTs, Weakly similar to heat shock protein 27 [ H. sapiens ]       81289   Hs.77443   T60048   g661885   ACTG2   actin, gamma 2, smooth muscle, enteric       77915   Hs.76422   T61323   g664360   PLA2G2A   phospholipase A2, group IIA (platelets, synovial fluid)       898092   Hs.75511   AA598794       CTGF   connective tissue growth factor       343646   Hs.2969   W69471       SKI   v-ski avian sarcoma viral oncogene homolog       134422   Hs.200499   R31679   g787522       ESTs                  
 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                   
               
               
                 Prostate Cancer Metastasis Minimum Segregation Set 2. 
               
               
                 19 genes (r = 0.988) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Clone 
                 UniGene 
                   
                   
                 Gene 
                   
               
               
                 ID 
                 Cluster 
                 Accession 
                 NID 
                 Symbol 
                 NAME 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 469954 
                 Hs.169449 
                 AA030029 
                 g1496255 
                 PRKCA 
                 protein kinase C, alpha 
               
               
                 308041 
                 Hs.3847 
                 W24429 
                 g1301379 
                 PNUTL1 
                 peanut ( Drosophila )-like 1 
               
               
                 83605 
                 Hs.50966 
                 T61078 
                 g664115 
                 CPS1 
                 carbamoyl-phosphate synthetase 1, mitochondrial 
               
               
                 123755 
                 Hs.45514 
                 R01304 
                 g751040 
                 ERG 
                 v-ets avian erythroblastosis virus E26 oncogene related 
               
               
                 784959 
                 Hs.90408 
                 AA447658 
                 g2161328 
                 NEO1 
                 neogenin (chicken) homolog 1 
               
               
                 130977 
                 Hs.23437 
                 R22926 
                 g777814 
                   
                   Homo sapiens  mRNA; cDNA DKFZp586G0623 (from clone DKFZp586G0623) 
               
               
                 80109 
                 Hs.198253 
                 T63324 
                 g667189 
                 HLA-DQA1 
                 major histocompatibility complex, class II, DQ alpha 1 
               
               
                 768370 
                 Hs.204354 
                 AA495846 
                 g2229167 
                 ARHB 
                 ras homolog gene family, member B 
               
               
                 795758 
                 Hs.179972 
                 AA460304 
                 g2185120 
                 G1P3 
                 interferon, alpha-inducible protein (clone IFI-6-16) 
               
               
                 839736 
                 Hs.1940 
                 AA504943 
                 g2241103 
                 CRYAB 
                 crystallin, alpha B 
               
               
                 783696 
                 Hs.75485 
                 AA446819 
                 g2159484 
                 OAT 
                 ornithine aminotransferase (gyrate atrophy) 
               
               
                 50506 
                 Hs.75465 
                 H17504 
                 g883744 
                 MAPK6 
                 mitogen-activated protein kinase 6 
               
               
                 773771 
                 Hs.85050 
                 AA427940 
                 g2112058 
                 PLN 
                 phospholamban 
               
               
                 813712 
                 Hs.181101 
                 AA453849 
                 g2167518 
                 ATP5F1 
                 ATP synthase, H+ transporting, mitochondrial F0 
               
               
                   
                   
                   
                   
                   
                 complex, subunit b, isoform 1 
               
               
                 502326 
                 Hs.184567 
                 AA156674 
                 g1728353 
                   
                 ESTs 
               
               
                 188036 
                 Hs.620 
                 H44784 
                 g920836 
                 BPAG1 
                 bullous pemphigoid antigen 1 (230/240 kD) 
               
               
                 840942 
                 Hs.814 
                 AA486627 
                 g2216791 
                 HLA-DPB1 
                 major histocompatibility complex, class II, DP beta 1 
               
               
                 208718 
                 Hs.78225 
                 H63077 
                 g1017878 
                 ANXA1 
                 annexin A1 
               
               
                 753104 
                 Hs.240217 
                 AA478553 
                 g2207187 
                 DCT 
                 dopachrome tautomerase (dopachrome delta-isomerase, 
               
               
                   
                   
                   
                   
                   
                 tyrosine-related protein 2) 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 2  
     Classification of Human Breast Cancers  
      A recent study on gene expression profiling of breast cancer identifies 70 genes whose expression pattern is strongly predictive of a short post-diagnosis and treatment interval to distant metastases (van&#39;t Veer, L. J., et al., “Gene expression profiling predicts clinical outcome of breast cancer,” Nature, 415: 530-536, 2002, incorporated herein by reference). The expression pattern of these 70 genes discriminates with 81% (optimized sensitivity threshold) or 83% (optimal accuracy threshold) accuracy the patient&#39;s prognosis in the group of 78 young women diagnosed with sporadic lymph-node-negative breast cancer. This group comprises 34 patients who developed distant metastases within 5 years and 44 patients who continued to be disease-free after a period of at least 5 years; they constitute a poor prognosis and good prognosis group, correspondingly.  
      We applied the methods of the present invention to further reduce the number of genes whose expression patterns represent genetic signatures of breast cancers with “poor prognosis” or “good prognosis.” Measurements of mRNA expression levels of 70 genes in established human breast carcinoma cell lines (MCF7; MDA-MB-435; MDA-MB-468; MDA-MB-23 1; MDA-MB-435Br1; MDA-MB-435BL3) and primary cultures of normal human breast epithelial cells were performed utilizing Q-PCR method, which generally is accepted as the current most reliable method of gene expression analysis and unambiguous confirmation of gene identity. Applying the methods of the present invention, for each breast cancer cell line, concordant sets of genes were identified exhibiting both positive and negative correlation between −fold expression changes in cancer cell lines versus control cell line and the poor prognosis group versus the good prognosis group. Minimum segregation sets were selected from corresponding concordance sets and individual phenotype association indices were calculated. Three top-performing breast cancer metastasis predictor gene clusters are listed in Tables 27-29, and corresponding phenotype association indices are presented in  FIGS. 34-36 .  
      A breast cancer poor prognosis predictor cluster comprising 6 genes was identified (r=0.981) using MDA-MB-468 cell line gene expression profile as a reference standard ( FIG. 34 ). 32 of 34 samples from the poor prognosis group had positive phenotype association indices, whereas 29 of 44 samples from the good prognosis group had negative phenotype association indices yielding an overall sample classification accuracy of 78%.  
               TABLE 27                          Breast Cancer Poor Prognosis Minimum Segregation Set 1.       6 genes (MDA-MB-468; Q-PCR) (r = 0.981)                         Systematic   Gene           name   name   Sequence description               NM_002019   FLT1   fms-related tyrosine kinase 1 (vascular               endothelial growth factor/vascular               permeability factor receptor)       U82987   BBC3   Bcl-2 binding component 3       NM_003239   TGFB3   transforming growth factor, beta 3       AF201951   MS4A7   high affinity immunoglobulin epsilon               receptor beta subunit       NM_000849   GSTM3   glutathione S-transferase M3 (brain)       NM_003862   FGF18   fibroblast growth factor 18                  
 
      A breast cancer good prognosis predictor cluster comprising 14 genes was identified (r=−0.952) using MDA-MB-435Br1 cell line gene expression profile as a reference standard ( FIG. 35 ). 30 of 34 samples from the poor prognosis group had negative phenotype association indices, whereas 34 of 44 samples from the good prognosis group had positive phenotype association indices yielding an overall sample classification accuracy of 82%.  
               TABLE 28                          Breast Cancer Good Prognosis Minimum Segregation Set 1.       MDA-MB-435Br1 (14 genes; Q-PCR) (r = −0.952)                         Systematic name   Gene name   Sequence description               AF201951   MS4A7   high affinity immunoglobulin epsilon               receptor beta subunit       NM_003239   TGFB3   transforming growth factor, beta 3       U82987   BBC3   Bcl-2 binding component 3       NM_001282   AP2B1   adaptor-related protein complex 2,               beta 1 subunit       NM_003748   ALDH4A1   aldehyde dehydrogenase 4 (glutamate               gamma-semialdehyde dehydrogenase;               pyrroline-5-carboxylate dehydro-               genase)       NM_018354   FLJ11190   hypothetical protein FLJ11190       NM_020188   DC13   DC13 protein       NM_003875   GMPS   guanine monphosphate synthetase       Contig57258_RC   AKAP2   ESTs       NM_000788   DCK   deoxycytidine kinase       Contig25991   ECT2   epithelial cell transforming               sequence 2 oncogene       Contig38288_RC       ESTs, Weakly similar to       NM_000436   OXCT   3-oxoacid CoA transferase       NM_000127   EXT1   exostoses (multiple) 1                  
 
      Another breast cancer good prognosis minimum segregation set 2 comprising 13 genes (r=−0.992) was identified using MCF7 cell line gene expression profile as a reference standard ( FIG. 36 ). 30 of 34 samples from the poor prognosis group had negative phenotype association indices, whereas 32 of 44 samples from the good prognosis group had positive phenotype association indices yielding overall sample classification accuracy of 79%.  
               TABLE 29                          Breast Cancer Good Prognosis Minimum Segregation Set 2.                     r = −0.992   System 1 (MCF7)                                     Locus Link           Systematic   Gene           Symbol   GenBank   UniGene   name   name   Gene Description               CEGP1       Hs.222399   NM_020974   CEGP1     Homo sapiens  CEGP1 protein (CEGP1), mRNA.       FGF18       Hs.49585   NM_003862   FGF18   fibroblast growth factor 18       GSTM3       Hs.2006   NM_000849   GSTM3   glutathione S-transferase M3 (brain)       TGFB3       Hs.2025   NM_003239   TGFB3   transforming growth factor, beta 3       CFFM4 or       Hs.11090   AF201951   MS4A7   high affinity immunoglobulin epsilon       MS4A7                   receptor beta subunit                                     AI918032   Hs.5521   Contig55377_RC   ESTs                                     AP2B1       Hs.74626   NM_001282   AP2B1   adaptor-related protein complex 2, beta 1 subunit       CCNE2       Hs.30464   NM_004702   CCNE2   cyclin E2       KIAA0175       Hs.184339   NM_014791   KIAA0175   KIAA0175 gene product       EXT1       Hs.184161   NM_000127   EXT1   exostoses (multiple) 1                                     AI813331   Hs.283127   Contig46218_RC   ESTs                                     PK428       Hs.44708   NM_003607   PK428   Ser-Thr protein kinase related to the myotonic                           dystrophy protein kinase                                     AI554061   Hs.309165   Contig38288_RC   ESTs, Weakly similar to quiescin [ H. sapiens ]                  
 
      To validate the classification accuracy using an independent data set, we tested performance of the 13 genes good prognosis predictor cluster (good prognosis minimum segregation set 2) on a set of 19 samples obtained from 11 breast cancer patients who developed distant metastases within five years after diagnosis and treatment and 8 patients who remained disease free for at least five years (van&#39;t Veer et al., 2002). As shown in  FIG. 37 , 9 of 11 samples from the poor prognosis group had negative phenotype association indices, whereas 6 of 8 samples from the good prognosis group had positive phenotype association indices yielding overall sample classification accuracy of 79%.  
     EXAMPLE 3  
     Classification of Human Ovarian Cancer  
      Lack of effective diagnostic and prognostic markers is generally considered a major problem in the clinical management of ovarian cancer—an epithelial neoplasm that has one of the worst prognoses among epithelial malignancies in women and is the leading cause of death from gynecologic cancer. The clinical utility of the most widely used biomarker of ovarian cancer, CA125, is largely limited to follow-up the response to therapy and progression of the disease and considered to be less efficient in diagnostic and prognostic applications (Meyer, T., Rustin, G. J. Br. J. Cancer, 82: 1535-1538, 2000, incorporated herein by reference).  
      We applied the methods of the present invention to identify gene expression profiles distinguishing poorly differentiated ovarian epithelial tumors, often exhibiting invasive, highly malignant phenotype, from less aggressive, well and moderately differentiated ovarian epithelial malignancies. Both clinical and cell line data sets utilized in this example were published in Welsh, J. B., et al., “Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer,” PNAS, 98: 1176-1181, 2001, incorporated herein by reference. As a starting point for identification of the concordant set of genes for established ovarian cancer cell lines and ovarian tumor tissue samples we utilized a set of the top 501 genes selected by a multidimensional statistical metric that was devised to identify genes with an expression pattern considered ideal for the molecular detection of epithelial ovarian cancer (Welsh et al., 2001). There determined that there was no significant correlation between the −fold changes in the expression levels of these 501 genes in the three cancer cell lines (SKOV8; MDA2774; CAOV3) compared to a control sample (HuOVR) and three poorly differentiated tumors (OVR — 1; OVR — 12; OVR — 27) compared to eleven moderately and well differentiated tumors (OVR — 1;  — 2;  — 5;  — 8;  — 10;  — 13;  — 16;  — 19;  — 22;  — 26;  — 28), (r=0.101).  
      According to the methods of present invention, we selected from the set of 501 genes two concordant sets of genes: concordant set 1 comprising 251 genes and exhibiting positive correlation (r=0.504) between cell lines and tissue samples data sets and concordant set 2, comprising 248 genes and exhibiting negative correlation (r=−0.296) between cell lines and clinical samples. We selected from concordance set 1 a set of 11 genes (ovarian cancer poor prognosis minimum segregation set 1) (ovarian cancer poor prognosis cluster—see Table 30) displaying a high positive correlation (r=0.988) between the cell lines and tissue samples data sets and exhibiting a 93% success rate in clinical sample classification based on individual phenotype association indices. As shown in  FIG. 38 , all three poorly differentiated tumors had positive phenotype association indices, whereas 10/11 well and moderately differentiated tumors displayed negative phenotype association indices.  
               TABLE 30                          Ovarian Cancer Poor Prognosis Minimum Segregation Set 1.       Poor Prognosis Predictor       Performance: 93% (13/14)       r = 0.988                     Affymetrix Probe Set           ID (HuFL6800)   Description               L22524_s_at   L22524, class B, 18 probes, 15 in L22524cds 462-734: 3 in           reverse Sequence, 46-197, Human matrilysin gene       U47077_at   U47077, class A, 20 probes, 20 in U47077 13025-13463, Human           DNA-dependent protein kinase catalytic subunit (DNA-PKcs)           mRNA, complete cds       U46006_s_at   U46006, class A, 20 probes, 20 in U46006 140-620, Human           smooth muscle LIM protein (h-SmLIM) mRNA, complete           cds. /gb = U46006 /ntype = RNA       L40357_at   L40357, class A, 20 probes, 20 in L40357mRNA 7-463,  Homo               sapiens  thyroid receptor interactor (TRIP7) mRNA, 3′ end of cds       M64098_at   M64098, class A, 20 probes, 20 in M64098 3873-4305, Human           high density lipoprotein binding protein (HBP) mRNA, complete           cds       D79993_at   D79993, class A, 20 probes, 20 in D79993 2741-3167, Human           mRNA for KIAA0171 gene, complete cds       U15085_at   U15085, class A, 20 probes, 20 in U15085 821-1289, Human           HLA-DMB mRNA, complete cds       U60975_at   U60975, class A, 20 probes, 20 in U60975 6398-6824, Human           hybrid receptor gp250 precursor mRNA, complete cds       M79462_at   M79462, class A, 20 probes, 20 in M79462 3853-4333, Human           PML-1 mRNA, complete CDS       Z23090_at   Z23090, class A, 20 probes, 17 in Z23090cds 277-589: 3 in           reverse Sequence, 1086-1098,  H. sapiens  mRNA for 28 kDa heat           shock protein       X03635_at   X03635, class C, 20 probes, 20 in all_X03635 5885-6402,           Human mRNA for oestrogen receptor                  
 
      Applying the methods of the present invention, we selected from concordance set 2 a set of 10 genes (ovarian cancer good prognosis minimum segregation set 1) (ovarian cancer good prognosis cluster—see Table 31) displaying a high negative correlation (r=−0.964) between the tumor cell lines and clinical samples data sets and exhibiting a 93% success rate in clinical sample classification based on individual phenotype association indices. As shown in  FIG. 39 , all three poorly differentiated tumors had negative phenotype association indices, whereas 10/11 well and moderately differentiated tumors displayed positive phenotype association indices.  
               TABLE 31                          Ovarian Cancer Good Prognosis Minimum Segregation Set 1       Good Prognosis Predictor       Performance: 93% (13/14)       r = −0.964                     Affymetrix           Probe Set ID       (HuFL6800)   Description               U90551_at   U90551, class A, 20 probes, 20 in U90551 1071-           1623, Human histone 2A-like protein (H2A/l)           mRNA, complete cds       L19779_at   L19779, class A, 20 probes, 20 in L19779 7-496,             Homo sapiens  histone H2A.2 mRNA, complete           cds       M90657_at   M90657, class A, 20 probes, 20 in M90657 581-           1163, Human tumor antigen (L6) mRNA, complete           cds       M13755_at   M13755, class A, 20 probes, 20 in M13755mRNA           33-591, Human interferon-induced 17-kDa/15-kDa           protein mRNA, complete cds       U90915_at   U90915, class A, 20 probes, 20 in U90915 122-           674, Human clone 23600 cytochrome c oxidase           subunit IV mRNA, complete cds       Z74792_s_at   Z74792, class A, 20 probes, 20 in Z74792mRNA           1470-1917,  H. sapiens  mRNA for CCAAT           transcription binding factor subunit gamma.       X99325_at   X99325, class C, 20 probes, 20 in all_X99325           1482-1927,  H. sapiens  mRNA for Ste20-like kinase       HG2614-   Collagen, Type Viii, Alpha 1       HT2710_at       J03242_s_at   J03242, class A, 20 probes, 20 in J03242 1155-           1324, Human insulin-like growth factor II mRNA,           complete cds       D86983_at   D86983, class A, 20 probes, 20 in D86983 5131-           5485, Human mRNA for KIAA0230 gene, partial           cds                  
 
     EXAMPLE 4  
     Classification of Human Lung Cancer  
      Lung cancer accounts for more than 150,000 cancer-related deaths every year in the United States, thus exceeding the combined mortality caused by breast, prostate, and colorectal cancers (Greenlee, R. T., Hill-Harmon, M. B., Murray, T., Thun, M. CA Cancer J. Clin. 51: 15-36, 2001, incorporated herein by reference). Late stage of cancer at diagnosis and lack of efficient diagnostic and prognostic biomarkers are significant factors that adversely affect the clinical management of lung cancer (Mountain, C. F. Revisions in the international system for staging lung cancer. Chest, 111:1710-1717, 1997; Ihde, D. C. Chemotherapy of lung cancer. N. Engl. J. Med., 327:1434-1441, 1992; Sugita, M., Geraci, M., Gao, B., Powell, R. L., Hirsch, F. R., Johnson, G., Lapadat, R., Gabrielson, E., Bremnes, R., Bunn, P. A., Franklin, W. A. Combined use of oligonucleotide and tissue microarrays identifies cancer/testis antigens as biomarkers in lung cancer. Cancer Res., 62:3971-3979, 2002). Non-small-cell lung carcinoma (NSCLC) is a clinically and histopathologically distinct major form of lung cancer and is further classified as adenocarcinoma (most common form of NSCLC), squamous cell carcinoma, and large-cell carcinoma (Travis, W. D., Travis, L. B., Devesa, S. S. Cancer, 75:191-202, 1995).  
      We applied the methods of the present invention to identify gene expression profiles distinguishing lung adenoracinoma samples from normal lung specimens as well as a highly malignant phenotype of lung adenocarcinoma, associated with short survival after diagnosis and therapy, from less aggressive lung cancers, associated with longer patient survival. Both clinical and cell line data sets utilized in this example were published (Clinical data: Bhattacharjee, A., Richards, W. G., Staunton, J., Li, C., Monti, S., Vasa, P., Ladd, C., Beheshti, J., Bueno, R., Gillette, M., Loda, M., Weber, G., Mark, E. J., Lander, E. S., Wong, W., Johnson, B. E., Golub, T. R., Sugarbaker, D. J., Meyerson, M. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. PNAS, 98: 13790-13795, 2001; incorporated herein by reference; Cell line data: Sugita, M., Geraci, M., Gao, B., Powell, R. L., Hirsch, F. R., Johnson, G., Lapadat, R., Gabrielson, E., Bremnes, R., Bunn, P. A., Franklin, W. A. Combined use of oligonucleotide and tissue microarrays identifies cancer/testis antigens as biomarkers in lung cancer. Cancer Res., 62:3971-3979, 2002; incorporated herein by reference. As a starting point for identification of the concordant set of genes for established lung cancer cell lines and lung cancer tissue samples we utilized a set of the 675 transcripts selected based on a statistical analysis of the quality of the dataset and variability of gene expression across dataset (Bhattacharje et al., 2001). Initial analysis showed that there was no significant correlation between the −fold changes in the expression levels of these 675 genes in the two NSCLC cancer cell lines (H647 and A549 cell lines) compared to a control sample (normal bronchial epithelial cell cultures obtained from a healthy 48-year-old donor) and 139 samples of lung adenoracinomas compared to the 17 normal lung specimens (r=0.163).  
      According to the methods of present invention, we selected from the set of 675 genes a concordant set of transcripts comprising 355 genes and exhibiting positive correlation (r=0.523) between cell lines and tissue samples data sets. Next we selected from the concordant set of 355 genes two minimum segregation sets of genes: a set of 13 genes (lung adenoracinoma minimum segregation set 1, also referred to as lung adenocarcinoma cluster 1—see Table 32) and a set of 26 genes (lung adenoracinoma minimum segregation set 2, also referred to as lung adenocarcinoma cluster 2—see Table 33) both displaying high positive correlation (r=0.979 and r=0.966, respectively) between the cell lines and tissue samples data sets ( FIGS. 40 and 41 ). For each minimum segregation set we calculated the individual phenotype association indices for 17 normal lung samples and 139 lung adenocarcinoma samples. After adjustment of the dataset by subtracting 0.52 from all the phenotype association indices, both gene clusters exhibited a 96% success rate in clinical sample classification based on individual phenotype association indices ( FIGS. 42 and 43 ). The adjustment was made following visual inspection of the raw data indicating that 0.52 was a useful threshold for discriminating normal lung samples from lung adenocarcinoma samples, and had the added benefit of allowing classification to be carried out according to the sign of the phenotype association index. Without wishing to be bound by theory, it appears likely that the adjustment was necessary because the published datasets used for constructing this example were derived from different groups using non-identical data reduction methods. As shown in  FIGS. 42 and 43 , 16/17 normal lung samples had negative phenotype association indices, whereas 134/139 of lung adenocarcinoma specimens displayed positive phenotype association indices. When scores from the two clusters were considered and a criterion of at least one positive phenotype association index was adopted for assigning a lung adenocarcinoma classification, the classification success rate was 99%. 16/17 (94%) normal lung samples had two negative phenotype association indices, whereas 131/139 of lung adenocarcinoma specimens displayed two positive phenotype association indices, seven of 139 had at least one positive phenotype association index, and only a single lung adenocarcinoma specimen had two negative phenotype association indices. Thus, 154/156 (99%) of clinical lung adenocarcinima samples were correctly classified using this strategy.  
               TABLE 32                          Lung adenocarcinoma minimum segregation set 1.       13 genes (r = 0.979)                     Affymetrix           Probe Set       ID (U95Av2)   Description               34342_s_at   secreted phosphoprotein 1 (osteopontin, bone sialo-           protein I, early T-lymphocyte activation 1)       2092_s_at   secreted phosphoprotein 1 (osteopontin, bone sialo-           protein I, early T-lymphocyte activation 1)       31798_at   Cluster Incl AA314825: EST186646  Homo sapiens             cDNA, 5end /clone = ATCC-111986 /clone_end =           5″ /gb = AA314825 /gi = 1967154 /ug =           Hs.1406 /len = 574″       668_s_at   matrix metalloproteinase 7 (matrilysin, uterine)       31599_f_at   melanoma antigen, family A, 6       39008_at   ceruloplasmin (ferroxidase)       31844_at   homogentisate 1,2-dioxygenase (homogentisate oxi-           dase)       31477_at   trefoil factor 3 (intestinal)       38825_at   fibrinogen, A alpha polypeptide       32306_g_at   collagen, type I, alpha 2       32773_at   Cluster Incl AA868382: ak41e04.s1  Homo sapiens             cDNA, 3 end /clone = IMAGE-1408542 /clone_end =           3″ /gb = AA868382 /gi = 2963827 /ug =           Hs.198253 /len = 936″       36623_at   Cluster Incl AB011406:  Homo sapiens  mRNA for alkalin           phosphatase, complete cds /cds = (176, 1750) /gb =           AB011406 /gi = 3401944 /ug = Hs.75431 /len = 2510       31870_at   CD37 antigen                  
 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                   
               
               
                 Lung adenocarcinoma minimum segregation set 2. 
               
               
                 26 genes (r = 0.966) 
               
            
           
           
               
               
            
               
                 Affymetrix Probe 
                   
               
               
                 Set ID (U95Av2) 
                 Description 
               
               
                   
               
               
                 33904_at 
                 claudin 3 
               
               
                 1481_at 
                 matrix metalloproteinase 12 (macrophage elastase) 
               
               
                 38261_at 
                 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 
               
               
                 1586_at 
                 insulin-like growth factor binding protein 3 
               
               
                 38066_at 
                 diaphorase (NADH/NADPH) (cytochrome b-5 reductase) 
               
               
                 34575_f_at 
                 melanoma antigen, family A, 5 
               
               
                 41583_at 
                 flap structure-specific endonuclease 1 
               
               
                 32787_at 
                 v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 
               
               
                 1788_s_at 
                 dual specificity phosphatase 4 
               
               
                 32805_at 
                 aldo-keto reductase family 1, member C1 (dihydrodiol 
               
               
                   
                 dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase) 
               
               
                 39260_at 
                 solute carrier family 16 (monocarboxylic acid transporters), member 
               
               
                   
                 4 
               
               
                 41748_at 
                 Cluster Incl AA196476: zp99g10.r1  Homo sapiens  cDNA, 5 
               
               
                   
                 end /clone = IMAGE-628386 /clone_end = 5″ /gb = 
               
               
                   
                 AA196476 /gi = 1792058 /ug = Hs.182421 /len = 697″ 
               
               
                 38656_s_at 
                 Cluster Incl W27939: 39g3  Homo sapiens  cDNA /gb = 
               
               
                   
                 W27939 /gi = 1307887 /ug = Hs.103834 /len = 862 
               
               
                 823_at 
                 small inducible cytokine subfamily D (Cys-X3-Cys), member 1 
               
               
                   
                 (fractalkine, neurotactin) 
               
               
                 32052_at 
                 hemoglobin, beta 
               
               
                 36979_at 
                 solute carrier family 2 (facilitated glucose transporter), member 3 
               
               
                 40367_at 
                 bone morphogenetic protein 2 
               
               
                 36937_s_at 
                 PDZ and LIM domain 1 (elfin) 
               
               
                 40567_at 
                 Tubulin, alpha, brain-specific 
               
               
                 33900_at 
                 follistatin-like 3 (secreted glycoprotein) 
               
               
                 34320_at 
                 Cluster Incl AL050224:  Homo sapiens  mRNA; cDNA DKFZp586L2123 (from 
               
               
                   
                 clone DKFZp586L2123) /cds = UNKNOWN /gb = AL050224 /gi = 
               
               
                   
                 4884466 /ug = Hs.29759 /len = 1250 
               
               
                 37027_at 
                 AHNAK nucleoprotein (desmoyokin) 
               
               
                 31622_f_at 
                 metallothionein 1F (functional) 
               
               
                 609_f_at 
                 metallothionein 1B (functional) 
               
               
                 37951_at 
                 deleted in liver cancer 1 
               
               
                 31687_f_at 
                 hemoglobin, beta 
               
               
                   
               
            
           
         
       
     
      Next we applied the methods of the present invention to identify gene expression profiles distinguishing highly malignant phenotype of lung adenocarcinoma, associated with short patient survival after diagnosis and therapy, from less aggressive lung cancers, associated with longer patient survival. Using the clinical data set and associated clinical history published in Bhattacharje et al., 2001, we selected two groups of adenocarcinoma patients having markedly distinct survival after diagnosis and therapy: poor prognosis group 1 comprising 34 patients with median survival of 8.5 months (range 0.1-17.3 months) and good prognosis group 2 comprising 16 patients with median survival of 84 months (range 75.4-106.1 months).  
      Applying the methods of the present invention, we selected from the set of 675 genes a concordant set of transcripts comprising 302 genes and exhibiting positive correlation (r=0.444) between cell lines data (NSCLC cell lines versus normal bronchial epithelial cells) and tissue samples data sets (poor prognosis samples versus good prognosis samples). We selected from the concordant set of 302 genes a set of 38 genes (lung adenocarcinoma poor prognosis predictor cluster 1—see Table 34) displaying high positive correlation (r=0.881) between the cell lines and tissue samples data sets (FIG.  
      44). This gene cluster exhibited a 64% success rate in clinical sample classification based on individual phenotype association indices ( FIG. 45 ). As shown in  FIG. 45 , 16/16 of the lung adenocarcinoma samples of the good prognosis group had negative phenotype association indices, whereas 16/34 of lung adenocarcinoma specimens of the poor prognosis group displayed positive phenotype association indices.  
               TABLE 34                          Lung adenocarcinoma poor prognosis predictor cluster 1.       38 genes (r = 0.881)                     Affymetrix Probe           Set ID (U95Av2)   Description               36990_at   ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)       33998_at   neurotensin       1481_at   matrix metalloproteinase 12 (macrophage elastase)       36555_at   synuclein, gamma (breast cancer-specific protein 1)       38389_at   2′,5′-oligoadenylate synthetase 1 (40-46 kD)       33128_s_at   Cluster Incl W68521: zd36f07.r1  Homo sapiens  cDNA, 5           end /clone = IMAGE-342757 /clone_end = 5″ /gb =           W68521 /gi = 1377410 /ug = Hs.83393 /len = 579″       40297_at   six transmembrane epithelial antigen of the prostate       41531_at   Cluster Incl AI445461: tj34g07.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2143452 /clone_end = 3″ /gb =           AI445461 /gi = 4288374 /ug = Hs.3337 /len = 775″       892_at   transmembrane 4 superfamily member 1       32821_at   Cluster Incl AI762213: wi54d04.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2394055 /clone_end = 3″ /gb =           AI762213 /gi = 5177880 /ug = Hs.204238 /len = 677″       1651_at   ubiquitin carrier protein E2-C       37921_at   neuronal pentraxin I       36302_f_at   melanoma antigen, family A, 4       32426_f_at   melanoma antigen, family A, 1 (directs expression of antigen MZ2-           E)       32607_at   brain acid-soluble protein 1       41471_at   Cluster Incl W72424: zd66a09.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-345592 /clone_end = 3″ /gb =           W72424 /gi = 1382379 /ug = Hs.112405 /len = 604″       41758_at   chromosome 22 open reading frame 5       38354_at   CCAAT/enhancer binding protein (C/EBP), beta       195_s_at   caspase 4, apoptosis-related cysteine protease       33267_at   Cluster Incl AF035315:  Homo sapiens  clone 23664 and 23905           mRNA sequence /cds = UNKNOWN /gb = AF035315 /gi =           2661077 /ug = Hs.180737 /len = 1331       39341_at   Cluster Incl AJ001902:  Homo sapiens  mRNA for TRIP6 (thyroid           receptor interacting protein) /cds = (72, 1502) /gb =           AJ001902 /gi = 2558591 /ug = Hs.119498 /len = 1653       34445_at   KIAA0471 gene product       36201_at   glyoxalase I       36736_f_at   phosphoserine phosphatase       1057_at   cellular retinoic acid-binding protein 2       32072_at   mesothelin       37811_at   calcium channel, voltage-dependent, alpha 2/delta subunit 2       41771_g_at   Cluster Incl AA420624: nc61c12.r1  Homo sapiens             cDNA /clone = IMAGE-745750 /gb = AA420624 /gi =           2094502 /ug = Hs.183109 /len = 533       41770_at   Cluster Incl AA420624: nc61c12.r1  Homo sapiens             cDNA /clone = IMAGE-745750 /gb = AA420624 /gi =           2094502 /ug = Hs.183109 /len = 533       41772_at   monoamine oxidase A       40004_at   sine oculis homeobox ( Drosophila ) homolog 1       40367_at   bone morphogenetic protein 2       40508_at   glutathione S-transferase A4       33754_at   thyroid transcription factor 1       32154_at   transcription factor AP-2 alpha (activating enhancer-binding protein           2 alpha)       37600_at   extracellular matrix protein 1       37874_at   flavin containing monooxygenase 5       37208_at   phosphoserine phosphatase-like                  
 
      Using the sample iteration and cluster reduction strategies described in the previous examples, we selected four additional sets of genes displaying high positive correlation between the cell lines (NSCLC cell lines versus normal bronchial epithelial cells) and tissue samples data sets (poor prognosis samples versus good prognosis samples) (see Tables 35-38) and thus having potential discriminating power in classification of lung adenocarcinoma samples.  
               TABLE 35                          Lung adenocarcinoma poor prognosis predictor cluster 2.       19 genes (r = 0.938)                     Affymetrix           Probe Set       ID (U95Av2)   Description               36555_at   synuclein, gamma (breast cancer-specific           protein 1)       41531_at   Cluster Incl AI445461: tj34g07.x1  Homo sapiens             cDNA, 3 end /clone = IMAGE-2143452 /clone_end =           3″ /gb = AI445461 /gi = 4288374 /ug =           Hs.3337 /len = 775″       1868_g_at   CASP8 and FADD-like apoptosis regulator       37921_at   neuronal pentraxin I       37918_at   integrin, beta 2 (antigen CD18 (p95), lympho-           cyte function-associated antigen 1; macrophage           antigen 1 (mac-1) beta subunit)       38422_s_at   four and a half LIM domains 2       39114_at   decidual protein induced by progesterone       34375_at   small inducible cytokine A2 (monocyte chemo-           tactic protein 1, homologous to mouse Sig-je)       36495_at   fructose-1,6-bisphosphatase 1       37187_at   GRO2 oncogene       37014_at   myxovirus (influenza) resistance 1, homolog           of murine (interferon-inducible protein p78)       925_at   interferon, gamma-inducible protein 30       39372_at   Cluster Incl W26480: 30b8  Homo sapiens             cDNA /gb = W26480 /gi = 1307179 /ug =           Hs.12214 /len = 854       32072_at   mesothelin       41771_g_at   Cluster Incl AA420624: nc61c12.r1  Homo sapiens             cDNA /clone = IMAGE-745750 /gb = AA420624 /gi =           2094502 /ug = Hs.183109 /len = 533       40508_at   glutathione S-transferase A4       41772_at   monoamine oxidase A       40004_at   sine oculis homeobox ( Drosophila ) homolog 1       37600_at   extracellular matrix protein 1                  
 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                   
               
               
                 Lung adenocarcinoma poor prognosis predictor cluster 3. 
               
               
                 23 genes (r = 0.891) 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set 
               
               
                 ID (U95Av2) 
                 Description 
               
               
                   
               
               
                 41106_at 
                 potassium intermediate/small conductance 
               
               
                   
                 calcium-activated channel, subfamily N, member 
               
               
                   
                 4 
               
               
                 1868_g_at 
                 CASP8 and FADD-like apoptosis regulator 
               
               
                 41471_at 
                 Cluster Incl W72424: zd66a09.s1  Homo sapiens   
               
               
                   
                 cDNA, 3 end /clone = IMAGE-345592 /clone_end = 
               
               
                   
                 3″ /gb = W72424 /gi = 1382379 /ug = 
               
               
                   
                 Hs.112405 /len = 604″ 
               
               
                 37921_at 
                 neuronal pentraxin I 
               
               
                 38422_s_at 
                 four and a half LIM domains 2 
               
               
                 39114_at 
                 decidual protein induced by progesterone 
               
               
                 34375_at 
                 small inducible cytokine A2 (monocyte chemo- 
               
               
                   
                 tactic protein 1, homologous to mouse Sig-je) 
               
               
                 36495_at 
                 fructose-1,6-bisphosphatase 1 
               
               
                 37187_at 
                 GRO2 oncogene 
               
               
                 37014_at 
                 myxovirus (influenza) resistance 1, homolog of 
               
               
                   
                 murine (interferon-inducible protein p78) 
               
               
                 925_at 
                 interferon, gamma-inducible protein 30 
               
               
                 35766_at 
                 keratin 18 
               
               
                 39372_at 
                 Cluster Incl W26480: 30b8  Homo sapiens   
               
               
                   
                 cDNA /gb = W26480 /gi = 1307179 /ug = 
               
               
                   
                 Hs.12214 /len = 854 
               
               
                 32072_at 
                 mesothelin 
               
               
                 40422_at 
                 insulin-like growth factor binding protein 2 
               
               
                   
                 (36 kD) 
               
               
                 41771_g_at 
                 Cluster Incl AA420624: nc61c12.r1  Homo sapiens   
               
               
                   
                 cDNA /clone = IMAGE-745750 /gb = AA420624 /gi = 
               
               
                   
                 2094502 /ug = Hs.183109 /len = 533 
               
               
                 40508_at 
                 glutathione S-transferase A4 
               
               
                 1741_s_at 
                 S37730 /FEATURE = cds /DEFINITION = S37712S4 
               
               
                   
                 insulin-like growth factor binding protein-2 
               
               
                   
                 [human, placenta, Genomic, 1342 nt, segment 4 of 4] 
               
               
                 41772_at 
                 monoamine oxidase A 
               
               
                 37874_at 
                 flavin containing monooxygenase 5 
               
               
                 37811_at 
                 calcium channel, voltage-dependent, alpha 2/ 
               
               
                   
                 delta subunit 2 
               
               
                 40004_at 
                 sine oculis homeobox ( Drosophila ) homolog 1 
               
               
                 37600_at 
                 extracellular matrix protein 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                   
               
               
                 Lung adenocarcinoma poor prognosis predictor cluster 4. 
               
               
                 10 genes (r = 0.872) 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set 
               
               
                 ID (U95Av2) 
                 Description 
               
               
                   
               
               
                 34342_s_at 
                 secreted phosphoprotein 1 (osteopontin, bone sialopro- 
               
               
                   
                 tein I, early T-lymphocyte activation 1) 
               
               
                 2092_s_at 
                 secreted phosphoprotein 1 (osteopontin, bone sialopro- 
               
               
                   
                 tein I, early T-lymphocyte activation 1) 
               
               
                 37019_at 
                 fibrinogen, B beta polypeptide 
               
               
                 38825_at 
                 fibrinogen, A alpha polypeptide 
               
               
                 37233_at 
                 oxidised low density lipoprotein (lectin-like) 
               
               
                   
                 receptor 1 
               
               
                 31512_at 
                 immunoglobulin kappa variable 1-13 
               
               
                 36736_f_at 
                 phosphoserine phosphatase 
               
               
                 37811_at 
                 calcium channel, voltage-dependent, alpha 2/delta 
               
               
                   
                 subunit 2 
               
               
                 40004_at 
                 sine oculis homeobox ( Drosophila ) homolog 1 
               
               
                 37874_at 
                 flavin containing monooxygenase 5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                   
               
               
                 Lung adenocarcinoma poor prognosis predictor cluster 5. 
               
               
                 6 genes (r = 0.918) 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set 
               
               
                 ID 
                 Description 
               
               
                   
               
               
                 34342_s_at 
                 secreted phosphoprotein 1 (osteopontin, bone sialoprotein 
               
               
                   
                 I, early T-lymphocyte activation 1) 
               
               
                 38825_at 
                 fibrinogen, A alpha polypeptide 
               
               
                 31512_at 
                 immunoglobulin kappa variable 1-13 
               
               
                 37811_at 
                 calcium channel, voltage-dependent, alpha 2/delta 
               
               
                   
                 subunit 2 
               
               
                 40004_at 
                 sine oculis homeobox ( Drosophila ) homolog 1 
               
               
                 37874_at 
                 flavin containing monooxygenase 5 
               
               
                   
               
            
           
         
       
     
      The scoring summary of the individual phenotype association indices calculated for each of the five poor prognosis predictor clusters are presented in Table 39 for the good prognosis patients and in Table 40 for the poor prognosis patients. Only a single patient in the good prognosis group had one positive association index. All the remaining 15 good prognosis patients had negative phenotype association indices for each of the five poor prognosis gene clusters (Table 39). In contrast, 30 of 34 poor prognosis patients had at least one positive association index and 27 of 34 poor prognosis patients scored at least two positive phenotype association indices (Table 40). Thus, applying the methods of the present invention and applying a criterion requiring at least 1 positive phenotype association index for poor prognosis classification, 45 of 50 (90%) adenocarcinoma patients in this data set could be correctly classified as having a good or a poor prognosis.  
               TABLE 39                          Scoring summary of the lung adenocarcinoma poor prognosis       gene clusters for good prognosis patients.                                             38 genes   19 genes   23 genes   10 genes   6 genes   Number of false                         Sample   Phenotype Association Indices   classifications                                                 AD187   −0.06452   −0.04784   0.452696   −0.00941   −0.23775   1       AD119   −0.29927   −0.33723   −0.1148   −0.28902   −0.23916   0       AD131   −0.17964   −0.48139   −0.33392   −0.401   −0.17498   0       AD163   −0.12353   −0.28925   −0.0734   −0.15033   −0.01296   0       AD170   −0.17682   −0.49435   −0.34161   −0.32239   −0.64159   0       AD186   −0.34093   −0.61548   −0.28551   −0.37547   −0.19218   0       AD203   −0.50111   −0.52408   −0.06856   −0.14395   −0.21014   0       AD250   −0.27238   −0.25103   −0.12624   −0.68264   −0.68955   0       AD305   −0.17459   −0.36628   −0.29005   −0.11941   −0.39534   0       AD308   −0.6101   −0.03024   −0.02817   −0.40192   −0.34151   0       AD317   −0.276   −0.56248   −0.16234   −0.57284   −0.51591   0       AD318   −0.08142   −0.60361   −0.52572   −0.30083   −0.54905   0       AD320   −0.09336   −0.40628   −0.09197   −0.16229   −0.29432   0       AD327   −0.05072   −0.11578   −0.1069   −0.11479   −0.49102   0       AD338   −0.49705   −0.45102   −0.26864   −0.803   −0.84779   0       AD367   −0.03213   −0.22574   −0.30494   −0.5605   −0.39852   0                  
 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                   
               
               
                 Scoring summary of the lung adenocarcinoma poor prognosis 
               
               
                 gene clusters for poor prognosis patients. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 38 genes 
                 19 genes 
                 23 genes 
                 10 genes 
                 6 genes 
                 Number of correct 
               
            
           
           
               
               
               
            
               
                 Sample 
                 Phenotype Association Indices 
                 classification 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 AD277 
                 0.234435 
                 0.410067 
                 0.736989 
                 0.574246 
                 0.712075 
                 5 
               
               
                 AD330 
                 0.413889 
                 0.175061 
                 0.101943 
                 0.382497 
                 0.242026 
                 5 
               
               
                 AD374 
                 0.055386 
                 0.455203 
                 0.549645 
                 0.002916 
                 0.052327 
                 5 
               
               
                 AD177 
                 0.304326 
                 0.55951 
                 0.423434 
                 −0.08411 
                 0.479041 
                 4 
               
               
                 AD258 
                 0.43388 
                 −0.05816 
                 0.293763 
                 0.558311 
                 0.70477 
                 4 
               
               
                 AD276 
                 0.171625 
                 −0.53343 
                 0.415923 
                 0.713297 
                 0.80945 
                 4 
               
               
                 AD287 
                 0.233826 
                 −0.14383 
                 0.281022 
                 0.069933 
                 0.221046 
                 4 
               
               
                 AD323 
                 −0.1194 
                 0.267964 
                 0.027922 
                 0.140244 
                 0.399934 
                 4 
               
               
                 AD352 
                 0.115964 
                 0.041747 
                 0.196362 
                 0.622718 
                 0.802551 
                 4 
               
               
                 AD157 
                 −0.08334 
                 0.179166 
                 0.102028 
                 −0.1272 
                 0.294908 
                 3 
               
               
                 AD164 
                 0.281754 
                 0.608169 
                 0.31086 
                 −0.10786 
                 −0.4293 
                 3 
               
               
                 AD208 
                 0.236001 
                 0.310463 
                 0.230929 
                 −0.23772 
                 −0.70165 
                 3 
               
               
                 AD221 
                 −0.23875 
                 −0.42763 
                 0.261846 
                 0.292941 
                 0.749037 
                 3 
               
               
                 AD236 
                 0.172613 
                 −0.4351 
                 0.155221 
                 −0.05824 
                 0.650534 
                 3 
               
               
                 AD275 
                 −0.04808 
                 0.203627 
                 0.111381 
                 0.050702 
                 −0.17309 
                 3 
               
               
                 AD296 
                 0.438626 
                 0.52086 
                 0.084982 
                 −0.57093 
                 −0.9214 
                 3 
               
               
                 AD301 
                 0.048676 
                 −0.41297 
                 −0.27021 
                 0.15905 
                 0.049724 
                 3 
               
               
                 AD043 
                 0.047335 
                 −0.00851 
                 0.357719 
                 −0.15053 
                 −0.23508 
                 2 
               
               
                 AD127 
                 −0.07916 
                 −0.3513 
                 0.273233 
                 −0.03922 
                 0.184294 
                 2 
               
               
                 AD262 
                 −0.05662 
                 0.287899 
                 0.423555 
                 −0.23891 
                 −0.12164 
                 2 
               
               
                 AD304 
                 −0.21516 
                 0.186401 
                 0.076621 
                 −0.25509 
                 −0.18305 
                 2 
               
               
                 AD332 
                 0.241748 
                 0.198359 
                 −0.20156 
                 −0.22034 
                 −0.06101 
                 2 
               
               
                 AD334 
                 0.234121 
                 −0.32246 
                 −0.47165 
                 0.357084 
                 −0.03519 
                 2 
               
               
                 AD346 
                 −0.54482 
                 −0.40513 
                 0.228292 
                 −0.22006 
                 0.355989 
                 2 
               
               
                 AD361 
                 −0.46304 
                 0.368086 
                 0.071209 
                 −0.455 
                 −0.48077 
                 2 
               
               
                 AD363 
                 −0.33631 
                 −0.1249 
                 −0.12018 
                 0.161188 
                 0.075687 
                 2 
               
               
                 AD384 
                 −0.20144 
                 −0.3584 
                 0.451957 
                 −0.13904 
                 0.870275 
                 2 
               
               
                 AD130 
                 −0.17359 
                 −0.26894 
                 0.414704 
                 −0.2768 
                 −0.41716 
                 1 
               
               
                 AD225 
                 −0.14786 
                 −0.2287 
                 0.072267 
                 −0.0685 
                 −0.35463 
                 1 
               
               
                 AD353 
                 −0.61406 
                 −0.52593 
                 0.187469 
                 −0.89949 
                 −0.97919 
                 1 
               
               
                 AD201 
                 −0.08499 
                 −0.4772 
                 −0.47199 
                 −0.23861 
                 −0.54777 
                 0 
               
               
                 AD252 
                 −0.07534 
                 −0.4901 
                 −0.35684 
                 −0.23247 
                 −0.15586 
                 0 
               
               
                 AD347 
                 −0.5658 
                 −0.52075 
                 −0.31889 
                 −0.60543 
                 −0.92335 
                 0 
               
               
                 AD366 
                 −0.34494 
                 −0.56913 
                 −0.24398 
                 −0.14348 
                 −0.43697 
                 0 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 5  
     Orthotopic Xenograft Gene Expression Profile as Predictive Reference of Expected Transcript Abundance Behavior in Clinical Samples and Use to Identify Gene Clusters with Clinically Useful Properties.  
      When human cancer cells derived from the metastatic tumors are injected into ectopic sites in nude mice most do not metastasize (1, 2). The host tissue environment influences metastatic ability of cancer cells in such a way that many human and animal tumors transplanted into nude mice metastasize only if placed in the orthotopic organ (3-8). Several orthotopic models of human cancer metastasis have been developed (9-15). The orthotopic model of human cancer metastasis in nude mice was utilized for in vivo selection of highly and poorly metastatic cell variants (6, 13-15). This approach was successfully applied for development of human prostate cancer cell variants with distinct metastatic potential (15). Experimental evidence indicates that enhancement of metastatic capability of human cancer cells transplanted orthotopically is associated with differential expression of several metastasis-associated genes that have been implicated earlier in certain key features of the metastatic phenotype (16). It is well established that even highly metastatic cells, when implanted ectopically, are not able to consistently produce metastasis.  
      Here we identified metastasis-associated gene expression signatures based on expression profiling human prostate carcinoma xenografts derived from the same highly metastatic variant implanted at orthotopic (metastasis promoting setting) and ectopic (metastasis suppressing setting) sites, demonstrating that distinct malignant behavior of highly metastatic cells associated with the site of inoculation in a nude mouse is dependent upon differential gene expression in prostate cancer cells implanted either orthotopically or ectopically. We utilized the Affymetrix GeneChip system to compare the expression profiles of 12,625 transcripts in highly metastatic variant PC-3MLN4 implanted at orthotopic (metastasis promoting setting) (“PC3MLN40R”) and ectopic (metastasis suppressing setting) (“PC3MLN4SC”) sites. PC-3MLN4 tumors growing in orthotopic metastasis-promoting setting appear to dramatically over-express a set of genes with well-established invasion-activation functions ( FIG. 46 ). Changes in expression for each transcript are plotted as Log10Fold Change Average expression level in PC-3MLN40R versus Average expression level in less metastatic parental PC30R and PC3MOR (recurrence signatures) ( FIG. 47A ) or versus Average expression level in PC3PC-3MLN4SC (invasion signatures) ( FIG. 47B ) and Log10Fold Change Average expression level in aggressive (recurrent or invasive) versus Average expression level in corresponding non-aggressive (non-recurrent or non-invasive) clinical phenotypes. Expression profiling of the 12,625 transcripts in the orthotopic and s.c. xenografts derived from the cell variants of the PC-3 lineage was carried out. Transcripts differentially expressed at the statistically significant level (p&lt;0.05; T-test) in the orthotopic PC-3M-LN4 tumors compared to the s.c. tumors of the same lineage as well as orthotopic tumors derived from the less metastatic parental PC-3M and PC-3 cell lines were identified using the Affymetrix MicroDB and Affymetrix DMT software. Similarly, transcripts differentially regulated in the 8 recurrent versus 13 non-recurrent ( FIG. 47A ) or 26 invasive versus 26 non-invasive ( FIG. 47B ) human prostate tumors at the statistically significant level (p&lt;0.05; T-test) were identified. The small clusters of genes exhibiting highly concordant gene expression patterns in the xenograft model and clinical setting were identified using the methods of the invention. In the first example ( FIG. 47A ), comparisons of the average fold expression changes in highly metastatic PC3MLN4 orthotopic xenografts versus less metastatic parental PC3 and PC3M orthotopic xenografts and 8 recurrent versus 13 non-recurrent primary carcinomas were carried out and a Pearson correlation coefficient was calculated for set of transcripts exhibiting concordant expression changes ( FIG. 47A ). In the second example ( FIG. 47B ), comparisons of the average fold expression changes in orthotopic versus s.c. PC3MLN4 xenografts and 26 invasive versus 26 non-invasive primary carcinomas were carried out and a Pearson correlation coefficient was calculated for set of transcripts exhibiting concordant expression changes ( FIG. 47B ). The transcript abundance levels of several genes encoding matrix metalloproteinases (MMP9; MMP10; MMP1; MMP14 [ FIG. 46A   1 - FIG. 46A   4 ]) as well as components of plasminogen activator (PA)/PA receptor &amp; plasminogen receptor system (uPA; tPA; uPA receptor; plasminogen receptor; PAI-1[FIGS.  46 B 1 -B 4 ]) are substantially higher in PC-3MLN4 orthotopic tumors versus PC-3MLN4 s.c. (ectopic) tumors, reflecting a plausible mechanistic association of the induction of multiple invasion-activating enzymes with enhanced metastatic potential of PC-3MLN4 tumors in orthotopic setting. Consistent with this idea, the transcript abundance levels for these genes were uniformly lower in orthotopic tumors derived from less metastatic parental PC-3 (“PC30R”) and PC-3M (“PC3MOR”) cells compared to the PC-3MLN4 orthotopic tumors ( FIGS. 46A &amp; 46B ). Decreased level of expression of protease and angiogenesis inhibitor Maspin in PC-3MLN4 orthotopic tumors ( FIG. 46C   4 ) provides an additional clinically relevant example of potential metastasis-promoting molecular alterations in this model since diminished level of Maspin was recently reported in clinical specimens of human prostate cancer (23, 24). Second, a functionally intriguing set of genes highlighted in this model is potentially relevant to metastatic affinity of human prostate carcinoma cells to the bone and represented by a constellation of adhesion molecules ( FIG. 46D ). Documented in this model is an increase in expression (in a metastasis-promoting setting) of non-epithelial cadherins such as osteoblast cadherins (OB-cadherin-1 and -2) as well as vascular endothelial cadherin (VE-cadherin) along with a concomitantly diminished level of expression of epithelial cadherin (E-cadherin) ( FIG. 46D ). These molecular aberrations identified in our model correlate with the clinical phenomenon described as a cadherin switching in human prostate carcinoma (25, 26). Interestingly, increased expression of the osteoblast cadherins in clinical prostate cancer specimens was associated with progression and metastasis of human prostate cancer (25, 26), supporting the notion that metastasis-associated molecular alterations identified in the model system are clinically relevant. Two other adhesion molecules expressed in PC-3MLN4 orthotopic tumors, MCAM and ALCAM (data not shown), share some common properties: they mediate both homotypic and heterotypic cell-cell adhesion crucial for metastasis of melanoma cells (27-30); they are expressed on activated leukocytes and on human endothelium (31-35). In addition, ALCAM expression was identified on bone marrow stromal and mesenchymal stem cells and implicated in bone marrow formation and hematopoiesis (31; 36-39). Interestingly, similarly to cadherins, ALCAM is capable to mediate cell-cell adhesion through homophilic ALCAM-ALCAM interactions (31, 40), thus, expression of ALCAM on human prostate carcinoma cells makes this molecule a viable candidate mediator of human prostate carcinoma homing to the bone. MCAM (MUC18) protein over-expression was reported recently in human prostate cancer cell lines, high-grade prostatic intraepithelial neoplasia (PIN), prostate carcinomas, and lymph node metastasis (41, 42).  
      Expression profiling experiments imply that human prostate carcinoma cells growing in orthotopic metastasis-promoting setting display many clinically relevant gene expression features. Highly aggressive clinically relevant biological behavior of human prostate cancer cells growing in the prostate of nude mice is particularly evident in a fluorescent orthotopic bone metastasis model recapitulating to a significant degree the clinical pattern of metastatic spread of advanced prostate cancer in men (12). Recent gene expression analysis experiments showed that molecular signatures of metastasis could be identified in primary solid tumors (43). We sought to determine whether human prostate carcinoma xenografts growing in the prostate of nude mice would carry the clinically relevant gene-expression signatures of metastasis. We compared the gene expression profiles of 9 metastatic and 23 primary human prostate tumors (the original clinical data were published in LaTulippe, E., Satagopan, J., Smith, A., Scher, H., Scardino, P., Reuter, V., Gerald, W. L. Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease. Cancer Res., 62: 4499-4506, 2002) to identify a broad spectrum of transcripts differentially regulated at the statistically significant level (p&lt;0.05) in metastatic human prostate cancer. Next, we compared a set of transcripts differentially regulated in clinical metastatic human prostate tumors with transcripts differentially regulated in orthotopic human prostate carcinoma xenografts versus subcutaneous (“s.c.”)(i.e., ectopic) tumors of the same lineage. This comparison identified a set of 131 genes that exhibited highly concordant behavior in clinical metastatic samples and orthotopic metastasis-promoting tumors (Pearson correlation coefficient, r=0.799;  FIG. 48A ; Table 41.0).  
               TABLE 41.0                          Prostate cancer metastasis segregation cluster comprising 131 genes                         Affymetrix   Change           Probe ID   direction in       (U95Av2)   metastasis   Description               33534_at   Up   Cluster Incl. X89426:  H. sapiens  mRNA for ESM-1               protein /cds = (55, 609) /gb = X89426 /gi =               1150418 /ug = Hs.41716 /len = 2006       33232_at   Up   Cluster Incl. AI017574: ou23f10.x1  Homo sapiens  cDNA,               3 end /clone = IMAGE-1627147 /clone_end = 3 /gb =               AI017574 /gi = 3231910 /ug = Hs.17409 /len = 501       34289_f_at   Up   Cluster Incl. D50920: Human mRNA for KIAA0130               gene, complete cds /cds = (73, 3042) /gb =               D50920 /gi = 1469182 /ug = Hs.23106 /len = 3468       38158_at   Up   Cluster Incl. D79987: Human mRNA for KIAA0165               gene, complete cds /cds = (1113, 6500) /gb =               D79987 /gi = 1136391 /ug = Hs.153479 /len = 6662       430_at   Up   X00737 /FEATURE = cds /DEFINITION = HSPNP Human               mRNA for purine nucleoside phosphorylase (PNP; EC               2.4.2.1)       907_at   Up   M13792 /FEATURE = cds /DEFINITION = HUMADAG               Human adenosine deaminase (ADA) gene, complete cds       34742_at   Up   Cluster Incl. Z23115:  H. sapiens  bcl-xL               mRNA /cds = (134, 835) /gb = Z23115 /gi =               510900 /ug = Hs.239744 /len = 926       1615_at   Up   Z23115 /FEATURE = cds /DEFINITION = HSBCLXL                 H. sapiens  bcl-xL mRNA       38110_at   Up   Cluster Incl. AF000652:  Homo sapiens  syntenin (sycl)               mRNA, complete cds /cds = (148, 1044) /gb =               AF000652 /gi = 2795862 /ug = Hs.8180 /len = 2162       38290_at   Up   Cluster Incl. AF037195:  Homo sapiens  regulator of G               protein signaling RGS14 mRNA, complete cds /cds =               (73, 1398) /gb = AF037195 /gi = 2708809 /ug =               Hs.9347 /len = 1531       34642_at   Up   Cluster Incl. U28964:  Homo sapiens  14-3-3 protein               mRNA, complete cds /cds = (126, 863) /gb =               U28964 /gi = 899458 /ug = Hs.75103 /len = 1030       36069_at   Up   Cluster Incl. AB007925:  Homo sapiens  mRNA for               KIAA0456 protein, partial cds /cds = (0,               3287) /gb = AB007925 /gi = 3413873 /ug =               Hs.5003 /len = 6305       1782_s_at   Up   M31303 /FEATURE = mRNA /DEFINITION =               HUMOP18A Human oncoprotein 18               (Op18) gene, complete cds       527_at   Up   U14518 /FEATURE = /DEFINITION = HSU14518 Human               centromere protein-A (CENP-A) mRNA, complete cds       1854_at   Up   X13293 /FEATURE = cds /DEFINITION = HSBMYB               Human mRNA for B-myb gene       40407_at   Up   Cluster Incl. U28386: Human nuclear localization               sequence receptor hSRP1alpha mRNA, complete               cds /cds = (132, 1721) /gb = U28386 /gi =               899538 /ug = Hs.159557 /len = 1976       36870_at   Up   Cluster Incl. AB018347:  Homo sapiens  mRNA for               KIAA0804 protein, partial cds /cds = (0,               3636) /gb = AB018347 /gi = 3882328 /ug =               Hs.7316 /len = 4216       1797_at   Up   U40343 /FEATURE = /DEFINITION = HSU40343 Human               CDK inhibitor p19INK4d mRNA, complete cds       1054_at   Up   M87339 /FEATURE = /DEFINITION = HUMACT1A               Human replication factor C, 37-kDa subunit mRNA,               complete cds       36922_at   Up   Cluster Incl. X59618:  H. sapiens  RR2 mRNA for small               subunit ribonucleotide reductase /cds = (194,               1363) /gb = X59618 /gi = 36154 /ug = Hs.75319 /len =               2475       40726_at   Up   Cluster Incl. U37426: Human kinesin-like spindle protein               HKSP (HKSP) mRNA, complete cds /cds = (90, 3260) /gb =               U37426 /gi = 1171152 /ug = Hs.8878 /len = 4858       34879_at   Up   Cluster Incl. AF007875:  Homo sapiens  dolichol               monophosphate mannose synthase (DPM1) mRNA,               partial cds /cds = (0, 761) /gb = AF007875 /gi =               2258417 /ug = Hs.5085 /len = 1054       39035_at   Up   Cluster Incl. AF006010: Human progestin induced protein               (DD5) mRNA, complete cds /cds = (33, 8423) /gb =               AF006010 /gi = 4101694 /ug = Hs.11469 /len = 8493       1624_at   Up   Stimulatory Gdp/Gtp Exchange Protein For C-Ki-Ras               P21 And Smg P21       34715_at   Up   Cluster Incl. U74612: Human hepatocyte nuclear factor-               3/fork head homolog 11A (HFH-11A) mRNA complete               cds /cds = (114, 2519) /gb = U74612 /gi = 1842252 /ug =               Hs.239 /len = 3474       1235_at   Up   M86400 /FEATURE = /DEFINITION = HUMPHPLA2               Human phospholipase A2 mRNA, complete cds       32683_at   Up   Cluster Incl. U18271: Human thymopoietin (TMPO)               gene /cds = (313, 2397) /gb = U18271 /gi =               2182141 /ug = Hs.170225 /len = 2796       41855_at   Up   Cluster Incl. AF030424:  Homo sapiens  histone               acetyltransferase 1 mRNA, complete cds /cds =               (36, 1295) /gb = AF030424 /gi = 2623155 /ug =               Hs.13340 /len = 1568       981_at   Up   X74794 /FEATURE = cds /DEFINITION = HSP1CDC21                 H. sapiens  P1-Cdc21 mRNA       39933_at   Up   Cluster Incl. X93921:  H. sapiens  mRNA for protein-               tyrosine-phosphatase (tissue type- testis) /cds =               (0, 968) /gb = X93921 /gi = 1418935 /ug =               Hs.3843 /len = 1471       34855_at   Up   Cluster Incl. X76770:  H. sapiens  PAP               mRNA /cds = UNKNOWN /gb = X76770 /gi =               556782 /ug = Hs.49007 /len = 1956       31597_r_at   Up   Cluster Incl. L36055: Human 4E-binding protein 1               mRNA, complete cds /cds = (0, 356) /gb =               L36055 /gi = 561629 /ug = Hs.198144 /len = 357       182_at   Up   U01062 /FEATURE = mRNA /DEFINITION = HUMIP3R3               Human type 3 inositol 1,4,5-trisphosphate               receptor (ITPR3) mRNA, complete cds       40051_at   Up   Cluster Incl. D31762: Human mRNA for KIAA0057               gene, complete cds /cds = (75, 1187) /gb =               D31762 /gi = 498149 /ug = Hs.153954 /len = 6974       1906_at   Up   Ras Inhibitor Inf       38480_s_at   Up   Cluster Incl. U66867: Human ubiquitin conjugating               enzyme 9 (hUBC9) mRNA, complete cds /cds = (806,               1282) /gb = U66867 /gi = 1561758 /ug =               Hs.84285 /len = 1823       40786_at   Up   Cluster Incl. U37352: Human protein phosphatase 2A               Balpha1 regulatory subunit mRNA, complete cds /cds =               (88, 1632) /gb = U37352 /gi = 1203811 /ug =               Hs.171734 /len = 4064       37729_at   Up   Cluster Incl. Y08614:  Homo sapiens  mRNA for CRM1               protein /cds = (38, 3253) /gb = Y08614 /gi =               5541866 /ug = Hs.79090 /len = 4148       38702_at   Up   Cluster Incl. AF070640:  Homo sapiens  clone 24781               mRNA sequence /cds = UNKNOWN /gb = AF070640 /gi =               3283913 /ug = Hs.108112 /len = 1583       32578_at   Up   Cluster Incl. AW005997: wz91c01.x1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-2566176 /clone_end =               3 /gb = AW005997 /gi = 5854775 /ug =               Hs.78185 /len = 702       890_at   Up   M74524 /FEATURE = /DEFINITION = HUMHHR6A               Human HHR6A (yeast RAD 6 homologue) mRNA,               complete cds       39337_at   Up   Cluster Incl. M37583: Human histone (H2A.Z) mRNA,               complete cds /cds = (106, 492) /gb = M37583 /gi =               184059 /ug = Hs.119192 /len = 873       34484_at   Up   Cluster Incl. AI961669: wt65e11.x1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-2512364 /clone_end =               3 /gb = AI961669 /gi = 5754382 /ug =               Hs.118249 /len = 565       41085_at   Up   Cluster Incl. AF025840:  Homo sapiens  DNA polymerase               epsilon subunit B (DPE2) mRNA, complete cds /cds =               (130, 1710) /gb = AF025840 /gi = 2697122 /ug =               Hs.99185 /len = 1807       40690_at   Up   Cluster Incl. X54942:  H. sapiens  ckshs2 mRNA for Cks1               protein homologue /cds = (95, 334) /gb = X54942 /gi =               29978 /ug = Hs.83758 /len = 612       38818_at   Up   Cluster Incl. Y08685:  H. sapiens  mRNA for serine               palmitoyltransferase, subunit I /cds = (0,               1421) /gb = Y08685 /gi = 2564246 /ug =               Hs.90458 /len = 1621       34795_at   Up   Cluster Incl. U84573:  Homo sapiens  lysyl hydroxylase               isoform 2 (PLOD2) mRNA, complete cds /cds = (0,               2213) /gb = U84573 /gi = 2138313 /ug = Hs.41270 /len =               3480       584_s_at   Up   M30938 /FEATURE = mRNA#1 /DEFINITION = HUMKUP               Human Ku (p70/p80) subunit mRNA, complete cds       41823_at   Up   Cluster Incl. AJ132258:  Homo sapiens  mRNA for staufen               protein, partial /cds = (35, 1525) /gb = AJ132258 /gi =               4572587 /ug = Hs.6113 /len = 3066       37445_at   Up   Cluster Incl. AB015633:  Homo sapiens  mRNA for type II               membrane protein, complete cds, clone-HP10481 /cds =               (104, 1435) /gb = AB015633 /gi = 4586843 /ug =               Hs.112986 /len = 1451       41569_at   Up   Cluster Incl. AI680675: tx40a08.x1  Homo sapiens  cDNA,               3 end /clone = IMAGE-2272022 /clone_end = 3 /gb =               AI680675 /gi = 4890857 /ug = Hs.44131 /len = 554       1515_at   Up   Rad2       39724_s_at   Up   Cluster Incl. U58087: Human Hs-cul-1 mRNA, complete               cds /cds = (124, 2382) /gb = U58087 /gi =               1381141 /ug = Hs.14541 /len = 2511       36492_at   Up   Cluster Incl. AI347155: tc04c11.x1  Homo sapiens  cDNA,               3 end /clone = IMAGE-2062868 /clone_end = 3 /gb =               AI347155 /gi = 4084361 /ug = Hs.5648 /len = 750       33877_s_at   Up   Cluster Incl. AB028990:  Homo sapiens  mRNA for               KIAA1067 protein, partial cds /cds = (0,               2072) /gb = AB028990 /gi = 5689470 /ug =               Hs.24375 /len = 4704       35810_at   Up   Cluster Incl. AI525393: PT1.1_07_A11.r  Homo sapiens                 cDNA, 5 end /clone_end = 5 /gb = AI525393 /gi =               4439528 /ug = Hs.6895 /len = 811       685_f_at   Up   K03460 /FEATURE = cds /DEFINITION = HUMTUBA2H               Human alpha-tubulin isotype H2-alpha gene, last exon       35165_at   Up   Cluster Incl. AF070582:  Homo sapiens  clone 24766               mRNA sequence /cds = UNKNOWN /gb = AF070582 /gi =               3387954 /ug = Hs.26118 /len = 1744       36178_at   Up   Cluster Incl. U23143: Human mitochondrial serine               hydroxymethyltransferase gene, nuclear encoded               mitochondrion protein, complete cds /cds = (0,               1451) /gb = U23143 /gi = 746435 /ug =               Hs.75069 /len = 1452       32657_at   Up   Cluster Incl. D25278: Human mRNA for KIAA0036               gene, complete cds /cds = (156, 1952) /gb =               D25278 /gi = 434780 /ug = Hs.169387 /len = 2535       38839_at   Up   Cluster Incl. AL096719:  Homo sapiens  mRNA; cDNA               DKFZp566N043 (from clone DKFZp566N043) /cds =               UNKNOWN /gb = AL096719 /gi = 5419854 /ug =               Hs.91747 /len = 2185       480_at   Up   U56816 /FEATURE = /DEFINITION = HSU56816 Human               kinase Myt1 (Myt1) mRNA, complete cds       982_at   Up   X74795 /FEATURE = cds /DEFINITION = HSP1CDC46                 H. sapiens  P1-Cdc46 mRNA       38094_at   Up   Cluster Incl. M65028: Human hnRNP type A/B protein               mRNA, complete cds /cds = (142, 996) /gb =               M65028 /gi = 337450 /ug = Hs.81361 /len = 1537       37717_at   Up   Cluster Incl. L03532: Human M4 protein mRNA,               complete cds /cds = (11, 2200) /gb =               L03532 /gi = 187280 /ug = Hs.79024 /len =               2457       36994_at   Up   Cluster Incl. M62762: Human vacuolar H+ ATPase               proton channel subunit mRNA, complete cds /cds =               (230, 697) /gb = M62762 /gi = 189675 /ug =               Hs.76159 /len = 1162       32573_at   Up   Cluster Incl. AL021546: Human DNA sequence from               BAC 15E1 on chromosome 12. Contains Cytochrome C               Oxidase Polypeptide VIa-liver precursor gene, 60S               ribosomal protein L31 pseudogene, pre-mRNA splicing               factor SRp30c gene, two putative genes, ESTs, STSs and               pu       32236_at   Up   Cluster Incl. AF032456:  Homo sapiens  ubiquitin               conjugating enzyme G2 (UBE2G2) mRNA, complete               cds /cds = (55, 552) /gb = AF032456 /gi =               3004908 /ug = Hs.192853 /len = 2890       38385_at   Down   Cluster Incl. S65738: actin depolymerizing factor [human,               fetal brain, mRNA, 1452 nt] /cds = (72, 569) /gb =               S65738 /gi = 415586 /ug = Hs.82306 /len = 1452       38982_at   Down   Cluster Incl. W28865: 53g9  Homo sapiens                 cDNA /gb = W28865 /gi = 1308876 /ug =               Hs.109875 /len = 926       36051_s_at   Down   Cluster Incl. X58199: Human mRNA for beta               adducin /cds = (322, 2502) /gb =               X58199 /gi = 29368 /ug = Hs.4852 /len = 2597       37298_at   Down   Cluster Incl. AF044671:  Homo sapiens  MM46 mRNA,               complete cds /cds = (78, 431) /gb =               AF044671 /gi = 4105274 /ug = Hs.7719 /len = 859       34643_at   Down   Cluster Incl. M58458: Human ribosomal protein S4               (RPS4X) isoform mRNA, complete cds /cds = (35,               826) /gb = M58458 /gi = 337509 /ug =               Hs.75344 /len = 888       32341_f_at   Down   Cluster Incl. U37230: Human ribosomal protein L23a               mRNA, complete cds /cds = (23, 493) /gb =               U37230 /gi = 1574941 /ug = Hs.184776 /len = 548       31956_f_at   Down   Cluster Incl. M17886: Human acidic ribosomal               phosphoprotein P1 mRNA, complete cds /cds =               (129, 473) /gb = M17886 /gi = 190233 /ug =               Hs.177592 /len = 512       31957_r_at   Down   Cluster Incl. M17886: Human acidic ribosomal               phosphoprotein P1 mRNA, complete cds /cds =               (129, 473) /gb = M17886 /gi = 190233 /ug =               Hs.177592 /len = 512       1488_at   Down   L77886 /FEATURE = /DEFINITION = HUMPTPC               Human protein tyrosine phosphatase mRNA, complete               cds       31861_at   Down   Cluster Incl. L14754: Human DNA-binding protein               (SMBP2) mRNA, complete cds /cds = (49,               3030) /gb = L14754 /gi = 401775 /ug =               Hs.1521 /len = 3892       31962_at   Down   Cluster Incl. L06499:  Homo sapiens  ribosomal protein               L37a (RPL37A) mRNA, complete cds /cds = (17,               295) /gb = L06499 /gi = 292438 /ug = Hs.184109 /len =               357       34864_at   Down   Cluster Incl. AF070638:  Homo sapiens  clone 24448               unknown mRNA, partial cds /cds = (0, 659) /gb =               AF070638 /gi = 3283909 /ug = Hs.4973 /len = 1348       32412_at   Down   Cluster Incl. M13934: Human ribosomal protein S14               gene, complete cds /cds = (2, 457) /gb =               M13934 /gi = 337498 /ug = Hs.3491 /len = 503       36980_at   Down   Cluster Incl. U03105: Human B4-2 protein mRNA,               complete cds /cds = (113, 1096) /gb =               U03105 /gi = 476094 /ug = Hs.75969 /len = 2061       33116_f_at   Down   Cluster Incl. AA977163: oq25a04.s1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-1587342 /clone_end =               3 /gb = AA977163 /gi = 3154609 /ug =               Hs.82148 /len = 524       35119_at   Down   Cluster Incl. X56932:  H. sapiens  mRNA for 23 kD highly               basic protein /cds = (17, 628) /gb = X56932 /gi =               23690 /ug = Hs.119122 /len = 672       31509_at   Down   Cluster Incl. X64707:  H. sapiens  BBC1               mRNA /cds = (51, 686) /gb = X64707 /gi =               29382 /ug = Hs.180842 /len = 942       31511_at   Down   Cluster Incl. U14971: Human ribosomal protein S9               mRNA, complete cds /cds = (35, 619) /gb =               U14971 /gi = 550022 /ug = Hs.180920 /len = 692       41138_at   Down   Cluster Incl. M16279: Human MIC2 mRNA, complete               cds /cds = (177, 734) /gb = M16279 /gi =               188542 /ug = Hs.177543 /len = 1238       33676_at   Down   Cluster Incl. X15940: Human mRNA for ribosomal               protein L31 /cds = (7, 384) /gb = X15940 /gi =               36129 /ug = Hs.184014 /len = 414       34592_at   Down   Cluster Incl. M13932: Human ribosomal protein S17               mRNA, complete cds /cds = (25, 432) /gb =               M13932 /gi = 337500 /ug = Hs.5174 /len = 477       38060_at   Down   Cluster Incl. AI541336: pec1.2-7.A07.r  Homo sapiens                 cDNA, 5 end /clone_end = 5 /gb = AI541336 /gi =               4458709 /ug = Hs.80595 /len = 717       32748_at   Down   Cluster Incl. AI557852: P6test.G05.r  Homo sapiens                 cDNA, 5 end /clone_end = 5 /gb = AI557852 /gi =               4490215 /ug = Hs.195453 /len = 693       883_s_at   Down   M54915 /FEATURE = /DEFINITION = HUMPIM1LE               Human h-pim-1 protein (h-pim-1) mRNA, complete cds       829_s_at   Down   U21689 /FEATURE = cds /DEFINITION = HSU21689               Human glutathione S-transferase-P1c gene, complete cds       37197_s_at   Down   Cluster Incl. AL050006:  Homo sapiens  mRNA; cDNA               DKFZp564A033 (from clone DKFZp564A033) /cds =               (0, 957) /gb = AL050006 /gi = 4884074 /ug =               Hs.7627 /len = 1252       31527_at   Down   Cluster Incl. X17206: Human mRNA for               LLRep3 /cds = (240, 905) /gb = X17206 /gi =               34391 /ug = Hs.182426 /len = 934       32276_at   Down   Cluster Incl. X03342: Human mRNA for ribosomal               protein L32 /cds = (34, 441) /gb = X03342 /gi =               36131 /ug = Hs.169793 /len = 505       683_at   Down   K02100 /FEATURE = mRNA /DEFINITION = HUMOTC               Human ornithine transcarbamylase (OTC) mRNA,               complete coding sequence       552_at   Down   U02570 /FEATURE = /DEFINITION = HSU02570 Human               CDC42 GTPase-activating protein mRNA, partial cds       1173_g_at   Down   Spermidine/Spermine N1-Acetyltransferase, Alt. Splice 2       31693_f_at   Down   Cluster Incl. Z80776:  H. sapiens  H2A/g gene /cds = (0,               392) /gb = Z80776 /gi = 1568542 /ug = Hs.239458 /len =               393       39916_r_at   Down   Cluster Incl. J02984: Human insulinoma rig-analog               mRNA encoding DNA-binding protein, complete               cds /cds = (29, 466) /gb = J02984 /gi = 184553 /ug =               Hs.133230 /len = 498       35852_at   Down   Cluster Incl. AB014558:  Homo sapiens  mRNA for               KIAA0658 protein, partial cds /cds = (0,               1770) /gb = AB014558 /gi = 3327129 /ug =               Hs.7278 /len = 4103       33619_at   Down   Cluster Incl. L01124: Human ribosomal protein S13               (RPS13) mRNA, complete cds /cds = (32, 487) /gb =               L01124 /gi = 307390 /ug = Hs.165590 /len = 530       36355_at   Down   Cluster Incl. M13903: Human involucrin               mRNA /cds = (0, 1757) /gb = M13903 /gi =               186520 /ug = Hs.157091 /len = 1758       32436_at   Down   Cluster Incl. U14968: Human ribosomal protein L27a               mRNA, complete cds /cds = (16, 462) /gb =               U14968 /gi = 550016 /ug = Hs.76064 /len = 507       38639_at   Down   Cluster Incl. AF040963:  Homo sapiens  Mad4 homolog               (Mad4) mRNA, complete cds /cds = (13, 642) /gb =               AF040963 /gi = 2792361 /ug = Hs.102402 /len = 879       37009_at   Down   Cluster Incl. AL035079: dJ53C18.1 (Catalase) /cds =               (74, 1657) /gb = AL035079 /gi = 4775614 /ug =               Hs.76359 /len = 2287       37027_at   Down   Cluster Incl. M80899: Human novel protein AHNAK               mRNA, partial sequence /cds = (0, 3835) /gb =               M80899 /gi = 178282 /ug = Hs.76549 /len = 4051       39294_at   Down   Cluster Incl. X16155: Human mRNA for chicken               ovalbumin upstream promoter transcription factor               (COUP-TF) /cds = (0, 1256) /gb = X16155 /gi =               30139 /ug = Hs.239468 /len = 1513       39713_at   Down   Cluster Incl. AJ132440:  Homo sapiens  mRNA for PLU-1               protein /cds = (89, 4723) /gb = AJ132440 /gi =               4902723 /ug = Hs.143323 /len = 6355       32587_at   Down   Cluster Incl. U07802: Human Tis11d gene, complete               cds /cds = (291, 1739) /gb = U07802 /gi =               984508 /ug = Hs.78909 /len = 3655       41402_at   Down   Cluster Incl. AL080121:  Homo sapiens  mRNA; cDNA               DKFZp564O0823 (from clone DKFZp564O0823) /cds =               (170, 904) /gb = AL080121 /gi = 5262554 /ug =               Hs.105460 /len = 2135       36899_at   Down   Cluster Incl. M97287: Human MAR/SAR DNA binding               protein (SATB1) mRNA, complete cds /cds = (214,               2505) /gb = M97287 /gi = 337810 /ug =               Hs.74592 /len = 2928       36039_s_at   Down   Cluster Incl. X93498:  H. sapiens  mRNA for 21-Glutamic               Acid-Rich Protein (21-GARP) /cds = UNKNOWN /gb =               X93498 /gi = 1673496 /ug = Hs.47438 /len = 1160       33657_at   Down   Cluster Incl. L38941:  Homo sapiens  ribosomal protein               L34 (RPL34) mRNA, complete cds /cds = (20, 373) /gb =               L38941 /gi = 1008855 /ug = Hs.179779 /len = 392       41721_at   Down   Cluster Incl. AA658877: nt84c12.s1  Homo sapiens                 cDNA /clone = IMAGE-1205206 /gb = AA658877 /gi =               2595031 /ug = Hs.181350 /len = 897       34775_at   Down   Cluster Incl. AF065388:  Homo sapiens  tetraspan NET-1               mRNA, complete cds /cds = (121, 846) /gb =               AF065388 /gi = 3152700 /ug = Hs.38972 /len = 1278       1022_f_at   Down   V00542 /FEATURE = mRNA /DEFINITION = HSIFR14               Messenger RNA for human leukocyte (alpha) interferon       35468_at   Down   Cluster Incl. AL050381:  Homo sapiens  mRNA; cDNA               DKFZp586B2023 (from clone DKFZp586B2023) /cds =               UNKNOWN /gb = AL050381 /gi = 4914611 /ug =               Hs.172639 /len = 1485       1147_at   Down   V-Erba Related Ear-3 Protein       34365_at   Down   Cluster Incl. AF042386:  Homo sapiens  cyclophilin-33B               (CYP-33) mRNA, complete cds /cds = (60, 950) /gb =               AF042386 /gi = 2828150 /ug = Hs.33251 /len = 1099       39273_at   Down   Cluster Incl. AL022718: dJ1052M9.3 (mouse DOC4               LIKE protein) /cds = (0, 4094) /gb =               AL022718 /gi = 3763969 /ug = Hs.23796 /len = 8728       33935_at   Down   Cluster Incl. AL035305:  H. sapiens  gene from PAC               102G20 /cds = (117, 803) /gb = AL035305 /gi =               4200223 /ug = Hs.27258 /len = 2435       36040_at   Down   Cluster Incl. AI337192: qx88h10.x1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-2009635 /clone_end =               3 /gb = AI337192 /gi = 4074119 /ug =               Hs.47438 /len = 925       39325_at   Down   Cluster Incl. U81523: Human endometrial bleeding               associated factor mRNA, complete cds /cds = (33,               1145) /gb = U81523 /gi = 2058537 /ug =               Hs.25195 /len = 1961       35546_at   Down   Cluster Incl. W28428: 49d8  Homo sapiens                 cDNA /gb = W28428 /gi = 1308583 /ug =               Hs.132153 /len = 812       32242_at   Down   Cluster Incl. AL038340: DKFZp566K192_s1  Homo                   sapiens  cDNA, 3 end /clone = DKFZp566K192 /clone —                 end = 3 /gb = AL038340 /gi = 5407591 /ug =               Hs.1940 /len = 746       762_f_at   Down   AB000905 /FEATURE = cds /DEFINITION = AB000905                 Homo sapiens  DNA for H4 histone, complete cds       41106_at   Down   Cluster Incl. AF022797:  Homo sapiens  intermediate               conductance calcium-activated potassium channel               (hKCa4) mRNA, complete cds /cds = (396, 1679) /gb =               AF022797 /gi = 2674355 /ug = Hs.10082 /len = 2238       38279_at   Down   Cluster Incl. D90150: Human Gx-alpha gene /cds =               (619, 1686) /gb = D90150 /gi = 219668 /ug =               Hs.92002 /len = 3289       1591_s_at   Down   J03242 /FEATURE = /DEFINITION = HUMGFIL2               Human insulin-lke growth factor II mRNA, complete cds                  
 
      Remarkably, when we compared the expression profiles of these 131 transcripts in orthotopic xenografts and individual clinical samples, we found that all metastatic prostate carcinomas have expression patterns highly similar to orthotopic xenografts as reflected in positive correlation of expression profiles, whereas all primary tumors displayed a negative correlation of expression profiles ( FIG. 49A ). We next attempted to refine the gene-expression signature associated with human prostate cancer metastasis to a small set of transcripts that would exhibit similar discrimination accuracy between metastatic and primary tumors. To achieve this we used the increase in correlation coefficient of gene expression profiles between orthotopic xenografts and clinical samples as a guide for reduction of transcripts number in a cluster ( FIGS. 48B , C, and D). Using this strategy we were able to identify several smaller clusters of co-regulated genes exhibiting highly concordant behavior in the model system and clinical samples (FIGS.  48 A-D and Tables 41.1, 41.2, 41 &amp; 42) and demonstrating highly accurate discrimination (at least 94%) between clinical samples of metastatic and primary human prostate carcinomas (FIGS.  49 A-D and Table 42).  
               TABLE 41.1                          Prostate cancer metastasis segregation cluster comprising 37 genes                             Change           Affymetrix   direction in       ID (U95Av2)   metastasis   Description               33534_at   Up   Cluster Incl. X89426:  H. sapiens  mRNA for ESM-1               protein /cds = (55, 609) /gb = X89426 /gi =               1150418 /ug = Hs.41716 /len = 2006       33232_at   Up   Cluster Incl. AI017574: ou23f10.x1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-1627147 /clone_end =               3 /gb = AI017574 /gi = 3231910 /ug =               Hs.17409 /len = 501       34289_f_at   Up   Cluster Incl. D50920: Human mRNA for KIAA0130               gene, complete cds /cds = (73, 3042) /gb =               D50920 /gi = 1469182 /ug = Hs.23106 /len =               3468       430_at   Up   X00737 /FEATURE = cds /DEFINITION = HSPNP               Human mRNA for purine nucleoside phosphorylase               (PNP; EC 2.4.2.1)       907_at   Up   M13792 /FEATURE = cds /DEFINITION = HUMADAG               Human adenosine deaminase (ADA) gene, complete cds       34742_at   Up   Cluster Incl. Z23115:  H. sapiens  bcl-xL               mRNA /cds = (134, 835) /gb = Z23115 /gi =               510900 /ug = Hs.239744 /len = 926       38110_at   Up   Cluster Incl. AF000652:  Homo sapiens  syntenin (sycl)               mRNA, complete cds /cds = (148, 1044) /gb =               AF000652 /gi = 2795862 /ug = Hs.8180 /len = 2162       38290_at   Up   Cluster Incl. AF037195:  Homo sapiens  regulator of G               protein signaling RGS14 mRNA, complete cds /cds =               (73, 1398) /gb = AF037195 /gi = 2708809 /ug =               Hs.9347 /len = 1531       36870_at   Up   Cluster Incl. AB018347:  Homo sapiens  mRNA for               KIAA0804 protein, partial cds /cds = (0,               3636) /gb = AB018347 /gi = 3882328 /ug =               Hs.7316 /len = 4216       1624_at   Up   Stimulatory Gdp/Gtp Exchange Protein For C-Ki-Ras               P21 And Smg P21       41855_at   Up   Cluster Incl. AF030424:  Homo sapiens  histone               acetyltransferase 1 mRNA, complete cds /cds =               (36, 1295) /gb = AF030424 /gi = 2623155 /ug =               Hs.13340 /len = 1568       36355_at   Down   Cluster Incl. M13903: Human involucrin               mRNA /cds = (0, 1757) /gb = M13903 /gi =               186520 /ug = Hs.157091 /len = 1758       32436_at   Down   Cluster Incl. U14968: Human ribosomal protein L27a               mRNA, complete cds /cds = (16, 462) /gb =               U14968 /gi = 550016 /ug = Hs.76064 /len = 507       38639_at   Down   Cluster Incl. AF040963:  Homo sapiens  Mad4 homolog               (Mad4) mRNA, complete cds /cds = (13, 642) /gb =               AF040963 /gi = 2792361 /ug = Hs.102402 /len = 879       37009_at   Down   Cluster Incl. AL035079: dJ53C18.1 (Catalase) /cds =               (74, 1657) /gb = AL035079 /gi = 4775614 /ug =               Hs.76359 /len = 2287       37027_at   Down   Cluster Incl. M80899: Human novel protein AHNAK               mRNA, partial sequence /cds = (0, 3835) /gb =               M80899 /gi = 178282 /ug = Hs.76549 /len = 4051       39294_at   Down   Cluster Incl. X16155: Human mRNA for chicken               ovalbumin upstream promoter transcription factor               (COUP-TF) /cds = (0, 1256) /gb = X16155 /gi =               30139 /ug = Hs.239468 /len = 1513       39713_at   Down   Cluster Incl. AJ132440:  Homo sapiens  mRNA for PLU-1               protein /cds = (89, 4723) /gb = AJ132440 /gi =               4902723 /ug = Hs.143323 /len = 6355       32587_at   Down   Cluster Incl. U07802: Human Tis11d gene, complete               cds /cds = (291, 1739) /gb = U07802 /gi =               984508 /ug = Hs.78909 /len = 3655       41402_at   Down   Cluster Incl. AL080121:  Homo sapiens  mRNA; cDNA               DKFZp564O0823 (from clone DKFZp564O0823) /cds =               (170, 904) /gb = AL080121 /gi = 5262554 /ug =               Hs.105460 /len = 2135       36039_s_at   Down   Cluster Incl. X93498:  H. sapiens  mRNA for 21-Glutamic               Acid-Rich Protein (21-GARP) /cds = UNKNOWN /gb =               X93498 /gi = 1673496 /ug = Hs.47438 /len = 1160       33657_at   Down   Cluster Incl. L38941:  Homo sapiens  ribosomal protein               L34 (RPL34) mRNA, complete cds /cds = (20, 373) /gb =               L38941 /gi = 1008855 /ug = Hs.179779 /len = 392       41721_at   Down   Cluster Incl. AA658877: nt84c12.s1  Homo sapiens                 cDNA /clone = IMAGE-1205206 /gb = AA658877 /gi =               2595031 /ug = Hs.181350 /len = 897       34775_at   Down   Cluster Incl. AF065388:  Homo sapiens  tetraspan NET-1               mRNA, complete cds /cds = (121, 846) /gb =               AF065388 /gi = 3152700 /ug = Hs.38972 /len = 1278       1022_f_at   Down   V00542 /FEATURE = mRNA /DEFINITION = HSIFR14               Messenger RNA for human leukocyte (alpha) interferon       35468_at   Down   Cluster Incl. AL050381:  Homo sapiens  mRNA; cDNA               DKFZp586B2023 (from clone DKFZp586B2023) /cds =               UNKNOWN /gb = AL050381 /gi = 4914611 /ug =               Hs.172639 /len = 1485       1147_at   Down   V-Erba Related Ear-3 Protein       34365_at   Down   Cluster Incl. AF042386:  Homo sapiens  cyclophilin-33B               (CYP-33) mRNA, complete cds /cds = (60, 950) /gb =               AF042386 /gi = 2828150 /ug = Hs.33251 /len = 1099       33935_at   Down   Cluster Incl. AL035305:  H. sapiens  gene from PAC               102G20 /cds = (117, 803) /gb = AL035305 /gi =               4200223 /ug = Hs.27258 /len = 2435       36040_at   Down   Cluster Incl. AI337192: qx88h10.x1  Homo sapiens                 cDNA, 3 end /clone = IMAGE-2009635 /clone_end =               3 /gb = AI337192 /gi = 4074119 /ug =               Hs.47438 /len = 925       39325_at   Down   Cluster Incl. U81523: Human endometrial bleeding               associated factor mRNA, complete cds /cds = (33,               1145) /gb = U81523 /gi = 2058537 /ug =               Hs.25195 /len = 1961       35546_at   Down   Cluster Incl. W28428: 49d8  Homo sapiens                 cDNA /gb = W28428 /gi = 1308583 /ug =               Hs.132153 /len = 812       32242_at   Down   Cluster Incl. AL038340: DKFZp566K192_s1  Homo                   sapiens  cDNA, 3 end /clone = DKFZp566K192 /clone —                 end = 3 /gb = AL038340 /gi = 5407591 /ug =               Hs.1940 /len = 746       762_f_at   Down   AB000905 /FEATURE = cds /DEFINITION = AB000905                 Homo sapiens  DNA for H4 histone, complete cds       41106_at   Down   Cluster Incl. AF022797:  Homo sapiens  intermediate               conductance calcium-activated potassium channel               (hKCa4) mRNA, complete cds /cds = (396, 1679) /gb =               AF022797 /gi = 2674355 /ug = Hs.10082 /len = 2238       38279_at   Down   Cluster Incl. D90150: Human Gx-alpha gene /cds =               (619, 1686) /gb = D90150 /gi = 219668 /ug =               Hs.92002 /len = 3289       1591_s_at   Down   J03242 /FEATURE = /DEFINITION = HUMGFIL2               Human insulin-lke growth factor II mRNA, complete cds                  
 
     
       
         
           
               
             
               
                 TABLE 41.2 
               
             
            
               
                   
               
               
                   
               
               
                 Prostate cancer metastasis segregation cluster comprising 12 genes 
               
            
           
           
               
               
               
            
               
                   
                 Change 
                   
               
               
                 Affymetrix 
                 direction in 
               
               
                 ID (U95Av2) 
                 metastasis 
                 Description 
               
               
                   
               
               
                 33534_at 
                 Up 
                 Cluster Incl. X89426:  H. sapiens  mRNA for ESM-1 
               
               
                   
                   
                 protein /cds = (55, 609) /gb = X89426 /gi = 
               
               
                   
                   
                 1150418 /ug = Hs.41716 /len = 2006 
               
               
                 33232_at 
                 Up 
                 Cluster Incl. AI017574: ou23f10.x1  Homo sapiens   
               
               
                   
                   
                 cDNA, 3 end /clone = IMAGE-1627147 /clone_end = 
               
               
                   
                   
                 3 /gb = AI017574 /gi = 3231910 /ug = 
               
               
                   
                   
                 Hs.17409 /len = 501 
               
               
                 34289_f_at 
                 Up 
                 Cluster Incl. D50920: Human mRNA for KIAA0130 
               
               
                   
                   
                 gene, complete cds /cds = (73, 3042) /gb = 
               
               
                   
                   
                 D50920 /gi = 1469182 /ug = Hs.23106 /len = 
               
               
                   
                   
                 3468 
               
               
                 430_at 
                 Up 
                 X00737 /FEATURE = cds /DEFINITION = HSPNP 
               
               
                   
                   
                 Human mRNA for purine nucleoside phosphorylase 
               
               
                   
                   
                 (PNP; EC 2.4.2.1) 
               
               
                 907_at 
                 Up 
                 M13792 /FEATURE = cds /DEFINITION = HUMADAG 
               
               
                   
                   
                 Human adenosine deaminase (ADA) gene, complete cds 
               
               
                 34742_at 
                 Up 
                 Cluster Incl. Z23115:  H. sapiens  bcl-xL mRNA /cds = 
               
               
                   
                   
                 (134, 835) /gb = Z23115 /gi = 510900 /ug = 
               
               
                   
                   
                 Hs.239744 /len = 926 
               
               
                 36040_at 
                 Down 
                 Cluster Incl. AI337192: qx88h10.x1  Homo sapiens   
               
               
                   
                   
                 cDNA, 3 end /clone = IMAGE-2009635 /clone_end = 
               
               
                   
                   
                 3 /gb = AI337192 /gi = 4074119 /ug = 
               
               
                   
                   
                 Hs.47438 /len = 925 
               
               
                 35546_at 
                 Down 
                 Cluster Incl. W28428: 49d8  Homo sapiens   
               
               
                   
                   
                 cDNA /gb = W28428 /gi = 1308583 /ug = 
               
               
                   
                   
                 Hs.132153 /len = 812 
               
               
                 762_f_at 
                 Down 
                 AB000905 /FEATURE = cds /DEFINITION = AB000905 
               
               
                   
                   
                   Homo sapiens  DNA for H4 histone, complete cds 
               
               
                 41106_at 
                 Down 
                 Cluster Incl. AF022797:  Homo sapiens  intermediate 
               
               
                   
                   
                 conductance calcium-activated potassium channel 
               
               
                   
                   
                 (hKCa4) mRNA, complete cds /cds = (396, 
               
               
                   
                   
                 1679) /gb = AF022797 /gi = 2674355 /ug = 
               
               
                   
                   
                 Hs.10082 /len = 2238 
               
               
                 38279_at 
                 Down 
                 Cluster Incl. D90150: Human Gx-alpha gene /cds = 
               
               
                   
                   
                 (619, 1686) /gb = D90150 /gi = 219668 /ug = 
               
               
                   
                   
                 Hs.92002 /len = 3289 
               
               
                 1591_s_at 
                 Down 
                 J03242 /FEATURE = /DEFINITION = HUMGFIL2 
               
               
                   
                   
                 Human insulin-lke growth factor II mRNA, complete 
               
               
                   
                   
                 cds 
               
               
                   
               
            
           
         
       
     
      Interestingly, the 9-gene molecular signature cluster ( FIG. 48D ; Tables 41&amp; 42) associated with human prostate cancer metastasis has several candidate markers and targets for mechanistic studies and/or drug development such as secreted proteins (ESM-] and EBAF), transcription regulators (CRIP1, TRAP100, NRF2F1), two enzymes playing a key role in the purine salvage pathway (NP and ADA), an apoptosis inhibitor (BCL-X L ), and a molecular chaperone (CRYAB).  
               TABLE 41                          The 9-gene molecular signature associated       with metastatic prostate cancer                                     GenBank   UniGene       Gene   Gene name   ID   ID               ESM1   Endothelial cell-specific   X89426   Hs.41716           molecule 1       CRIP1   Cysteine-rich protein 1   AI0175174   Hs.17409       TRAP100   Thyroid hormone receptor-   D50920   Hs.23106           associated protein       NP   Nucleoside phosphorylase   X00737   Hs.75514       ADA   Adenosine deaminase   M13792   Hs.1217       BCL2L1   BCL2-like 1   Z23115   Hs.305890       NRF2F1   Nuclear receptor subfamily 2,   X16155   Hs.421993           group F, member 1       EBAF   Endometrial bleeding associated   U81523   Hs.25195           factor       CRYAB   Crystallin, alpha B   AL038340   Hs.391270                  
 
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                   
               
               
                 Classification accuracy of metastasis segregation clusters 
               
            
           
           
               
               
               
               
               
            
               
                 Number of 
                   
                   
                   
                   
               
               
                 genes in 
                 Correlation 
                 Performance 
                 Performance 
                 Overall 
               
               
                 cluster 
                 coefficient 
                 (metastases) 
                 (primary tumors) 
                 performance 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 131 
                 genes 
                 r = 0.799 
                 9 of 9 (100%) 
                 23 of 23 (100%) 
                 32 of 32 (100%) 
               
               
                 37 
                 genes 
                 r = 0.938 
                 9 of 9 (100%) 
                 21 of 23 (91%) 
                 30 of 32 (94%) 
               
               
                 15 
                 genes 
                 r = 0.958 
                 9 of 9 (100%) 
                 21 of 23 (91%) 
                 30 of 32 (94%) 
               
               
                 12 
                 genes 
                 r = 0.990 
                 9 of 9 (100%) 
                 21 of 23 (91%) 
                 30 of 32 (94%) 
               
               
                 9 
                 genes 
                 r = 0.973 
                 9 of 9 (100%) 
                 21 of 23 (91%) 
                 30 of 32 (94%) 
               
               
                 14 
                 genes 
                 r = 0.937 
                 9 of 9 (100%) 
                 22 of 23 (96%) 
                 31 Of 32 (97%) 
               
               
                   
               
            
           
         
       
     
      To further test the potential clinical relevance of the models, we attempted to utilize expression profiling of highly metastatic orthotopic human prostate carcinoma xenografts for identification of gene expression correlates of clinically significant phenotypes such as invasive behavior and recurrence propensity of human prostate tumors (the original clinical data utilized in these examples were recently published in Singh, D., Febbo, P. G., Ross, K., Jackson, D. G., Manola, C. L., Tamayo, P., Renshaw, A. A., D&#39;Amico, A. V., Richie, J. P., Lander, E. S., Loda, M., Kantoff, P. W., Golub, T. R., Sellers, W. R. Gene expression correlates of clinical prostate cancer behavior. Cancer Cell, 1: 203-209, 2002). Using gene expression profiles of metastasis-promoting orthotopic xenografts as a predictive reference of expected transcript abundance behavior in clinical samples, we identified a five-gene cluster (Table 43) of co-regulated transcripts discriminating with 75% accuracy invasive versus non-invasive human prostate tumors ( FIGS. 47B and 50A ).  
               TABLE 43                          The 5-gene molecular fingerprint associated with       invasive phenotype of human prostate cancer                             Gene   Gene name   GenBank ID   UniGene ID               HRASLS3   HRAS-like suppressor 3   X92814   Hs.37189           EST   AI986201   Hs.355812       KIAA0962   KIAA0962 protein   AB023179   Hs.9059       SLC29A2   Solute carrier family 29   AF034102   Hs.32951       KIAA0557   KIAA0557 protein   AB011129   Hs.101414                  
 
      20 of 26 samples (77%) obtained from the patients with invasive prostate cancer defined by histology as having positive surgical margins (“PSM”) and/or extra-capsular penetration (“PCP”) exhibited a positive correlation coefficient of expression of the five-gene cluster (Table 43) compared to orthotopic xenografts. In contrast, 19 of 26 samples (73%) from the patients with organ-confined disease showed a negative correlation coefficient of expression of the five-gene cluster (Table 43) compared to orthotopic xenografts ( FIG. 50A ). Furthermore, using this strategy we identified an eight-gene cluster (Table 44) of co-regulated transcripts discriminating with 90% accuracy human prostate tumors exhibiting recurrent or non-recurrent clinical behavior ( FIGS. 47A&amp; 50B ).  
               TABLE 44                          The 8-gene molecular fingerprint predicting       recurrent phenotype of human prostate cancer                             Gene   Gene name   GenBank ID   UniGene ID               MGC5466   Hypothetical protein MGC5466   U90904   Hs.83724       CHAF1A   Chromatin assembly factor 1,   U20979   Hs.79018           subunit A       CDS2   CDP-diacylglycerol synthase 2   Y16521   Hs.24812       STX7   Syntaxin 7   U77942   Hs.427065       IER3   Immediate early response 3   S81914   Hs.76095       GLUL   Glutamate-ammonia ligase   X59834   Hs.170171       MYBPC1   Myosin binding protein C   X73114   Hs.169849       SOX9   SRY-box 9   Z46629   Hs.2316                  
 
      In this example we compared a set of transcripts differentially regulated in recurrent versus non-recurrent human prostate tumors with transcripts differentially regulated in orthotopic human prostate carcinoma xenografts derived from highly metastatic PC3MLN4 cell variant versus orthotopic tumors of the less metastatic parental lineages, PC3 and PC3M.  FIG. 50B  illustrates application of the eight-gene cluster (Table 44) to characterize clinical prostate cancer samples according to their propensity for recurrence after therapy. The expression pattern of the genes in the recurrence predictor cluster was analyzed in each of twenty-one separate clinical samples. The analysis produces a quantitative phenotype association index (plotted on the Y-axis) for each of the twenty-one clinical prostate cancer samples. Tumors that are likely to recur are expected to have positive phenotype association indices reflecting positive correlation of gene expression with metastasis-promoting orthotopic xenografts, while those that are unlikely to recur are expected to have negative association indices.  
       FIG. 50B  shows the phenotype association indices for eight samples from patients who later had recurrence as bars 1 through 8, while the association indices for thirteen samples from patients whose tumors did not recur is shown as bars 12 through 24. Eight of the eight samples (or 100%) from patients who later experienced recurrence had positive phenotype association indices and so were properly classified. Eleven of the thirteen samples (or 84.6%) from patients whose tumors did not recur had negative phenotype association indices and so were properly classified as non-recurrent tumors. Thus, overall, nineteen of the twenty-one samples (or 90.5%) were properly classified using an eight-gene recurrence predictor cluster.  
      Next we compared a set of transcripts differentially regulated in recurrent versus non-recurrent human prostate tumors with transcripts differentially regulated in orthotopic human prostate carcinoma xenografts derived from highly metastatic PC3MLN4 cell variant versus subcutaneous (“s.c.”) ectopic tumors of the same lineage. This comparison identified a set of 25 genes ( FIGS. 52A  &amp; B &amp; Table 45) that exhibited highly concordant behavior in clinical recurrent samples and orthotopic metastasis-promoting tumors (Pearson correlation coefficient, r=0.862;  FIG. 52B ).  
               TABLE 45                          The 25-gene molecular signature predicting       recurrent prostate cancer                                     GenBank           Gene   Gene name   ID   UniGene ID               ETS1   v-ets erythroblastosis virus   X14798   Hs.18063           E26 oncogene homolog 1       MGC5466   Hypothetical protein MGC5466   U90904   Hs.83724       CA2   carbonic anhydrase II   J03037   Hs.155097       LRP2   Megalin   U33837   Hs.153595       EPHA3   receptor tyrosine kinase HEK   M83941   Hs.123642       Wnt5A   proto-oncogene Wnt5A   L20861   Hs.152213       ADRA1A   adrenergic, alpha-1A-, receptor   D32202   Hs.52931           EST   R38263   Hs.375190       CDS2   CDP-diacylglycerol synthase   Y16521   Hs.24812           EST   AL050002   Hs.94795       STX7   syntaxin 7   U77942   Hs.427065       RANBP3   RAN binding protein 3   Y08698   Hs.176657       FSTL1   follistatin-like 1   U06863   Hs.433622       ZFP36L2   zinc finger protein 36   U07802   Hs.78909       GGT2   gamma-glutamyltransferase 2   M30474   Hs.289098       KIAA0476   KIAA0476 protein   AB007945   Hs.6684       ITPR1   inositol 1,4,5-trisphosphate   D26070   Hs.198443           receptor, type 1       ITCH   Itchy homolog E3 ubiquitin   AF038564   Hs.98074           protein ligase       CD44   CD44 antigen   L05424   Hs.169610       TNRC15   Trinucleotide repeat containing   AB014542   Hs.323317           15       MXI1   MAX interacting protein 1   L07648   Hs.118630       TCF2   transcription factor 2, hepatic   X58840   Hs.169853       KCNN4   intermediate conductance   AF022797   Hs.10082           calcium-activated potassium           channel       APS   Adaptor protein   AB000520   Hs.105052       SOX9   SRY-box 9   Z46629   Hs.2316                  
 
      When we compared the expression profiles of these 25 transcripts in orthotopic xenografts and individual clinical samples, we found that all recurrent prostate carcinomas have expression patterns highly similar to orthotopic xenografts as reflected in positive correlation of expression profiles, whereas 12 of 13 non-recurrent tumors displayed a negative correlation of expression profiles ( FIG. 53 ). We next attempted to refine the gene-expression signature associated with human prostate cancer metastasis to a smaller set of transcripts that would exhibit similar discrimination accuracy between recurrent and non-recurrent tumors. To achieve this we used the increase in correlation coefficient of gene expression profiles between orthotopic xenografts and clinical samples as a guide for reducing the number of genes in the cluster (cf.  FIGS. 52B &amp; 55 ). Using this strategy we identified a smaller cluster of 12 co-regulated genes ( FIG. 54  &amp; Table 46) exhibiting highly concordant behavior in the model system and clinical samples (r=0.992;  FIG. 55 ) and demonstrating highly accurate discrimination (20 of 21 samples, or 95% were correctly classified) between clinical samples of recurrent and non-recurrent human prostate carcinomas ( FIG. 56 ).  
               TABLE 46                          The 12-gene molecular signature predicting       recurrent prostate cancer                                     GenBank   UniGene       Gene   Gene name   ID   ID               MGC5466   Hypothetical protein MGC5466   U90904   Hs.83724       EPHA3   Receptor tyrosine kinase HEK   M83941   Hs.123642       Wnt5A   Proto-oncogene Wnt5A   L20861   Hs.152213       CDS2   CDP-diacylglycerol synthase   Y16521   Hs.24812           EST   AL050002   Hs.94795       STX7   Syntaxin 7   U77942   Hs.427065       RANBP3   RAN binding protein 3   Y08698   Hs.176657       KIAA0476   KIAA0476 protein   AB007945   Hs.6684       ITPR1   Inositol 1,4,5-trisphosphate   D26070   Hs.198443           receptor, type 1       MXI1   MAX interacting protein 1   L07648   Hs.118630       TCF2   Transcription factor 2, hepatic   X58840   Hs.169853       KCNN4   Intermediate conductance calcium-   AF022797   Hs.10082           activated potassium channel                  
 
      In conclusion, using gene expression profiles of metastasis-promoting orthotopic xenografts as a predictive reference of expected transcript abundance behavior in clinical samples, we identified clusters of co-regulated genes discriminating with 75-100% accuracy among metastatic versus primary, invasive versus non-invasive, and recurrent versus non-recurrent human prostate tumors. Our data indicate that human prostate cancer cells derived from metastatic lesions have stable “genetic memory” of metastatic behavior and that genetic signatures associated with metastatic phenotype could be revived by growth in a metastasis-promoting orthotopic environment. The genetic signatures of metastatic prostate cancer have the ability to be used as nucleic acid-based and/or protein-based clinical prognostic and diagnostic tests useful in clinical management of prostate cancer patients, and as a source of targets for novel therapeutic approaches for disease management.  
     EXAMPLE 6  
     Selection of the Gene Clusters with Clinically Useful Properties Using the Best-Fit Sample(S) as a Reference Standard.  
      Application of the present invention for identification of gene clusters with useful clinical properties was not limited by the availability of the suitable reference standard such as the appropriate cell lines and/or in vivo model systems. When a suitable reference standard was not readily available an algorithm utilizing the expression profile(s) of the best-fit sample(s) as a reference standard was applied for selection of the minimum segregation set of genes. As the first step of such analysis we compared the gene expression profiles of two distinct sets of samples that are subjects of classification (for example, metastatic and non-metastatic human breast tumors) to identify a broad spectrum of transcripts differentially regulated at a statistically significant level (p&lt;0.05) in metastatic human breast cancer. If desirable, further criteria such as a particular cut-off based on fold expression changes (e.g., 2-fold, 3-fold, etc.) can be applied for selecting differentially expressed genes. Next, we calculated the average expression values for each transcript of the differentially expressed genes in the metastatic and non-metastatic tumors and determined the average fold expression change in metastatic versus non-metastatic tumors (“average” metastatic expression profile). We then determined the individual expression profiles for each sample within the two classification groups by calculating fold expression change for each transcript of the differentially expressed class of genes in a given sample by dividing an individual expression value of a gene by the average expression value for a particular gene across the entire data set. At the next step, we determined the individual phenotype association indices across the entire data set by calculating the Pearson correlation coefficient between the “average” metastatic expression profile and individual expression profiles. Next, the selection of the best-fit sample(s) was performed based on a highest positive and/or negative value(s) of the individual phenotype association index. The expression profile(s) of the best-fit sample(s) was utilized to refine the gene-expression signature associated with a particular phenotype to a small set of transcripts that would exhibit high discrimination accuracy between metastatic and non-metastatic tumors. To achieve this we used the increase in correlation coefficient of gene expression profiles between the “average” metastatic expression profile and an expression profile(s) of the best-fit sample(s) as a guide for reducing the number of members within a cluster.  
     EXAMPLE 7  
     Selection of the Gene Clusters Discriminating Between Invasive and Non-Invasive Human Prostate Cancer  
      The methods of the invention were used along with the data reported by Singh, et al. (2002) to identify gene clusters associated with an invasive phenotype. These data were the supplemental data reported in Singh, D., Febbo, P. G., et al., “Gene Expression Correlates of Clinical Prostate Cancer Behavior,”  Cancer Cell March  2002 1:203-209, incorporated herein by reference. The clinical human prostate tumor samples were divided into two groups, invasive and non-invasive, as reported in Singh, et al. (2002). Invasive phenotype was assessed by determining the presence or absence of positive surgical margins (“PSM”) and positive or negative capsular penetration (“PCP”). The reference set was obtained following the procedures described above in part B, using the supplemental data reported in Singh, et al. (2002) for 26 invasive (identified as having positive surgical margins and/or positive capsular penetration) and 26 non-invasive (identified as having no evidence of positive surgical margins and/or positive capsular penetration) human prostate tumors. Thus, the first reference set was obtained by using the Affymetrix MicroDB (version 3.0) and Affymetrix Data Mining Tools (DMT) (version 3.0) data analysis software to identify genes that were differentially regulated in invasive group compared to non-invasive group of patients at the statistically significant level (p&lt;0.05; Student T-test). Candidate genes were included in the first reference set if they were identified by the DMT software as having p values of 0.05 or less both for up-regulated and down-regulated genes. 114 genes were identified as being members of the reference set (Table 47).  
               TABLE 47                          114 genes differentially regulated in 26 invasive       versus 26 non-invasive human prostate tumors.                     Affymetrix           Probe Set ID       (U95Av2)   Description               40635_at   Cluster Incl. AF089750:  Homo sapiens  flotillin-1 mRNA, complete           cds /cds = (164, 1447) /gb = AF089750 /gi = 3599572 /ug =           Hs.179986 /len = 1796       36993_at   Cluster Incl. M33210: Human colony stimulating factor 1 receptor           (CSF1R) gene /cds = (0, 283) /gb = M33210 /gi = 532592 /ug =           Hs.76144 /len = 2206       38682_at   Cluster Incl. AF045581:  Homo sapiens  BRCA1 associated protein 1           (BAP1) mRNA, complete cds /cds = (39, 2228) /gb = AF045581 /gi =           2854120 /ug = Hs.106674 /len = 3506       38260_at   Cluster Incl. AL050306: Human DNA sequence from clone 475B7 on           chromosome Xq12.1-13. Contains the 3 part of the gene for a novel           KIAA0615 and KIAA0323 LIKE protein, the gene for a novel protein,           ESTs, STSs, GSSs and two putative CpG islands /cds = (48,           2201) /gb = AL050306 /gi = 5419784 /ug = Hs.90625 /len = 2395       41725_at   Cluster Incl. U89896:  Homo sapiens  casein kinase I gamma 2 mRNA,           complete cds /cds = (239, 1486) /gb = U89896 /gi = 1890117 /ug =           Hs.181390 /len = 1749       34880_at   Cluster Incl. AC002115: Human DNA from overlapping chromosome           19 cosmids R31396, F25451, and R31076 containing COX6B and           UPKA, genomic sequence /cds = (336, 1355) /gb = AC002115 /gi =           2098573 /ug = Hs.5086 /len = 1473       32140_at   Cluster Incl. Y08110:  H. sapiens  mRNA for mosaic protein           LR11 /cds = (80, 6724) /gb = Y08110 /gi = 1552323 /ug =           Hs.166294 /len = 6840       35704_at   Cluster Incl. X92814:  H. sapiens  mRNA for rat HREV107-like           protein /cds = (407, 895) /gb = X92814 /gi = 1054751 /ug =           Hs.37189 /len = 1070       32212_at   Cluster Incl. AL049703: Human gene from PAC 179D3, chromosome           X, isoform of mitochondrial apoptosis inducing factor, AIF,           AF100928 /cds = (96, 1925) /gb = AL049703 /gi = 4678806 /ug =           Hs.18720 /len = 2121       1385_at   M77349 /FEATURE = /DEFINITION = HUMTGFBIG Human           transforming growth factor-beta induced gene product (BIGH3)           mRNA, complete cds       37585_at   Cluster Incl. X13482: Human mRNA for U2 snRNP-specific A           protein /cds = (56, 823) /gb = X13482 /gi = 37546 /ug =           Hs.80506 /len = 1033       41869_at   Cluster Incl. U78310:  Homo sapiens  pescadillo mRNA, complete           cds /cds = (58, 1824) /gb = U78310 /gi = 2194202 /ug =           Hs.13501 /len = 2235       33833_at   Cluster Incl. J05243: Human nonerythroid alpha-spectrin (SPTAN1)           mRNA, complete cds /cds = (102, 7520) /gb = J05243 /gi =           179105 /ug = Hs.237180 /len = 7787       38794_at   Cluster Incl. X53390: Human mRNA for upstream binding factor           (hUBF) /cds = (147, 2441) /gb = X53390 /gi = 509240 /ug =           Hs.89781 /len = 3097       33915_at   Cluster Incl. W22655: 71B9  Homo sapiens  cDNA /clone = (not-           directional) /gb = W22655 /gi = 1299488 /ug = Hs.26070 /len = 761       35905_s_at   Cluster Incl. U34995: Human normal keratinocyte subtraction library           mRNA, clone H22a, complete sequence /cds = UNKNOWN /gb =           U34995 /gi = 1497857 /ug = Hs.195188 /len = 1626       39798_at   Cluster Incl. R87876: yo45h01.r1  Homo sapiens  cDNA, 5 end /clone =           IMAGE-180913 /clone_end = 5 /gb = R87876 /gi = 946689 /ug =           Hs.153177 /len = 483       1878_g_at   M13194 /FEATURE = mRNA /DEFINITION = HUMERCC1 Human           excision repair protein (ERCC1) mRNA, complete cds, clone pcDE       41116_at   Cluster Incl. AI799802: wc43d09.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2321393 /clone_end = 3 /gb =           AI799802 /gi = 5365274 /ug = Hs.101516 /len = 688       35961_at   Cluster Incl. AL049390:  Homo sapiens  mRNA; cDNA DKFZp586O1318           (from clone DKFZp586O1318) /cds = UNKNOWN /gb = AL049390 /gi =           4500184 /ug = Hs.22689 /len = 2322       37390_at   Cluster Incl. D86977: Human mRNA for KIAA0224 gene, complete           cds /cds = (136, 3819) /gb = D86977 /gi = 1504027 /ug = Hs.78054 /len = 4226       38841_at   Cluster Incl. AF068195:  Homo sapiens  putative glialblastoma cell           differentiation-related protein (GBDR1) mRNA, complete cds /cds =           (58, 1062) /gb = AF068195 /gi = 3192872 /ug = Hs.9194 /len = 1493       35787_at   Cluster Incl. AI986201: wr81a01.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2494056 /clone_end = 3 /gb = AI986201 /gi =           5813478 /ug = Hs.66881 /len = 814       39379_at   Cluster Incl. AL049397:  Homo sapiens  mRNA; cDNA           DKFZp586C1019 (from clone DKFZp586C1019) /cds =           UNKNOWN /gb = AL049397 /gi = 4500188 /ug =           Hs.12314 /len = 1720       928_at   L02785 /FEATURE = /DEFINITION = HUMDRA  Homo sapiens  colon           mucosa-associated (DRA) mRNA, complete cds       37349_r_at   Cluster Incl. AI817618: wk39f01.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2417785 /clone_end = 3 /gb =           AI817618 /gi = 5436697 /ug = Hs.77558 /len = 734       32933_r_at   Cluster Incl. AL050122:  Homo sapiens  mRNA; cDNA DKFZp586E121           (from clone DKFZp586E121) /cds = UNKNOWN /gb =           AL050122 /gi = 4884330 /ug = Hs.227742 /len = 1843       34909_at   Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185107 from           7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi =           3924668 /ug = Hs.128653 /len = 1767       AFFX-   U18530 SGD: YEL018W Yeast  S. cerevisiae  Protein of unknown       YEL018w/_at   function       37054_at   Cluster Incl. J04739: Human bactericidal permeability increasing           protein (BPI) mRNA, complete cds /cds = (30, 1493) /gb =           J04739 /gi = 179528 /ug = Hs.89535 /len = 1813       38871_at   Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10 protein /cds =           (690, 1391) /gb = AJ006288 /gi = 4049459 /ug = Hs.193516 /len = 1877       37800_r_at   Cluster Incl. AI263099: qz35b09.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2028857 /clone_end = 3 /gb =           AI263099 /gi = 3871302 /ug = Hs.126261 /len = 838       37236_at   Cluster Incl. M11437: Human kininogen gene /cds = (0,           1934) /gb = M11437 /gi = 186752 /ug = Hs.77741 /len = 1935       38198_at   Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert           cDNA clone EUROIMAGE 566443 /cds = UNKNOWN /gb =           AL079275 /gi = 5102578 /ug = Hs.157078 /len = 2082       35640_at   Cluster Incl. D14822: Human chimeric mRNA derived from AML1           gene and MTG8(ETO) gene, partial sequence /cds = (0,           597) /gb = D14822 /gi = 467498 /ug = Hs.31551 /len = 799       39828_at   Cluster Incl. AA477714: zu44e09.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-740872 /clone_end = 3 /gb =           AA477714 /gi = 2206348 /ug = Hs.111554 /len = 588       38938_at   Cluster Incl. AI816413: au47f05.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2517921 /clone_end = 3 /gb =           AI816413 /gi = 5431959 /ug = Hs.210862 /len = 586       39654_at   Cluster Incl. S67156: ASP = aspartoacylase [human, kidney, mRNA,           1435 nt] /cds = (158, 1099) /gb = S67156 /gi = 455833 /ug =           Hs.32042 /len = 1417       1393_at   L20348 /FEATURE = expanded_cds /DEFINITION = HUMOMDLN04             Homo sapiens  oncomodulin gene, exon 5       35920_at   Cluster Incl. N55205: yv44g05.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-245624 /clone_end = 3 /gb =           N55205 /gi = 1198084 /ug = Hs.20205 /len = 458       31368_at   Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb =           W27967 /gi = 1307915 /ug = Hs.136154 /len = 755       39912_at   Cluster Incl. AB006179:  Homo sapiens  mRNA for heparin-sulfate 6-           sulfotransferase, complete cds /cds = (111, 1343) /gb =           AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051       35489_at   Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic           acid hydrolase alpha subunit (PPH alpha) mRNA, complete           cds /cds = (9, 2249) /gb = M82962 /gi = 535474 /ug =           Hs.179704 /len = 2902       34486_at   Cluster Incl. U88897: Human endogenous retroviral H D2 leader region,           protease region, and integrase/envelope region mRNA sequence /cds =           UNKNOWN /gb = U88897 /gi = 2104917 /ug = Hs.11828 /len = 1004       32596_at   Cluster Incl. W25828: 13g2  Homo sapiens  cDNA /gb =           W25828 /gi = 1305951 /ug = Hs.79362 /len = 744       34057_at   Cluster Incl. U84392: Human Na+-dependent purine specific transporter           mRNA, complete cds /cds = (59, 2035) /gb = U84392 /gi = 2731438 /ug =           Hs.193665 /len = 2459       31759_at   Cluster Incl. W26220: 22d9  Homo sapiens  cDNA /gb = W26220 /gi =           1306631 /ug = Hs.136089 /len = 687       1485_at   L36642 /FEATURE = mRNA /DEFINITION = HUMRPTK  Homo               sapiens  receptor protein-tyrosine kinase (HEK11) mRNA, complete cds       39475_at   Cluster Incl. L37199:  Homo sapiens  (clone cD24-1) Huntingtons           disease candidate region mRNA fragment /cds = UNKNOWN /gb =           L37199 /gi = 600520 /ug = Hs.117487 /len = 1356       33012_at   Cluster Incl. L09753:  Homo sapiens  CD30 ligand mRNA, complete           cds /cds = (114, 818) /gb = L09753 /gi = 349277 /ug =           Hs.1313 /len = 1906       1321_s_at   U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor-           associated membrane protein homolog (TMP) mRNA, complete cds       32387_at   Cluster Incl. AB017494:  Homo sapiens  mRNA for LCAT-like           lysophospholipase (LLPL), complete cds /cds = (32,           1270) /gb = AB017494 /gi = 4589719 /ug = Hs.227221 /len = 1400       35565_at   Cluster Incl. U79301: Human clone 23842 mRNA           sequence /cds = UNKNOWN /gb = U79301 /gi =           1710286 /ug = Hs.135617 /len = 1582       1832_at   M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human           colorectal mutant cancer protein mRNA, complete cds       39924_at   Cluster Incl. AB020660:  Homo sapiens  mRNA for KIAA0853 protein,           partial cds /cds = (0, 2905) /gb = AB020660 /gi = 4240194 /ug =           Hs.136102 /len = 4363       39281_at   Cluster Incl. AB002378: Human mRNA for KIAA0380 gene, complete           cds /cds = (745, 5313) /gb = AB002378 /gi = 2224700 /ug =           Hs.239022 /len = 5790       34976_at   Cluster Incl. M60052: Human histidine-rich calcium binding protein           (HRC) mRNA, complete cds /cds = (170, 2269) /gb = M60052 /gi =           183918 /ug = Hs.1480 /len = 2365       39642_at   Cluster Incl. AL080199:  Homo sapiens  mRNA; cDNA DKFZp434E082           (from clone DKFZp434E082) /cds = UNKNOWN /gb = AL080199 /gi =           5262682 /ug = Hs.30504 /len = 1034       33615_at   Cluster Incl. X64994:  H. sapiens  HGMP07I gene for olfactory           receptor /cds = (0, 944) /gb = X64994 /gi = 32085 /ug =           Hs.163670 /len = 945       32054_at   Cluster Incl. AF048732:  Homo sapiens  cyclin T2b mRNA, complete           cds /cds = (0, 2192) /gb = AF048732 /gi = 2981199 /ug =           Hs.155478 /len = 2193       36383_at   Cluster Incl. M17254: Human erg2 gene encoding erg2 protein,           complete cds /cds = (0, 1388) /gb = M17254 /gi = 182186 /ug =           Hs.159432 /len = 1389       154_at   X07024 /FEATURE = cds /DEFINITION = HSCCG1 Human X           chromosome mRNA for CCG1 protein inv. in cell proliferation       39882_at   Cluster Incl. U66035: Human X-linked deafness dystonia protein (DDP)           mRNA, complete cds /cds = (35, 328) /gb = U66035 /gi =           3123842 /ug = Hs.125565 /len = 1169       35452_at   Cluster Incl. AL109690:  Homo sapiens  mRNA full length insert cDNA           clone EUROIMAGE 190711 /cds = UNKNOWN /gb = AL109690 /gi =           5689787 /ug = Hs.169950 /len = 2031       39926_at   Cluster Incl. U59913: Human chromosome 5 Mad homolog Smad5           mRNA, complete cds /cds = (130, 1527) /gb = U59913 /gi =           1654324 /ug = Hs.37501 /len = 2205       39246_at   Cluster Incl. Z75330:  H. sapiens  mRNA for nuclear protein           SA-1 /cds = (400, 4176) /gb = Z75330 /gi = 2204212 /ug =           Hs.234435 /len = 4337       40248_at   Cluster Incl. AL022165: dJ71L16.5 (KIAA0267 LIKE putative           Na(+)/H(+) exchanger) /cds = (0, 1852) /gb = AL022165 /gi =           3281985 /ug = Hs.154353 /len = 3487       31446_s_at   Cluster Incl. D89501: Human PBI gene, complete cds /cds = (14,           418) /gb = D89501 /gi = 1854451 /ug = Hs.166099 /len = 576       37937_at   Cluster Incl. AJ005257:  Homo sapiens  partial mRNA for beta-           transducin family protein (putative) /cds = (0, 262) /gb =           AJ005257 /gi = 3043442 /ug = Hs.85570 /len = 1349       39914_r_at   Cluster Incl. W28976: 54e5  Homo sapiens  cDNA /gb =           W28976 /gi = 1308924 /ug = Hs.133151 /len = 903       37514_s_at   Cluster Incl. AB008047:  Homo sapiens  sMAP mRNA for small MBL-           associated protein, complete cds /cds = (26, 583) /gb =           AB008047 /gi = 5002493 /ug = Hs.119983 /len = 725       971_s_at   Y00083 /FEATURE = cds /DEFINITION = HSGTSF Human mRNA for           glioblastoma-derived T-cell suppressor factor G-TsF (transforming           growth factor-beta2, TGF-beta2)       41863_at   Cluster Incl. AF070623:  Homo sapiens  clone 24468 mRNA           sequence /cds = UNKNOWN /gb = AF070623 /gi =           3283889 /ug = Hs.13423 /len = 1226       39304_g_at   Cluster Incl. Y14153:  Homo sapiens  mRNA for beta-transducin           repeat containing protein /cds = (69, 1778) /gb =           Y14153 /gi = 2995193 /ug = Hs.239742 /len = 2141       35003_at   Cluster Incl. AA534868: nf82b01.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-926377 /clone_end = 3 /gb =           AA534868 /gi = 2279121 /ug = Hs.152400 /len = 595       34059_at   Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-1086587 /clone_end = 3 /gb =           AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522       41112_at   Cluster Incl. AB011129:  Homo sapiens  mRNA for KIAA0557 protein,           partial cds /cds = (0, 1482) /gb = AB011129 /gi = 3043637 /ug =           Hs.101414 /len = 5627       31922_i_at   Cluster Incl. U60269: Human endogenous retrovirus HERV-K(HML6)           proviral clone HML6.17 putative polymerase and envelope genes,           partial cds, and 3LTR /cds = (0, 491) /gb = U60269 /gi =           1408208 /ug = Hs.159902 /len = 492       2023_g_at   M77198 /FEATURE = /DEFINITION = HUMRPKB Human rac protein           kinase beta mRNA, complete cds       40919_at   Cluster Incl. M81830: Human somatostatin receptor isoform 2 (SSTR2)           gene, complete cds /cds = (0, 1109) /gb = M81830 /gi = 307435 /ug =           Hs.184841 /len = 1110       677_s_at   J04430 /FEATURE = mRNA /DEFINITION = HUMACP5 Human           tartrate-resistant acid phosphatase type 5 mRNA, complete cds       41291_at   Cluster Incl. AC004528:  Homo sapiens  chromosome 19, cosmid           R32184 /cds = (0, 1589) /gb = AC004528 /gi = 3025444 /ug =           Hs.238519 /len = 1590       32746_at   Cluster Incl. AF015451:  Homo sapiens  Usurpin-beta mRNA, complete           cds /cds = (0, 1388) /gb = AF015451 /gi = 3133282 /ug =           Hs.195175 /len = 1389       39364_s_at   Cluster Incl. Y18207:  Homo sapiens  mRNA for protein phosphatase 1           (PPP1R5) /cds = (91, 1044) /gb = Y18207 /gi = 3805818 /ug =           Hs.12112 /len = 1158       135_g_at   X95632 /FEATURE = cds /DEFINITION = HSARGBPIA  H. sapiens             mRNA for Arg protein tyrosine kinase-binding protein       37785_at   Cluster Incl. U69563: U69563  Homo sapiens  cDNA /clone =           25050 /gb = U69563 /gi = 2731394 /ug = Hs.124940 /len = 1657       39190_s_at   Cluster Incl. AC002126:  Homo sapiens  DNA from chromosome 19-           cosmids R30102-R29350-R27740 containing MEF2B, genomic           sequence /cds = (0, 307) /gb = AC002126 /gi = 2329908 /ug =           Hs.125220 /len = 308       41550_at   Cluster Incl. AF091071:  Homo sapiens  clone 192 Rer1 mRNA,           complete cds /cds = (76, 696) /gb = AF091071 /gi =           3859979 /ug = Hs.40500 /len = 1400       40240_at   Cluster Incl. AC004131:  Homo sapiens  Chromosome 16 BAC clone           CIT987SK-A-69G12 /cds = (0, 1211) /gb = AC004131 /gi =           3342217 /ug = Hs.154050 /len = 1887       38224_at   Cluster Incl. U71300: Human snRNA activating protein complex 50 kD           subunit (SNAP50) mRNA, complete cds /cds = (14, 1249) /gb =           U71300 /gi = 1619945 /ug = Hs.164915 /len = 1848       41534_at   Cluster Incl. AB006755:  Homo sapiens  mRNA for PCDH7 (BH-Pcdh)a,           complete cds /cds = (1010, 4219) /gb = AB006755 /gi =           2979417 /ug = Hs.34073 /len = 4648       1569_r_at   L42243 /FEATURE = exon#3 /DEFINITION = HUMIFNAM08  Homo               sapiens  (clone 51H8) alternatively spliced interferon receptor           (IFNAR2) gene, exon 9 and complete cds s       35960_at   Cluster Incl. AF031416:  Homo sapiens  IkB kinase beta subunit mRNA,           complete cds /cds = (0, 2270) /gb = AF031416 /gi = 3213216 /ug =           Hs.226573 /len = 2271       32149_at   Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone = IMAGE-           996282 /gb = AA532495 /gi = 2276749 /ug = Hs.183752 /len = 549       1668_s_at   L15409 /FEATURE = /DEFINITION = HUMHIPLIND  Homo sapiens             (clone g7) von Hippel-Lindau disease tumor suppressor mRNA           sequence       32877_i_at   Cluster Incl. AA524802: nh33h11.s1  Homo sapiens             cDNA /clone = IMAGE-954213 /gb = AA524802 /gi =           2265730 /ug = Hs.203907 /len = 500       37152_at   Cluster Incl. L07592: Human peroxisome proliferator activated receptor           mRNA, complete cds /cds = (337, 1662) /gb = L07592 /gi = 190229 /ug =           Hs.106415 /len = 3301       33155_at   Cluster Incl. M95740: Human alpha-L-iduronidase gene /cds = (0,           1961) /gb = M95740 /gi = 178412 /ug = Hs.89560 /len = 2234       34031_i_at   Cluster Incl. U90268: Human Krit1 mRNA, complete cds /cds = (25,           1614) /gb = U90268 /gi = 2149601 /ug = Hs.93810 /len = 1986       39504_at   Cluster Incl. AF014643:  Homo sapiens  connexin46.6 (Cx46.6) gene,           complete cds /cds = (28, 1338) /gb = AF014643 /gi = 2738576 /ug =           Hs.100072 /len = 2087       40975_s_at   Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin-           interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb =           AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565       40241_at   Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA,           complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug =           Hs.154095 /len = 3908       33723_at   Cluster Incl. AL049346:  Homo sapiens  mRNA; cDNA DKFZp566B213           (from clone DKFZp566B213) /cds = UNKNOWN /gb = AL049346 /gi =           4500130 /ug = Hs.194051 /len = 1554       1459_at   M68941 /FEATURE = mRNA /DEFINITION = HUMPTYPH Human           protein-tyrosine phosphatase mRNA, complete cds       40033_at   Cluster Incl. AL022328: Human DNA sequence from clone 402G11 on           chromosome 22q13.31-13.33 Contains genes for SAPK3 (stress-           activated protein kinase 3), PRKM11 (protein kinase mitogen-activated           11), KIAA0315, ESTs, GSSs and CpG islands /cds = (11, 1105) /gb =           AL022328 /gi = 5263010 /ug = Hs.57732 /len = 2341       39661_s_at   Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside           transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb =           AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522       37629_at   Cluster Incl. M55268: Human casein kinase II alpha subunit mRNA,           complete cds /cds = (163, 1215) /gb = M55268 /gi = 177837 /ug =           Hs.82201 /len = 1677       1624_at   Stimulatory Gdp/Gtp Exchange Protein For C-Ki-Ras P21 And Smg P21       1903_at   Ras-Related Protein Rap1b       33170_at   Cluster Incl. AB023179:  Homo sapiens  mRNA for KIAA0962 protein,           partial cds /cds = (0, 1893) /gb = AB023179 /gi = 4589567 /ug =           Hs.9059 /len = 5460       33175_at   Cluster Incl. AA156237: z150c09.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-505360 /clone_end = 3 /gb =           AA156237 /gi = 1727855 /ug = Hs.90804 /len = 644       38044_at   Cluster Incl. AF035283:  Homo sapiens  clone 23916 mRNA           sequence /cds = UNKNOWN /gb = AF035283 /gi =           2661034 /ug = Hs.8022 /len = 2022       40440_at   Cluster Incl. AL080119:  Homo sapiens  mRNA; cDNA DKFZp564M2423 (from           clone DKFZp564M2423) /cds = (85, 1248) /gb = AL080119 /gi =           5262550 /ug = Hs.165998 /len = 2183       35254_at   Cluster Incl. AB007447:  Homo sapiens  mRNA for Fln29, complete           cds /cds = (54, 1802) /gb = AB007447 /gi = 2463530 /ug =           Hs.5148 /len = 2618                  
 
      Next, we calculated phenotype association indices for all 52 samples and determined that this gene cluster exhibited a 77% success rate in clinical sample classification based on individual phenotype association indices (Table 48). As shown in Table 48, 22/26 (or 85%) of the invasive prostate cancer samples had positive phenotype association indices, whereas 18/26 (or 69%) of non-invasive prostate cancer samples displayed negative phenotype association indices. Overall, 40 of 52 samples (or 77%) were correctly classified.  
               TABLE 48                          Classification accuracy of the prostate cancer invasion clusters                                     r value                       (Phenotype           Association   Invasive   Non-invasive       Cluster   Index)   tumors   tumors   Overall                                             114   genes   0.704   22/26 (85%)   18/26 (69%)   40/52 (77%)       53   genes   0.893   22/26 (85%)   17/26 (65%)   39/52 (75%)       39   genes   0.972   22/26 (85%)   18/26 (69%)   40/52 (77%)       26   genes   0.994   23/26 (88%)   17/26 (65%)   40/52 (77%)       24   genes   0.997   21/26 (81%)   17/26 (65%)   38/52 (73%)       22   genes   0.995   21/26 (81%)   18/26 (69%)   39/52 (75%)                  
 
      Next, we identified a single best-fit invasive prostate cancer sample displaying the correlation coefficient of 0.704 to the average expression profile of the 26 invasive prostate cancer samples. The expression profile of this single best-fit invasive prostate cancer sample was utilized as a second reference set.  
      The concordance set was obtained by selecting only those genes having a consistent direction of the differential expression in both the first and the second reference sets (i.e., greater gene expression difference in the invasive cf. the non-invasive samples and greater gene expression in the best-fit tumor sample cf. the average expression value across the entire data set or vice-versa). The concordance set comprised of 107 genes (r=0.721). A minimum segregation set was selected following the procedures described in above. Scatter plots were generated of the log 10  transformed average −fold expression change in the first reference set and average −fold expression change in the second reference set (in case of a single best-fit tumor it was the log 10  transformed ratio of the expression value for a gene to the average expression value across the entire data set). For the samples of the first reference set, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who had invasive tumors and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who had non-invasive tumors. A minimum segregation set was identified by selecting a subset of the highly correlated genes between two reference sets from the invasiveness concordance set. Using this approach we identified five gene clusters discriminating with high accuracy between invasive and non-invasive human prostate tumors. The members of these invasion predictors or invasion minimum segregation sets (invasion minimum segregation gene clusters) are listed in Tables 49-54. The classification performance for each of these gene clusters is presented in the Table 48.  
               TABLE 49                          53-gene signature of invasive prostate cancer                     Affymetrix           Probe Set ID       (U95Av2)   Description               1878_g_at   M13194 /FEATURE = mRNA /DEFINITION = HUMERCC1 Human           excision repair protein (ERCC1) mRNA, complete cds, clone pcDE       33833_at   Cluster Incl. J05243: Human nonerythroid alpha-spectrin (SPTAN1)           mRNA, complete cds /cds = (102, 7520) /gb = J05243 /gi =           179105 /ug = Hs.237180 /len = 7787       33915_at   Cluster Incl. W22655: 71B9  Homo sapiens  cDNA /clone = (not-           directional) /gb = W22655 /gi = 1299488 /ug = Hs.26070 /len = 761       35787_at   Cluster Incl. AI986201: wr81a01.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2494056 /clone_end = 3 /gb =           AI986201 /gi = 5813478 /ug = Hs.66881 /len = 814       37390_at   Cluster Incl. D86977: Human mRNA for KIAA0224 gene, complete           cds /cds = (136, 3819) /gb = D86977 /gi = 1504027 /ug =           Hs.78054 /len = 4226       38260_at   Cluster Incl. AL050306: Human DNA sequence from clone 475B7 on           chromosome Xq12.1-13. Contains the 3 part of the gene for a novel           KIAA0615 and KIAA0323 LIKE protein, the gene for a novel protein,           ESTs, STSs, GSSs and two putative CpG islands /cds = (48,           2201) /gb = AL050306 /gi = 5419784 /ug = Hs.90625 /len = 2395       38794_at   Cluster Incl. X53390: Human mRNA for upstream binding factor           (hUBF) /cds = (147, 2441) /gb = X53390 /gi = 509240 /ug =           Hs.89781 /len = 3097       38841_at   Cluster Incl. AF068195:  Homo sapiens  putative glialblastoma cell           differentiation-related protein (GBDR1) mRNA, complete cds /cds =           (58, 1062) /gb = AF068195 /gi = 3192872 /ug = Hs.9194 /len = 1493       39379_at   Cluster Incl. AL049397:  Homo sapiens  mRNA; cDNA DKFZp586C1019           (from clone DKFZp586C1019) /cds = UNKNOWN /gb = AL049397 /gi =           4500188 /ug = Hs.12314 /len = 1720       40635_at   Cluster Incl. AF089750:  Homo sapiens  flotillin-1 mRNA, complete           cds /cds = (164, 1447) /gb = AF089750 /gi = 3599572 /ug =           Hs.179986 /len = 1796       41116_at   Cluster Incl. AI799802: wc43d09.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2321393 /clone_end = 3 /gb =           AI799802 /gi = 5365274 /ug = Hs.101516 /len = 688       41869_at   Cluster Incl. U78310:  Homo sapiens  pescadillo mRNA, complete           cds /cds = (58, 1824) /gb = U78310 /gi = 2194202 /ug =           Hs.13501 /len = 2235       1321_s_at   U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor-           associated membrane protein homolog (TMP) mRNA, complete cds       154_at   X07024 /FEATURE = cds /DEFINITION = HSCCG1 Human X           chromosome mRNA for CCG1 protein inv. in cell proliferation       1569_r_at   L42243 /FEATURE = exon#3 /DEFINITION = HUMIFNAM08  Homo               sapiens  (clone 51H8) alternatively spliced interferon receptor (IFNAR2)           gene, exon 9 and complete cds s       1668_s_at   L15409 /FEATURE = /DEFINITION = HUMHIPLIND  Homo sapiens             (clone g7) von Hippel-Lindau disease tumor suppressor mRNA sequence       1832_at   M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human           colorectal mutant cancer protein mRNA, complete cds       1903_at   Ras-Related Protein Rap1b       31368_at   Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb =           W27967 /gi = 1307915 /ug = Hs.136154 /len = 755       31446_s_at   Cluster Incl. D89501: Human PBI gene, complete cds /cds = (14,           418) /gb = D89501 /gi = 1854451 /ug = Hs.166099 /len = 576       31922_i_at   Cluster Incl. U60269: Human endogenous retrovirus HERV-K(HML6)           proviral clone HML6.17 putative polymerase and envelope genes, partial           cds, and 3LTR /cds = (0, 491) /gb = U60269 /gi = 1408208 /ug =           Hs.159902 /len = 492       32054_at   Cluster Incl. AF048732:  Homo sapiens  cyclin T2b mRNA, complete           cds /cds = (0, 2192) /gb = AF048732 /gi = 2981199 /ug =           Hs.155478 /len = 2193       32149_at   Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone =           IMAGE-996282 /gb = AA532495 /gi = 2276749 /ug =           Hs.183752 /len = 549       32596_at   Cluster Incl. W25828: 13g2  Homo sapiens  cDNA /gb =           W25828 /gi = 1305951 /ug = Hs.79362 /len = 744       33615_at   Cluster Incl. X64994:  H. sapiens  HGMP07I gene for olfactory           receptor /cds = (0, 944) /gb = X64994 /gi = 32085 /ug =           Hs.163670 /len = 945       33723_at   Cluster Incl. AL049346:  Homo sapiens  mRNA; cDNA DKFZp566B213           (from clone DKFZp566B213) /cds = UNKNOWN /gb =           AL049346 /gi = 4500130 /ug = Hs.194051 /len = 1554       34057_at   Cluster Incl. U84392: Human Na+-dependent purine specific transporter           mRNA, complete cds /cds = (59, 2035) /gb = U84392 /gi = 2731438 /ug =           Hs.193665 /len = 2459       34059_at   Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3           end /clone = IMAGE-1086587 /clone_end = 3 /gb =           AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522       34486_at   Cluster Incl. U88897: Human endogenous retroviral H D2 leader region,           protease region, and integrase/envelope region mRNA sequence /cds =           UNKNOWN /gb = U88897 /gi = 2104917 /ug = Hs.11828 /len = 1004       34909_at   Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185107 from           7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi = 3924668 /ug =           Hs.128653 /len = 1767       35489_at   Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic acid           hydrolase alpha subunit (PPH alpha) mRNA, complete cds /cds = (9,           2249) /gb = M82962 /gi = 535474 /ug = Hs.179704 /len = 2902       35565_at   Cluster Incl. U79301: Human clone 23842 mRNA sequence /cds =           UNKNOWN /gb = U79301 /gi = 1710286 /ug = Hs.135617 /len = 1582       35640_at   Cluster Incl. D14822: Human chimeric mRNA derived from AML1 gene           and MTGS(ETO) gene, partial sequence /cds = (0, 597) /gb =           D14822 /gi = 467498 /ug = Hs.31551 /len = 799       35960_at   Cluster Incl. AF031416:  Homo sapiens  IkB kinase beta subunit mRNA,           complete cds /cds = (0, 2270) /gb = AF031416 /gi = 3213216 /ug =           Hs.226573 /len = 2271       37054_at   Cluster Incl. J04739: Human bactericidal permeability increasing protein           (BPI) mRNA, complete cds /cds = (30, 1493) /gb = J04739 /gi =           179528 /ug = Hs.89535 /len = 1813       37785_at   Cluster Incl. U69563: U69563  Homo sapiens  cDNA /clone = 25050 /gb =           U69563 /gi = 2731394 /ug = Hs.124940 /len = 1657       38198_at   Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert cDNA           clone EUROIMAGE 566443 /cds = UNKNOWN /gb = AL079275 /gi =           5102578 /ug = Hs.157078 /len = 2082       38871_at   Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10           protein /cds = (690, 1391) /gb = AJ006288 /gi =           4049459 /ug = Hs.193516 /len = 1877       38938_at   Cluster Incl. AI816413: au47f05.x1  Homo sapiens  cDNA, 3           end /clone = IMAGE-2517921 /clone_end = 3 /gb =           AI816413 /gi = 5431959 /ug = Hs.210862 /len = 586       39304_g_at   Cluster Incl. Y14153:  Homo sapiens  mRNA for beta-transducin repeat           containing protein /cds = (69, 1778) /gb = Y14153 /gi =           2995193 /ug = Hs.239742 /len = 2141       39364_s_at   Cluster Incl. Y18207:  Homo sapiens  mRNA for protein phosphatase 1           (PPP1R5) /cds = (91, 1044) /gb = Y18207 /gi = 3805818 /ug =           Hs.12112 /len = 1158       39475_at   Cluster Incl. L37199:  Homo sapiens  (clone cD24-1) Huntingtons disease           candidate region mRNA fragment /cds = UNKNOWN /gb = L37199 /gi =           600520 /ug = Hs.117487 /len = 1356       39661_s_at   Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside           transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb =           AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522       39882_at   Cluster Incl. U66035: Human X-linked deafness dystonia protein (DDP)           mRNA, complete cds /cds = (35, 328) /gb = U66035 /gi = 3123842 /ug =           Hs.125565 /len = 1169       39912_at   Cluster Incl. AB006179:  Homo sapiens  mRNA for heparan-sulfate 6-           sulfotransferase, complete cds /cds = (111, 1343) /gb =           AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051       39924_at   Cluster Incl. AB020660:  Homo sapiens  mRNA for KIAA0853 protein,           partial cds /cds = (0, 2905) /gb = AB020660 /gi = 4240194 /ug =           Hs.136102 /len = 4363       39926_at   Cluster Incl. U59913: Human chromosome 5 Mad homolog Smad5           mRNA, complete cds /cds = (130, 1527) /gb = U59913 /gi =           1654324 /ug = Hs.37501 /len = 2205       40241_at   Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA,           complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug =           Hs.154095 /len = 3908       40975_s_at   Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin-           interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb =           AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565       41112_at   Cluster Incl. AB011129:  Homo sapiens  mRNA for KIAA0557 protein,           partial cds /cds = (0, 1482) /gb = AB011129 /gi = 3043637 /ug =           Hs.101414 /len = 5627       41550_at   Cluster Incl. AF091071:  Homo sapiens  clone 192 Rer1 mRNA, complete           cds /cds = (76, 696) /gb = AF091071 /gi = 3859979 /ug =           Hs.40500 /len = 1400       677_s_at   J04430 /FEATURE = mRNA /DEFINITION = HUMACP5 Human tartrate-           resistant acid phosphatase type 5 mRNA, complete cds       971_s_at   Y00083 /FEATURE = cds /DEFINITION = HSGTSF Human mRNA for           glioblastoma-derived T-cell suppressor factor G-TsF (transforming           growth factor-beta2, TGF-beta2)                  
 
     
       
         
           
               
             
               
                 TABLE 50 
               
             
            
               
                   
               
               
                   
               
               
                 39-gene signature of invasive prostate cancer 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set ID 
               
               
                 (U95Av2) 
                 Description 
               
               
                   
               
               
                 1878_g_at 
                 M13194 /FEATURE = mRNA /DEFINITION = HUMERCC1 Human 
               
               
                   
                 excision repair protein (ERCC1) mRNA, complete cds, clone pcDE 
               
               
                 33833_at 
                 Cluster Incl. J05243: Human nonerythroid alpha-spectrin (SPTAN1) 
               
               
                   
                 mRNA, complete cds /cds = (102, 7520) /gb = J05243 /gi = 
               
               
                   
                 179105 /ug = Hs.237180 /len = 7787 
               
               
                 33915_at 
                 Cluster Incl. W22655: 71B9  Homo sapiens  cDNA /clone = (not-direc- 
               
               
                   
                 tional) /gb = W22655 /gi = 1299488 /ug = Hs.26070 /len = 761 
               
               
                 35787_at 
                 Cluster Incl. AI986201: wr81a01.x1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-2494056 /clone_end = 3 /gb = 
               
               
                   
                 AI986201 /gi = 5813478 /ug = Hs.66881 /len = 814 
               
               
                 37390_at 
                 Cluster Incl. D86977: Human mRNA for KIAA0224 gene, complete 
               
               
                   
                 cds /cds = (136, 3819) /gb = D86977 /gi = 1504027 /ug = 
               
               
                   
                 Hs.78054 /len = 4226 
               
               
                 38260_at 
                 Cluster Incl. AL050306: Human DNA sequence from clone 475B7 on 
               
               
                   
                 chromosome Xq12.1-13. Contains the 3 part of the gene for a novel 
               
               
                   
                 KIAA0615 and KIAA0323 LIKE protein, the gene for a novel protein, 
               
               
                   
                 ESTs, STSs, GSSs and two putative CpG islands /cds = (48, 
               
               
                   
                 2201) /gb = AL050306 /gi = 5419784 /ug = Hs.90625 /len = 2395 
               
               
                 38794_at 
                 Cluster Incl. X53390: Human mRNA for upstream binding factor 
               
               
                   
                 (hUBF) /cds = (147, 2441) /gb = X53390 /gi = 509240 /ug = 
               
               
                   
                 Hs.89781 /len = 3097 
               
               
                 38841_at 
                 Cluster Incl. AF068195:  Homo sapiens  putative glialblastoma cell 
               
               
                   
                 differentiation-related protein (GBDR1) mRNA, complete cds /cds = 
               
               
                   
                 (58, 1062) /gb = AF068195 /gi = 3192872 /ug = Hs.9194 /len = 1493 
               
               
                 39379_at 
                 Cluster Incl. AL049397:  Homo sapiens  mRNA; cDNA DKFZp586C1019 
               
               
                   
                 (from clone DKFZp586C1019) /cds = UNKNOWN /gb = AL049397 /gi = 
               
               
                   
                 4500188 /ug = Hs.12314 /len = 1720 
               
               
                 40635_at 
                 Cluster Incl. AF089750:  Homo sapiens  flotillin-1 mRNA, complete 
               
               
                   
                 cds /cds = (164, 1447) /gb = AF089750 /gi = 3599572 /ug = 
               
               
                   
                 Hs.179986 /len = 1796 
               
               
                 41116_at 
                 Cluster Incl. AI799802: wc43d09.x1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-2321393 /clone_end = 3 /gb = 
               
               
                   
                 AI799802 /gi = 5365274 /ug = Hs.101516 /len = 688 
               
               
                 41869_at 
                 Cluster Incl. U78310:  Homo sapiens  pescadillo mRNA, complete 
               
               
                   
                 cds /cds = (58, 1824) /gb = U78310 /gi = 2194202 /ug = 
               
               
                   
                 Hs.13501 /len = 2235 
               
               
                 1321_s_at 
                 U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor- 
               
               
                   
                 associated membrane protein homolog (TMP) mRNA, complete cds 
               
               
                 1668_s_at 
                 L15409 /FEATURE = /DEFINITION = HUMHIPLIND  Homo sapiens   
               
               
                   
                 (clone g7) von Hippel-Lindau disease tumor suppressor mRNA sequence 
               
               
                 1832_at 
                 M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human colorectal 
               
               
                   
                 mutant cancer protein mRNA, complete cds 
               
               
                 1903_at 
                 Ras-Related Protein Rap1b 
               
               
                 31368_at 
                 Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb = 
               
               
                   
                 W27967 /gi = 1307915 /ug = Hs.136154 /len = 755 
               
               
                 31446_s_at 
                 Cluster Incl. D89501: Human PBI gene, complete cds /cds = (14, 
               
               
                   
                 418) /gb = D89501 /gi = 1854451 /ug = Hs.166099 /len = 576 
               
               
                 31922_i_at 
                 Cluster Incl. U60269: Human endogenous retrovirus HERV-K(HML6) 
               
               
                   
                 proviral clone HML6.17 putative polymerase and envelope genes, partial 
               
               
                   
                 cds, and 3LTR /cds = (0, 491) /gb = U60269 /gi = 1408208 /ug = 
               
               
                   
                 Hs.159902 /len = 492 
               
               
                 32054_at 
                 Cluster Incl. AF048732:  Homo sapiens  cyclin T2b mRNA, complete 
               
               
                   
                 cds /cds = (0, 2192) /gb = AF048732 /gi = 2981199 /ug = 
               
               
                   
                 Hs.155478 /len = 2193 
               
               
                 32149_at 
                 Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone = IMAGE- 
               
               
                   
                 996282 /gb = AA532495 /gi = 2276749 /ug = Hs.183752 /len = 549 
               
               
                 33723_at 
                 Cluster Incl. AL049346:  Homo sapiens  mRNA; cDNA DKFZp566B213 
               
               
                   
                 (from clone DKFZp566B213) /cds = UNKNOWN /gb = AL049346 /gi = 
               
               
                   
                 4500130 /ug = Hs.194051 /len = 1554 
               
               
                 34059_at 
                 Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-1086587 /clone_end = 3 /gb = 
               
               
                   
                 AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522 
               
               
                 34909_at 
                 Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185107 from 
               
               
                   
                 7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi = 
               
               
                   
                 3924668 /ug = Hs.128653 /len = 1767 
               
               
                 35489_at 
                 Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic acid 
               
               
                   
                 hydrolase alpha subunit (PPH alpha) mRNA, complete cds /cds = (9, 
               
               
                   
                 2249) /gb = M82962 /gi = 535474 /ug = Hs.179704 /len = 2902 
               
               
                 35640_at 
                 Cluster Incl. D14822: Human chimeric mRNA derived from AML1 gene 
               
               
                   
                 and MTGS(ETO) gene, partial sequence /cds = (0, 597) /gb = 
               
               
                   
                 D14822 /gi = 467498 /ug = Hs.31551 /len = 799 
               
               
                 37054_at 
                 Cluster Incl. J04739: Human bactericidal permeability increasing protein 
               
               
                   
                 (BPI) mRNA, complete cds /cds = (30, 1493) /gb = J04739 /gi = 
               
               
                   
                 179528 /ug = Hs.89535 /len = 1813 
               
               
                 37785_at 
                 Cluster Incl. U69563: U69563  Homo sapiens  cDNA /clone = 
               
               
                   
                 25050 /gb = U69563 /gi = 2731394 /ug = Hs.124940 /len = 1657 
               
               
                 38198_at 
                 Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert cDNA 
               
               
                   
                 clone EUROIMAGE 566443 /cds = UNKNOWN /gb = AL079275 /gi = 
               
               
                   
                 5102578 /ug = Hs.157078 /len = 2082 
               
               
                 38871_at 
                 Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10 protein /cds = 
               
               
                   
                 (690, 1391) /gb = AJ006288 /gi = 4049459 /ug = Hs.193516 /len = 1877 
               
               
                 39475_at 
                 Cluster Incl. L37199:  Homo sapiens  (clone cD24-1) Huntingtons disease 
               
               
                   
                 candidate region mRNA fragment /cds = UNKNOWN /gb = L37199 /gi = 
               
               
                   
                 600520 /ug = Hs.117487 /len = 1356 
               
               
                 39661_s_at 
                 Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside 
               
               
                   
                 transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb = 
               
               
                   
                 AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522 
               
               
                 39882_at 
                 Cluster Incl. U66035: Human X-linked deafness dystonia protein (DDP) 
               
               
                   
                 mRNA, complete cds /cds = (35, 328) /gb = U66035 /gi = 3123842 /ug = 
               
               
                   
                 Hs.125565 /len = 1169 
               
               
                 39912_at 
                 Cluster Incl. AB006179:  Homo sapiens  mRNA for heparan-sulfate 6- 
               
               
                   
                 sulfotransferase, complete cds /cds = (111, 1343) /gb = 
               
               
                   
                 AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051 
               
               
                 40241_at 
                 Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA, 
               
               
                   
                 complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug = 
               
               
                   
                 Hs.154095 /len = 3908 
               
               
                 40975_s_at 
                 Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin- 
               
               
                   
                 interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb = 
               
               
                   
                 AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565 
               
               
                 41550_at 
                 Cluster Incl. AF091071:  Homo sapiens  clone 192 Rer1 mRNA, complete 
               
               
                   
                 cds /cds = (76, 696) /gb = AF091071 /gi = 3859979 /ug = 
               
               
                   
                 Hs.40500 /len = 1400 
               
               
                 677_s_at 
                 J04430 /FEATURE = mRNA /DEFINITION = HUMACP5 Human tartrate- 
               
               
                   
                 resistant acid phosphatase type 5 mRNA, complete cds 
               
               
                 971_s_at 
                 Y00083 /FEATURE = cds /DEFINITION = HSGTSF Human mRNA for 
               
               
                   
                 glioblastoma-derived T-cell suppressor factor G-TsF (transforming growth 
               
               
                   
                 factor-beta2, TGF-beta2) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 51 
               
             
            
               
                   
               
               
                   
               
               
                 26-gene signature of invasive prostate cancer 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set ID 
               
               
                 (U95Av2) 
                 Description 
               
               
                   
               
               
                 36993_at 
                 Cluster Incl. M33210: Human colony stimulating factor 1 receptor 
               
               
                   
                 (CSF1R) gene /cds = (0, 283) /gb = M33210 /gi = 532592 /ug = 
               
               
                   
                 Hs.76144 /len = 2206 
               
               
                 38682_at 
                 Cluster Incl. AF045581:  Homo sapiens  BRCA1 associated protein 1 
               
               
                   
                 (BAP1) mRNA, complete cds /cds = (39, 2228) /gb = AF045581 /gi = 
               
               
                   
                 2854120 /ug = Hs.106674 /len = 3506 
               
               
                 41725_at 
                 Cluster Incl. U89896:  Homo sapiens  casein kinase I gamma 2 mRNA, 
               
               
                   
                 complete cds /cds = (239, 1486) /gb = U89896 /gi = 1890117 /ug = 
               
               
                   
                 Hs.181390 /len = 1749 
               
               
                 32212_at 
                 Cluster Incl. AL049703: Human gene from PAC 179D3, chromosome 
               
               
                   
                 X, isoform of mitochondrial apoptosis inducing factor, AIF, 
               
               
                   
                 AF100928 /cds = (96, 1925) /gb = AL049703 /gi = 4678806 /ug = 
               
               
                   
                 Hs.18720 /len = 2121 
               
               
                 1385_at 
                 M77349 /FEATURE = /DEFINITION = HUMTGFBIG Human 
               
               
                   
                 transforming growth factor-beta induced gene product (BIGH3) 
               
               
                   
                 mRNA, complete cds 
               
               
                 37585_at 
                 Cluster Incl. X13482: Human mRNA for U2 snRNP-specific A 
               
               
                   
                 protein /cds = (56, 823) /gb = X13482 /gi = 37546 /ug = 
               
               
                   
                 Hs.80506 /len = 1033 
               
               
                 1903_at 
                 Ras-Related Protein Rap1b 
               
               
                 39661_s_at 
                 Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside 
               
               
                   
                 transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb = 
               
               
                   
                 AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522 
               
               
                 40241_at 
                 Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA, 
               
               
                   
                 complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug = 
               
               
                   
                 Hs.154095 /len = 3908 
               
               
                 40975_s_at 
                 Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin- 
               
               
                   
                 interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb = 
               
               
                   
                 AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565 
               
               
                 32149_at 
                 Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone = 
               
               
                   
                 IMAGE-996282 /gb = AA532495 /gi = 2276749 /ug = Hs.183752 /len = 549 
               
               
                 39190_s_at 
                 Cluster Incl. AC002126:  Homo sapiens  DNA from chromosome 19- 
               
               
                   
                 cosmids R30102-R29350-R27740 containing MEF2B, genomic 
               
               
                   
                 sequence /cds = (0, 307) /gb = AC002126 /gi = 2329908 /ug = 
               
               
                   
                 Hs.125220 /len = 308 
               
               
                 32746_at 
                 Cluster Incl. AF015451:  Homo sapiens  Usurpin-beta mRNA, complete 
               
               
                   
                 cds /cds = (0, 1388) /gb = AF015451 /gi = 3133282 /ug = 
               
               
                   
                 Hs.195175 /len = 1389 
               
               
                 34059_at 
                 Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-1086587 /clone_end = 3 /gb = 
               
               
                   
                 AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522 
               
               
                 39914_r_at 
                 Cluster Incl. W28976: 54e5  Homo sapiens  cDNA /gb = 
               
               
                   
                 W28976 /gi = 1308924 /ug = Hs.133151 /len = 903 
               
               
                 32054_at 
                 Cluster Incl. AF048732:  Homo sapiens  cyclin T2b mRNA, complete 
               
               
                   
                 cds /cds = (0, 2192) /gb = AF048732 /gi = 2981199 /ug = 
               
               
                   
                 Hs.155478 /len = 2193 
               
               
                 1832_at 
                 M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human 
               
               
                   
                 colorectal mutant cancer protein mRNA, complete cds 
               
               
                 1321_s_at 
                 U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor- 
               
               
                   
                 associated membrane protein homolog (TMP) mRNA, complete cds 
               
               
                 35489_at 
                 Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic 
               
               
                   
                 acid hydrolase alpha subunit (PPH alpha) mRNA, complete 
               
               
                   
                 cds /cds = (9, 2249) /gb = M82962 /gi = 535474 /ug = 
               
               
                   
                 Hs.179704 /len = 2902 
               
               
                 39912_at 
                 Cluster Incl. AB006179:  Homo sapiens  mRNA for heparan-sulfate 6- 
               
               
                   
                 sulfotransferase, complete cds /cds = (111, 1343) /gb = 
               
               
                   
                 AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051 
               
               
                 31368_at 
                 Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb = 
               
               
                   
                 W27967 /gi = 1307915 /ug = Hs.136154 /len = 755 
               
               
                 35640_at 
                 Cluster Incl. D14822: Human chimeric mRNA derived from AML1 
               
               
                   
                 gene and MTG8(ETO) gene, partial sequence /cds = (0, 
               
               
                   
                 597) /gb = D14822 /gi = 467498 /ug = Hs.31551 /len = 799 
               
               
                 38198_at 
                 Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert cDNA 
               
               
                   
                 clone EUROIMAGE 566443 /cds = UNKNOWN /gb = AL079275 /gi = 
               
               
                   
                 5102578 /ug = Hs.157078 /len = 2082 
               
               
                 38871_at 
                 Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10 
               
               
                   
                 protein /cds = (690, 1391) /gb = AJ006288 /gi = 
               
               
                   
                 4049459 /ug = Hs.193516 /len = 1877 
               
               
                 37054_at 
                 Cluster Incl. J04739: Human bactericidal permeability increasing 
               
               
                   
                 protein (BPI) mRNA, complete cds /cds = (30, 1493) /gb = 
               
               
                   
                 J04739 /gi = 179528 /ug = Hs.89535 /len = 1813 
               
               
                 34909_at 
                 Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185I07 from 
               
               
                   
                 7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi = 
               
               
                   
                 3924668 /ug = Hs.128653/len = 1767 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 52 
               
             
            
               
                   
               
               
                   
               
               
                 24-gene signature of invasive prostate cancer 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set ID 
               
               
                 (U95Av2) 
                 Description 
               
               
                   
               
               
                 40635_at 
                 Cluster Incl. AF089750:  Homo sapiens  flotillin-1 mRNA, complete 
               
               
                   
                 cds /cds = (164, 1447) /gb = AF089750 /gi = 3599572 /ug = 
               
               
                   
                 Hs.179986 /len = 1796 
               
               
                 38260_at 
                 Cluster Incl. AL050306: Human DNA sequence from clone 475B7 on 
               
               
                   
                 chromosome Xq12.1-13. Contains the 3 part of the gene for a novel 
               
               
                   
                 KIAA0615 and KIAA0323 LIKE protein, the gene for a novel protein, 
               
               
                   
                 ESTs, STSs, GSSs and two putative CpG islands /cds = (48, 
               
               
                   
                 2201) /gb = AL050306 /gi = 5419784 /ug = Hs.90625 /len = 2395 
               
               
                 41869_at 
                 Cluster Incl. U78310:  Homo sapiens  pescadillo mRNA, complete 
               
               
                   
                 cds /cds = (58, 1824) /gb = U78310 /gi = 2194202 /ug = 
               
               
                   
                 Hs.13501 /len = 2235 
               
               
                 1878_g_at 
                 M13194 /FEATURE = mRNA /DEFINITION = HUMERCC1 Human 
               
               
                   
                 excision repair protein (ERCC1) mRNA, complete cds, clone pcDE 
               
               
                 41116_at 
                 Cluster Incl. AI799802: wc43d09.x1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-2321393 /clone_end = 3 /gb = 
               
               
                   
                 AI799802 /gi = 5365274 /ug = Hs.101516 /len = 688 
               
               
                 37390_at 
                 Cluster Incl. D86977: Human mRNA for KIAA0224 gene, complete 
               
               
                   
                 cds /cds = (136, 3819) /gb = D86977 /gi = 1504027 /ug = 
               
               
                   
                 Hs.78054 /len = 4226 
               
               
                 38841_at 
                 Cluster Incl. AF068195:  Homo sapiens  putative glialblastoma cell 
               
               
                   
                 differentiation-related protein (GBDR1) mRNA, complete cds /cds = 
               
               
                   
                 (58, 1062) /gb = AF068195 /gi = 3192872 /ug = Hs.9194 /len = 1493 
               
               
                 35787_at 
                 Cluster Incl. AI986201: wr81a01.x1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-2494056 /clone_end = 3 /gb = 
               
               
                   
                 AI986201 /gi = 5813478 /ug = Hs.66881 /len = 814 
               
               
                 1903_at 
                 Ras-Related Protein Rap1b 
               
               
                 39661_s_at 
                 Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside 
               
               
                   
                 transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb = 
               
               
                   
                 AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522 
               
               
                 40241_at 
                 Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA, 
               
               
                   
                 complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug = 
               
               
                   
                 Hs.154095 /len = 3908 
               
               
                 40975_s_at 
                 Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin- 
               
               
                   
                 interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb = 
               
               
                   
                 AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565 
               
               
                 32149_at 
                 Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone = 
               
               
                   
                 IMAGE-996282 /gb = AA532495 /gi = 2276749 /ug = Hs.183752 /len = 549 
               
               
                 34059_at 
                 Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-1086587 /clone_end = 3 /gb = 
               
               
                   
                 AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522 
               
               
                 1832_at 
                 M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human 
               
               
                   
                 colorectal mutant cancer protein mRNA, complete cds 
               
               
                 1321_s_at 
                 U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor- 
               
               
                   
                 associated membrane protein homolog (TMP) mRNA, complete cds 
               
               
                 35489_at 
                 Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic acid 
               
               
                   
                 hydrolase alpha subunit (PPH alpha) mRNA, complete cds /cds = (9, 
               
               
                   
                 2249) /gb = M82962 /gi = 535474 /ug = Hs.179704 /len = 2902 
               
               
                 39912_at 
                 Cluster Incl. AB006179:  Homo sapiens  mRNA for heparan-sulfate 6- 
               
               
                   
                 sulfotransferase, complete cds /cds = (111, 1343) /gb = 
               
               
                   
                 AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051 
               
               
                 31368_at 
                 Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb = 
               
               
                   
                 W27967 /gi = 1307915 /ug = Hs.136154 /len = 755 
               
               
                 35640_at 
                 Cluster Incl. D14822: Human chimeric mRNA derived from AML1 gene 
               
               
                   
                 and MTGS(ETO) gene, partial sequence /cds = (0, 597) /gb = 
               
               
                   
                 D14822 /gi = 467498 /ug = Hs.31551 /len = 799 
               
               
                 38198_at 
                 Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert cDNA 
               
               
                   
                 clone EUROIMAGE 566443 /cds = UNKNOWN /gb = AL079275 /gi = 
               
               
                   
                 5102578 /ug = Hs.157078 /len = 2082 
               
               
                 38871_at 
                 Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10 
               
               
                   
                 protein /cds = (690, 1391) /gb = AJ006288 /gi = 
               
               
                   
                 4049459 /ug = Hs.193516 /len = 1877 
               
               
                 37054_at 
                 Cluster Incl. J04739: Human bactericidal permeability increasing protein 
               
               
                   
                 (BPI) mRNA, complete cds /cds = (30, 1493) /gb = J04739 /gi = 
               
               
                   
                 179528 /ug = Hs.89535 /len = 1813 
               
               
                 34909_at 
                 Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185107 from 
               
               
                   
                 7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi = 
               
               
                   
                 3924668 /ug = Hs.128653 /len = 1767 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 53 
               
             
            
               
                   
               
               
                   
               
               
                 22-gene-signature of invasive prostate cancer 
               
            
           
           
               
               
            
               
                 Affymetrix 
                   
               
               
                 Probe Set ID 
               
               
                 (U95Av2) 
                 Description 
               
               
                   
               
               
                 40635_at 
                 Cluster Incl. AF089750:  Homo sapiens  flotillin-1 mRNA, complete 
               
               
                   
                 cds /cds = (164, 1447) /gb = AF089750 /gi = 3599572 /ug = 
               
               
                   
                 Hs.179986 /len = 1796 
               
               
                 38260_at 
                 Cluster Incl. AL050306: Human DNA sequence from clone 475B7 on 
               
               
                   
                 chromosome Xq12.1-13. Contains the 3 part of the gene for a novel 
               
               
                   
                 KIAA0615 and KIAA0323 LIKE protein, the gene for a novel protein, 
               
               
                   
                 ESTs, STSs, GSSs and two putative CpG islands /cds = (48, 2201) /gb = 
               
               
                   
                 AL050306 /gi = 5419784 /ug = Hs.90625 /len = 2395 
               
               
                 33833_at 
                 Cluster Incl. J05243: Human nonerythroid alpha-spectrin (SPTAN1) 
               
               
                   
                 mRNA, complete cds /cds = (102, 7520) /gb = J05243 /gi = 
               
               
                   
                 179105 /ug = Hs.237180 /len = 7787 
               
               
                 38794_at 
                 Cluster Incl. X53390: Human mRNA for upstream binding factor 
               
               
                   
                 (hUBF) /cds = (147, 2441) /gb = X53390 /gi = 509240 /ug = 
               
               
                   
                 Hs.89781 /len = 3097 
               
               
                 33915_at 
                 Cluster Incl. W22655: 71B9  Homo sapiens  cDNA /clone = (not- 
               
               
                   
                 directional) /gb = W22655 /gi = 1299488 /ug = Hs.26070 /len = 761 
               
               
                 39379_at 
                 Cluster Incl. AL049397:  Homo sapiens  mRNA; cDNA DKFZp586C1019 
               
               
                   
                 (from clone DKFZp586C1019) /cds = UNKNOWN /gb = AL049397 /gi = 
               
               
                   
                 4500188 /ug = Hs.12314 /len = 1720 
               
               
                 1903_at 
                 Ras-Related Protein Rap1b 
               
               
                 39661_s_at 
                 Cluster Incl. AF034102:  Homo sapiens  NBMPR-insensitive nucleoside 
               
               
                   
                 transporter ei (ENT2) mRNA, complete cds /cds = (237, 1607) /gb = 
               
               
                   
                 AF034102 /gi = 2811136 /ug = Hs.32951 /len = 2522 
               
               
                 40241_at 
                 Cluster Incl. U09850: Human zinc finger protein (ZNF143) mRNA, 
               
               
                   
                 complete cds /cds = (37, 1917) /gb = U09850 /gi = 495571 /ug = 
               
               
                   
                 Hs.154095 /len = 3908 
               
               
                 40975_s_at 
                 Cluster Incl. AL050258: Novel human mRNA similar to mouse tuftelin- 
               
               
                   
                 interacting protein 10 mRNA, AF097181 /cds = (263, 2776) /gb = 
               
               
                   
                 AL050258 /gi = 4886426 /ug = Hs.20225 /len = 3565 
               
               
                 32149_at 
                 Cluster Incl. AA532495: nj54a10.s1  Homo sapiens  cDNA /clone = IMAGE- 
               
               
                   
                 996282 /gb = AA532495 /gi = 2276749 /ug = Hs.183752 /len = 549 
               
               
                 34059_at 
                 Cluster Incl. AA586695: nn42h06.s1  Homo sapiens  cDNA, 3 
               
               
                   
                 end /clone = IMAGE-1086587 /clone_end = 3 /gb = 
               
               
                   
                 AA586695 /gi = 2397509 /ug = Hs.193956 /len = 522 
               
               
                 1832_at 
                 M62397 /FEATURE = /DEFINITION = HUMCRCMUT Human 
               
               
                   
                 colorectal mutant cancer protein mRNA, complete cds 
               
               
                 1321_s_at 
                 U43916 /FEATURE = /DEFINITION = HSU43916 Human tumor- 
               
               
                   
                 associated membrane protein homolog (TMP) mRNA, complete cds 
               
               
                 35489_at 
                 Cluster Incl. M82962: Human N-benzoyl-L-tyrosyl-p-amino-benzoic acid 
               
               
                   
                 hydrolase alpha subunit (PPH alpha) mRNA, complete cds /cds = (9, 
               
               
                   
                 2249) /gb = M82962 /gi = 535474 /ug = Hs.179704 /len = 2902 
               
               
                 39912_at 
                 Cluster Incl. AB006179:  Homo sapiens  mRNA for heparan-sulfate 6- 
               
               
                   
                 sulfotransferase, complete cds /cds = (111, 1343) /gb = 
               
               
                   
                 AB006179 /gi = 3073774 /ug = Hs.132884 /len = 2051 
               
               
                 31368_at 
                 Cluster Incl. W27967: 40b10  Homo sapiens  cDNA /gb = 
               
               
                   
                 W27967 /gi = 1307915 /ug = Hs.136154 /len = 755 
               
               
                 35640_at 
                 Cluster Incl. D14822: Human chimeric mRNA derived from AML1 gene 
               
               
                   
                 and MTG8(ETO) gene, partial sequence /cds = (0, 597) /gb = 
               
               
                   
                 D14822 /gi = 467498 /ug = Hs.31551 /len = 799 
               
               
                 38198_at 
                 Cluster Incl. AL079275:  Homo sapiens  mRNA full length insert cDNA 
               
               
                   
                 clone EUROIMAGE 566443 /cds = UNKNOWN /gb = AL079275 /gi = 
               
               
                   
                 5102578 /ug = Hs.157078 /len = 2082 
               
               
                 38871_at 
                 Cluster Incl. AJ006288:  Homo sapiens  mRNA for bcl-10 
               
               
                   
                 protein /cds = (690, 1391) /gb = AJ006288 /gi = 
               
               
                   
                 4049459 /ug = Hs.193516 /len = 1877 
               
               
                 37054_at 
                 Cluster Incl. J04739: Human bactericidal permeability increasing protein 
               
               
                   
                 (BPI) mRNA, complete cds /cds = (30, 1493) /gb = J04739 /gi = 
               
               
                   
                 179528 /ug = Hs.89535 /len = 1813 
               
               
                 34909_at 
                 Cluster Incl. AC004990:  Homo sapiens  PAC clone DJ1185I07 from 
               
               
                   
                 7q11.23-q21 /cds = (0, 1766) /gb = AC004990 /gi = 
               
               
                   
                 3924668 /ug = Hs.128653 /len = 1767 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 8  
     Selection of the Gene Clusters Discriminating Between Metastatic and Non-Metastatic Human Breast Cancer.  
      In this example we utilized gene expression data and associated clinical information published in the recent study on gene expression profiling of breast cancer (van&#39;t Veer, L. J., et al., “Gene expression profiling predicts clinical outcome of breast cancer,” Nature, 415: 530-536, 2002, incorporated herein by reference). This study identifies 70 genes whose expression pattern is strongly predictive of a short post-diagnosis and treatment interval to distant metastases (van&#39;t Veer, L. J., et al., 2002). The expression pattern of these 70 genes discriminate with 81% (optimized sensitivity threshold) or 83% (optimal accuracy threshold) accuracy the patient&#39;s prognosis in the group of 78 young women diagnosed with sporadic lymph-node-negative breast cancer (this group comprises of 34 patients who developed distant metastases within 5 years and 44 patients who continued to be disease-free after a period of at least 5 years; they constitute a poor prognosis and good prognosis group, correspondingly). The authors described in this paper the second independent groups of breast cancer patients comprising 11 patients who developed distant metastases within 5 years and 8 patients who continued to be disease-free after a period of at least 5 years. We applied the method of the present invention to further reduce the number of genes whose expression patterns represent genetic signatures of breast cancer with “poor prognosis” or “good prognosis.” In our example we utilized the data derived from a group of 19 patients as a training set of samples, and the data derived from a group of 78 patients as a test set of samples.  
      Using the methods of present invention, we calculated the phenotype association indices for 19 samples of the training set and determined that this gene cluster exhibited a 84% success rate in clinical sample classification based on individual phenotype association indices (Table 54). As shown in Table 54, 7/8 (or 88%) of the good prognosis breast cancer samples had negative phenotype association indices, whereas 9/11 (or 82%) of poor prognosis breast cancer samples displayed negative phenotype association indices. Overall, 16 of 19 samples (or 84%) were correctly classified.  
               TABLE 54                          Classification accuracy of the breast cancer       prognosis predictor gene clusters                                 Cluster   r value   Good prognosis   Poor prognosis   Overall                                                     70   genes       7/8   (88%)   9/11   (82%)   16/19 (84%)       19   genes   0.984   7/8   (88%)   9/11   (82%)   16/19 (84%)       19   genes   0.984   29/44   (66%)   28/34   (82%)   57/78 (73%)       9   genes   0.984   7/8   (88%)   10/11   (91%)   17/19 (89%)       9   genes   0.984   32/44   (73%)   28/34   (82%)   60/78 (77%)       22   genes   0.975   7/8   (88%)   10/11   (91%)   17/19 (89%)       22   genes   0.975   29/44   (66%)   29/34   (85%)   58/78 (74%)       12   genes   0.989   7/8   (88%)   10/11   (91%)   17/19 (89%)       12   genes   0.989   31/44   (70%)   28/34   (82%)   59/78 (76%)                  
 
      Next, we identified two best-fit poor prognosis breast cancer samples displaying the correlation coefficient of 0.751 and 0.832 to the average expression profile of the 11 poor prognosis breast cancer samples. The average expression profile of the 11 poor prognosis breast cancer samples was utilized as a first reference set. The average expression profile of these two best-fit poor prognosis breast cancer samples was utilized as a second reference set.  
      The concordance set was obtained by selecting only those genes having a consistent direction of the differential expression in both the first and the second reference sets (i.e., greater gene expression difference in the poor prognosis cf. the good prognosis samples and greater gene expression in the best-fit tumor sample cf. the average expression value across the entire data set or vice-versa). The concordance set comprised of 44 genes (r=0.950). A minimum segregation set was selected following the procedures described above. Scatter plots were generated of the log 10  transformed average −fold expression change in the first reference set and average −fold expression change in the second reference set (in case of a single best-fit tumor it was the log 10  transformed ratio of the expression value for a gene to the average expression value across the entire data set). For the samples of the first reference set, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who had invasive tumors and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who had non-invasive tumors. A minimum segregation set was identified by selecting a subset of the highly correlated genes between two reference sets from the concordance set. Using this approach we identified two gene clusters (19-gene cluster and 9-gene cluster) discriminating with high accuracy between poor prognosis and good prognosis human breast tumors in both training and test sets of clinical samples. These two breast cancer metastasis predictors or poor prognosis minimum segregation sets are listed in Tables 55 &amp; 56. The classification performance for each of these gene clusters is presented in the Table 54.  
               TABLE 55                          19-gene signature of breast cancer prognosis predictor (r = 0.984)                             Gene ID (Chip identified in van&#39;t               Veer, L. J., et al., 2002)   Sequence Name                       Contig55725_RC   EST           NM_005915   MCM6           Contig46218_RC   EST           NM_001809   CENPA           NM_016359   LOC51203           NM_002073   GNAZ           NM_014321   ORC6L           NM_016448   L2DTL           NM_002916   RFC4           NM_003875   GMPS           NM_014791   KIAA0175           Contig28552_RC   EST           NM_003981   PRC1           AL137718   DIAPH3           NM_000849   GSTM3           NM_003862   FGF18           NM_004994   MMP9           NM_003239   TGFB3           NM_020974   CEGP1                      
 
     
       
         
           
               
             
               
                 TABLE 56 
               
             
            
               
                   
               
               
                   
               
               
                 9-gene signature of breast cancer 
               
               
                 prognosis predictor (r = 0.984) 
               
            
           
           
               
               
               
            
               
                   
                 Gene ID (Chip identified in van&#39;t 
                   
               
               
                   
                 Veer, L.J., et al.,2002) 
                 Sequence Name 
               
               
                   
                   
               
               
                   
                 Contig55725_RC 
                 EST 
               
               
                   
                 NM_005915 
                 MCM6 
               
               
                   
                 Contig46218_RC 
                 EST 
               
               
                   
                 NM_003875 
                 GMPS 
               
               
                   
                 NM_000849 
                 GSTM3 
               
               
                   
                 NM_003862 
                 FGF18 
               
               
                   
                 NM_004994 
                 MMP9 
               
               
                   
                 NM_003239 
                 TGFB3 
               
               
                   
                 NM_020974 
                 CEGP1 
               
               
                   
                   
               
            
           
         
       
     
      In the next example, the average expression profile of all 19 breast cancer samples obtained from 11 patients with poor prognosis and 8 patients with good prognosis was utilized as a first reference set. Next, we calculated the individual phenotype association indices and identified a single best-fit poor prognosis breast cancer sample displaying the correlation coefficient of 0.677 to the average expression profile of the 19 breast cancer samples. The average expression profile of this single best-fit poor prognosis breast cancer sample was utilized as a second reference set.  
      The concordance set was obtained by selecting only those genes having a consistent direction of the differential expression in both the first and the second reference sets (i.e., greater gene expression difference in the poor prognosis cf. the good prognosis samples and greater gene expression in the best-fit tumor sample cf. the average expression value across the entire data set or vice-versa). The concordance set comprised of 47 genes (r=0.822). A minimum segregation set was selected following the procedures described in the introduction to the Detailed Description of the Preferred Embodiments and the Materials &amp; Methods sections. Scatter plots were generated of the log 10  transformed average −fold expression change in the first reference set and average −fold expression change in the second reference set (in case of a single best-fit tumor it was the log 10  transformed ratio of the expression value for a gene to the average expression value across the entire data set). For the samples of the first reference set, &lt;expression&gt; 1  corresponds to the average expression value for gene x over all samples from patients who had invasive tumors and &lt;expression&gt; 2  corresponds to the average expression value for gene x over all samples from patients who had non-invasive tumors. A minimum segregation set was identified by selecting a subset of the highly correlated genes between two reference sets from the concordance set. Using this approach we identified two gene clusters (22-gene cluster and 12-gene cluster) discriminating with high accuracy between poor prognosis and good prognosis human breast tumors in both training and test sets of clinical samples. These two breast cancer metastasis predictors or poor prognosis minimum segregation sets are listed in Tables 57 &amp; 58. The classification performance for each of these gene clusters is presented in the Table 54.  
               TABLE 57                          22-gene signature of breast cancer prognosis predictor (r = 0.975)                             Gene ID (Chip identified in van&#39;t               Veer, L. J., et al., 2002)   Sequence Name                       NM_005915   MCM6           Contig46218_RC   EST           AA555029_RC   EST           NM_016359   LOC51203           Contig56457_RC   TMEFF1           NM_007036   ESM1           NM_007203   AKAP2           AF073519   SERF1A           NM_015984   UCH37           NM_014321   ORC6L           U82987   BBC3           Contig2399_RC   SM-20           NM_003882   WISP1           AB037863   KIAA1442           Contig63649_RC   EST           Contig20217_RC   EST           AF055033   IGFBP5           NM_003862   FGF18           NM_003239   TGFB3           NM_000849   GSTM3           NM_000599   IGFBP5           NM_020974   CEGP1                      
 
     
       
         
           
               
             
               
                 TABLE 58 
               
             
            
               
                   
               
               
                   
               
               
                 12-gene signature of breast cancer prognosis predictor (r = 0.989) 
               
            
           
           
               
               
               
            
               
                   
                 Gene ID (Chip identified in van&#39;t 
                   
               
               
                   
                 Veer, L. J., et al., 2002) 
                 Sequence Name 
               
               
                   
                   
               
               
                   
                 NM_005915 
                 MCM6 
               
               
                   
                 NM_007036 
                 ESM1 
               
               
                   
                 NM_007203 
                 AKAP2 
               
               
                   
                 AF073519 
                 SERF1A 
               
               
                   
                 NM_015984 
                 UCH37 
               
               
                   
                 NM_014321 
                 ORC6L 
               
               
                   
                 AF055033 
                 IGFBP5 
               
               
                   
                 NM_003862 
                 FGF18 
               
               
                   
                 NM_003239 
                 TGFB3 
               
               
                   
                 NM_000849 
                 GSTM3 
               
               
                   
                 NM_000599 
                 IGFBP5 
               
               
                   
                 NM_020974 
                 CEGP1 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 9  
     Selection of the Gene Clusters Predicting Good and Poor Prognosis of Human Lung Carcinoma  
      We applied the methods of the present invention to identify gene expression profiles distinguishing lung adenocarcinoma samples from normal lung specimens as well as highly malignant phenotype of lung adenocarcinoma, associated with short survival after diagnosis and therapy, from less aggressive lung cancers, associated with longer patient&#39;s survival. Clinical data set utilized in this example was published (Bhattacharjee, A., Richards, W. G., Staunton, J., Li, C., Monti, S., Vasa, P., Ladd, C., Beheshti, J., Bueno, R., Gillette, M., Loda, M., Weber, G., Mark, E. J., Lander, E. S., Wong, W., Johnson, B. E., Golub, T. R., Sugarbaker, D. J., Meyerson, M. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. PNAS, 98: 13790-13795, 2001; incorporated herein by reference).  
      Using the clinical data set and associated clinical history (Bhattacharje et al., 2001), we selected two groups of adenocarcinoma patients having markedly distinct survival after diagnosis and therapy: poor prognosis group 1 comprising 34 patients with the median survival of 8.5 month (range 0.1-17.3 month) and good prognosis group 2 comprising 16 patients with the median survival of 84 month (range 75.4-106.1 month). As a starting point, we utilized a set of the 675 transcripts selected based on a statistical analysis of the quality of the dataset and variability of gene expression across the dataset (Bhattacharje et al., 2001). Applying methods of the present invention, we identified a set of 38 genes displaying at least a 2-fold difference in the average values of the mRNA expression levels between 34 poor prognosis samples versus 16 good prognosis samples (Table 59).  
               TABLE 59                          38 genes differentially regulated in human lung adenocarcinomas       exhibiting poor and good clinical outcomes after the therapy.                     Affymetrix           Probe Set       ID (U95Av2)   Description               1665_s_at   Endothelial Cell Growth Factor 1       38428_at   matrix metalloproteinase 1 (interstitial collagenase)       40544_g_at   achaete-scute complex ( Drosophila ) homolog-like 1       34898_at   amphiregulin (schwannoma-derived growth factor)       1482_g_at   matrix metalloproteinase 12 (macrophage elastase)       35175_f_at   eukaryotic translation elongation factor 1 alpha 2       1481_at   matrix metalloproteinase 12 (macrophage elastase)       38389_at   2′,5′-oligoadenylate synthetase 1 (40-46 kD)       40543_at   achaete-scute complex ( Drosophila ) homolog-like 1       408_at   GRO1 oncogene (melanoma growth stimulating activity,           alpha)       40004_at   sine oculis homeobox ( Drosophila ) homolog 1       35938_at   phospholipase A2, group IVA (cytosolic, calcium-           dependent)       37874_at   flavin containing monooxygenase 5       33754_at   thyroid transcription factor 1       38790_at   epoxide hydrolase 1, microsomal (xenobiotic)       32275_at   secretory leukocyte protease inhibitor           (antileukoproteinase)       32081_at   citron (rho-interacting, serine/threonine kinase 21)       32154_at   transcription factor AP-2 alpha (activating           enhancer-binding protein 2 alpha)       206_at   cathepsin E       36623_at   Cluster Incl AB011406:  Homo sapiens  mRNA for alkalin           phosphatase, complete cds /cds = (176, 1750) /gb =           AB011406 /gi = 3401944 /ug = Hs.75431 /len = 2510       37576_at   Purkinje cell protein 4       37811_at   calcium channel, voltage-dependent, alpha 2/delta           subunit 2       39681_at   zinc finger protein 145 (Kruppel-like, expressed in           promyelocytic leukemia)       1270_at   RAP1, GTPase activating protein 1       32570_at   hydroxyprostaglandin dehydrogenase 15-(NAD)       37600_at   extracellular matrix protein 1       31844_at   homogentisate 1,2-dioxygenase (homogentisate           oxidase)       35834_at   alpha-2-glycoprotein 1, zinc       36681_at   apolipoprotein D       37430_at   arachidonate 15-lipoxygenase, second type       36680_at   amylase, alpha 2B; pancreatic       40031_at   aldehyde dehydrogenase 3       38773_at   carbonyl reductase 1       765_s_at   lectin, galactoside-binding, soluble, 4 (galectin 4)       37209_g_at   phosphoserine phosphatase-like       36736_f_at   phosphoserine phosphatase       41069_at   chondromodulin I precursor       37208_at   phosphoserine phosphatase-like                  
 
      Next, we calculated the phenotype association indices for all 50 samples and determined that this gene cluster exhibited a 72% success rate in clinical sample classification based on individual phenotype association indices (Table 60). As shown in Table 60, 12/16 (or 75%) of the lung adenocarcinoma samples of the good prognosis group had negative phenotype association indices, whereas 24/34 (or 71%) of lung adenocarcinoma specimens of the poor prognosis group displayed positive phenotype association indices. Overall, 36 of 50 samples (or 72%) were correctly classified.  
               TABLE 60                          Classification accuracy of lung adenocarcinoma       prognosis predictor clusters                                 Cluster   r value   Poor prognosis   Good Prognosis   Overall               38 genes   0.771   24/34 (71%)   12/16 (75%)   36/50 (72%)       26 genes   0.938   13/34 (38%)   15/16 (94%)   28/50 (56%)       15 genes   0.942   28/34 (82%)   11/16 (69%)   39/50 (78%)                  
 
      Next, we identified 8 best-fit poor prognosis samples displaying the correlation coefficient of 0.3 or higher to the average expression profile of the 34 poor prognosis samples. We calculated the average expression profile for these 8 best-fit poor prognosis samples by dividing the average expression value for each gene in the 8 samples of the best-fir set by the average expression value across the entire data set.  
      Next, we selected from an initial set of 38 genes a set of 26 genes (lung adenocarcinoma poor prognosis predictor cluster 1—see Table 61) displaying high positive correlation (r=0.938) between the best-fit tumors and poor prognosis samples data sets. This gene cluster exhibited a 56% success rate in clinical sample classification based on individual phenotype association indices (Table 60). As shown in Table 60, 15/16 (or 94%) of the lung adenocarcinoma samples of the good prognosis group had negative phenotype association indices, whereas 13/34 of lung adenocarcinoma specimens of the poor prognosis group displayed positive phenotype association indices. Overall, 28 of 50 samples (or 56%) were correctly classified.  
               TABLE 61                          26 genes of the lung adenocarcinoma       poor prognosis predictor cluster 1.                     Affymetrix           Probe Set       ID (U95Av2)   Description               1665_s_at   Endothelial Cell Growth Factor 1       38428_at   matrix metalloproteinase 1 (interstitial           collagenase)       40544_g_at   achaete-scute complex ( Drosophila ) homolog-like 1       1482_g_at   matrix metalloproteinase 12 (macrophage elastase)       1481_at   matrix metalloproteinase 12 (macrophage elastase)       38389_at   2′,5′-oligoadenylate synthetase 1 (40-46 kD)       40543_at   achaete-scute complex ( Drosophila ) homolog-like 1       408_at   GRO1 oncogene (melanoma growth stimulating activity,           alpha)       35938_at   phospholipase A2, group IVA (cytosolic, calcium-           dependent)       37874_at   flavin containing monooxygenase 5       33754_at   thyroid transcription factor 1       38790_at   epoxide hydrolase 1, microsomal (xenobiotic)       32275_at   secretory leukocyte protease inhibitor           (antileukoproteinase)       32081_at   citron (rho-interacting, serine/threonine kinase 21)       206_at   cathepsin E       36623_at   Cluster Incl AB011406:  Homo sapiens  mRNA for alkalin           phosphatase, complete cds /cds = (176, 1750) /gb =           AB011406 /gi = 3401944 /ug = Hs.75431 /len = 2510       37576_at   Purkinje cell protein 4       37811_at   calcium channel, voltage-dependent, alpha 2/delta           subunit 2       32570_at   hydroxyprostaglandin dehydrogenase 15-(NAD)       37600_at   extracellular matrix protein 1       31844_at   homogentisate 1,2-dioxygenase (homogentisate           oxidase)       36681_at   apolipoprotein D       36680_at   amylase, alpha 2B; pancreatic       38773_at   carbonyl reductase 1       37209_g_at   phosphoserine phosphatase-like       36736_f_at   phosphoserine phosphatase                  
 
      To improve the classification accuracy, we selected from an initial set of 38 genes a set of 15 genes (lung adenocarcinoma poor prognosis predictor cluster 2—see Table 62) displaying high positive correlation (r=0.942) between the best-fit tumors and poor prognosis samples data sets.  
               TABLE 62                          15 genes of the lung adenocarcinoma       poor prognosis predictor cluster 2.                     Affymetrix           Probe Set ID       (U95Av2)   Description               1665_s_at   Endothelial Cell Growth Factor 1       38428_at   matrix metalloproteinase 1 (interstitial collagenase)       40544_g_at   achaete-scute complex ( Drosophila ) homolog-like 1       1482_g_at   matrix metalloproteinase 12 (macrophage elastase)       1481_at   matrix metalloproteinase 12 (macrophage elastase)       38389_at   2′,5′-oligoadenylate synthetase 1 (40-46 kD)       40543_at   achaete-scute complex ( Drosophila ) homolog-like 1       408_at   GRO1 oncogene (melanoma growth stimulating activity,           alpha)       35938_at   phospholipase A2, group IVA (cytosolic, calcium-           dependent)       39681_at   zinc finger protein 145 (Kruppel-like, expressed in           promyelocytic leukemia)       35834_at   alpha-2-glycoprotein 1, zinc       40031_at   aldehyde dehydrogenase 3       765_s_at   lectin, galactoside-binding, soluble, 4 (galectin 4)       41069_at   chondromodulin I precursor       37208_at   phosphoserine phosphatase-like                  
 
      This gene cluster exhibited a 78% success rate in clinical sample classification based on individual phenotype association indices (Table 60). As shown in Table 60, 11/16 (or 69%) of the lung adenocarcinoma samples of the good prognosis group had negative phenotype association indices, whereas 28/34 (or 82%) of lung adenocarcinoma specimens of the poor prognosis group displayed positive phenotype association indices. Overall, 39 of 50 samples (or 78%) were correctly classified.  
     EXAMPLE 10  
     Selection of the Gene Clusters Associated with Metastatic Cancer  
      The methods of the present invention were used along with the data reported by Ramaswamy et al. (2003) to identify gene clusters distinguishing between the human primary adenocarcinomas of diverse origin and metastatic adenocarcinoma lesions. These data were the supplemental data reported in Ramaswamy, S., Ross, K. N., Lander, E. S., Golub, T. R. “A molecular signature of metastasis in primary solid tumors,” Nature Genetics, January 2003, 33: 49-54, incorporated herein by reference. Ramaswamy et al. (2003) identified the 17-gene cluster expression profile of which distinguishes 12 metastatic adenocarcinoma nodules of diverse origin and 64 human primary adenocarcinomas of diverse origin (lung, breast, prostate, colorectal, uterus, ovary). Both metastatic lesions and primary adenocarcinomas were representing the same diverse spectrum of tumor types obtained from different individuals (Ramaswamy et al., 2003).  
      The expression profile of the 17-gene cluster in metastatic versus primary tumors was utilized as a first reference set.  
      Next, we calculated the phenotype association indices for all 76 samples and determined that this gene cluster exhibited a 45% success rate in clinical sample classification based on individual phenotype association indices (Table 63). As shown in Table 63, 12/12 (or 100%) of the metastatic samples had positive phenotype association indices, whereas 22/64 (or 34%) of primary tumor samples displayed negative phenotype association indices. Overall, 34 of 76 samples (or 45%) were correctly classified.  
               TABLE 63                          Classification accuracy of the metastases segregation gene       clusters (r = 0.000 discrimination threshold)                                     r   Primary tumors   Primary                                                             Cluster   value   Breast   Colon   Lung   Prostate   Uterus   Ovary   tumors   Metastases   Overall               17   0.964   2 of 11   4 of 11   3 of 11   8 of 10   5 of 10   0 of 11   22/64   12/12   34/76       genes                               (34%)   (100%)   (45%)       12   0.991   3 of 11   5 of 11   0 of 11   8 of 10   6 of 10   0 of 11   22/64   12/12   34/76       genes                               (34%)   (100%)   (45%)       11   0.992   8 of 11   6 of 11   6 of 11   4 of 10   6 of 10   2 of 11   32/64   12/12   44/76       genes                               (50%)   (100%)   (58%)        8   0.989   3 of 11   7 of 11   1 of 11   8 of 10   6 of 10   1 of 11   26/64   12/12   38/76       genes                               (41%)   (100%)   (50%)        7   0.993   7 of 11   6 of 11   7 of 11   6 of 10   7 of 10   2 of 11   35/64   12/12   47/76       genes                               (55%)   (100%)   (62%)                  
 
      The classification accuracy of the 17-gene cluster was much improved when the discrimination threshold was set at the level of 0.400 of a correlation coefficient. As shown in Table 64, 12/12 (or 100%) of the metastatic samples had phenotype association indices higher than 0.400, whereas 48/64 (or 75%) of primary tumor samples displayed phenotype association indices lower than 0.400. Overall, 60 of 76 samples (or 79%) were correctly classified.  
               TABLE 64                          Classification accuracy of the metastases segregation gene       clusters (r = 0.400 discrimination threshold)                                     r   Primary tumors   Primary                                                             Cluster   value   Breast   Colon   Lung   Prostate   Uterus   Ovary   tumors   Metastases   Overall               17   0.964    9 of 11   7 of 11   8 of 11   8 of 10   8 of 10   8 of 11   48/64   12/12   60/76       genes                               (75%)   (100%)   (79%)       12   0.991   10 of 11   7 of 11   7 of 11   8 of 10   8 of 10   3 of 11   43/64   12/12   55/76       genes                               (67%)   (100%)   (72%)       11   0.992   11 of 11   7 of 11   8 of 11   8 of 10   8 of 10   8 of 11   50/64   12/12   62/76       genes                               (78%)   (100%)   (82%)        8   0.989    8 of 11   7 of 11   7 of 11   8 of 10   7 of 10   5 of 11   42/64   12/12   54/76       genes                               (66%)   (100%)   (71%)        7   0.993   11 of 11   7 of 11   8 of 11   8 of 10   7 of 10   7 of 11   49/64   12/12   61/76       genes                               (77%)   (100%)   (80%)                  
 
      Next, we identified three best-fit metastatic samples displaying the correlation coefficient of 0.870, 0.923, and 0.874 to the average expression profile of the 12 metastatic samples. The average expression profile of these three best-fit metastatic samples was utilized as a second reference set.  
      The expression profile of the best-fit samples was utilized to refine the gene-expression signature associated with a metastatic phenotype to a small set of transcripts that would exhibit high discrimination accuracy between metastatic lesions and primary tumors. Thus, selecting a subset of the highly correlated genes between two reference sets identified a minimum segregation set suitable for clinical samples classification. Using this approach we identified four gene clusters discriminating with high accuracy between metastatic lesions and primary tumors. The members of these metastases minimum segregation sets (metastases minimum segregation gene clusters) are listed in Tables 65-68. The classification performance for each of these gene clusters is presented in the Tables 63 and 64.  
               TABLE 65                       12-gene signature of metastases       Affymetrix Probe ID (U95Av2)                                            J03464_s_at           L37747_s_at           RC_AA430032_at           X85372_at           RC_AA608850_at           HG110-HT110_s_at           Z74615_at           U23946_at           D43968_at           U48959_at           D17408_s_at           D00654_at                      
 
     
       
         
           
               
             
               
                 TABLE 66 
               
               
                   
               
               
                   
               
               
                 11-gene signature of metastases 
               
               
                 Affymetrix Probe ID (U95Av2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 J03464_s_at 
               
               
                   
                 L37747_s_at 
               
               
                   
                 RC_AA430032_at 
               
               
                   
                 X85372_at 
               
               
                   
                 RC_AA608850_at 
               
               
                   
                 HG110-HT110_s_at 
               
               
                   
                 Z74615_at 
               
               
                   
                 U23946_at 
               
               
                   
                 D43968_at 
               
               
                   
                 M83664_at 
               
               
                   
                 AF001548_rna1_at 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 67 
               
               
                   
               
               
                   
               
               
                 8-gene signature of metastases 
               
               
                 Affymetrix Probe ID (U95Av2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 J03464_s_at 
               
               
                   
                 L37747_s_at 
               
               
                   
                 RC_AA430032_at 
               
               
                   
                 U23946_at 
               
               
                   
                 D43968_at 
               
               
                   
                 U48959_at 
               
               
                   
                 D17408_s_at 
               
               
                   
                 D00654_at 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 68 
               
               
                   
               
               
                   
               
               
                 7-gene signature of metastases 
               
               
                 Gene ID (Chip identified in van&#39;t Veer 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 J03464_s_at 
               
               
                   
                 L37747_s_at 
               
               
                   
                 RC_AA430032_at 
               
               
                   
                 U23946_at 
               
               
                   
                 D43968_at 
               
               
                   
                 M83664_at 
               
               
                   
                 AF001548_rna1_at 
               
               
                   
                   
               
            
           
         
       
     
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     EXAMPLE 11  
     Use of Expression Data with Other Metrics to Predict Prostate Cancer Patient Survival  
      Introduction  
      Critical clinical need in development of reliable prognostic markers suitable for stratification of prostate cancer patients is clearly demonstrated by the results of a recent randomized study of the therapeutic efficacy of surgery versus watch and wait strategy demonstrating only modest 6.6% absolute reduction in mortality after prostatectomy compared to observation, despite the association of surgery with a 50% reduction in hazard ration of death from prostate cancer (1). It appears that a measurable clinical benefit of surgery is limited to poorly defined sub-population of prostate cancer patients. Therefore, an improved ability to identify a sub-group of prostate cancer patients who would benefit from therapy should have a significant immediate positive clinical and socioeconomic impact.  
      Widely used biochemical, histopathological, and clinical criteria such as PSA level, Gleason score, the clinical tumor stage and molecular genetic approaches assaying loss of tumor suppressors or gain of oncogenes (2) had only limited success with respect to prostate cancer patients stratification and demonstrated a significant variability in predictive value among different clinical laboratories and hospitals. Furthermore, best existing markers cannot reliably identify at the time of diagnosis a poor prognosis group of prostate cancer patients that ultimately would fail therapy (3). Classification nomograms that incorporate measurements of several individual pre- and postoperative parameters are generally recognized as most efficient clinically useful models currently available for prediction of the probability of relapse-free survival after therapy of individual prostate cancer patients (4-7). However, one of the significant deficiencies of these classification systems is that they have only limited utility in predicting the differences in outcomes readily observed between patients diagnosed with prostate cancers exhibiting similar clinical, histopathological, and biochemical features. Therefore, a critical clinical need exists to improve the classification accuracy of prostate cancer patients with respect to clinical outcome after therapy.  
      Expression profiling of prostate tumor samples using oligonucleotide or cDNA microarray technology revealed gene expression signatures associated with human prostate cancer (8-19), including potential prostate cancer prognosis markers (9, 14, 16, 17). However, one of the major limitations of these studies was that the same clinical data set was utilized for both signature discovery and validation. Furthermore, usually only a single or few hits were validated using independent methods and independent clinical data sets, thus diminishing the potential advantage of the use of a panel of markers over a single marker in diagnostic and/or prognostic applications.  
      Here we applied a microarray-based gene expression profiling approach to identify molecular signatures distinguishing sub-groups of patients with differing outcome and develop a stratification algorithm demonstrating high discrimination accuracy between sub-groups of prostate cancer patients with distinct clinical outcome after therapy in a training set of 21 prostate cancer patients. To validate a potential clinical utility of discovered genetic signatures, we confirmed the discrimination power of proposed prostate cancer prognosis stratification algorithm using an independent set of 79 clinical tumor samples.  
      Our data indicate that identified molecular signatures provide the bases for developing clinical prognostic tests suitable for stratification of prostate cancer patients at the time of diagnosis with respect to likelihood of negative or positive clinical outcome after therapy. Our results provide experimental evidence of a transcriptional resemblance between metastatic human prostate carcinoma xenografts in nude mice and primary prostate tumors from patients subsequently developing relapse after therapy. These data suggest that genetically defined metastasis-promoting features of primary tumors are one of the major contributing factors of aggressive clinical behavior and unfavorable prognosis in prostate cancer patients.  
      Materials and Methods  
      Clinical Samples. We utilized in our experiments two independent sets of clinical samples for signature discovery (training outcome set of 21 samples) and validation (validation outcome set of 79 samples). Original gene expression profiles of the training set of 21 clinical samples analyzed in this study were recently reported (14). Primary gene expression data files of clinical samples as well as associated clinical information were provided by Dr. W. Sellers and can be found at http://www-genome.wi.mit.edu/cancer/.  
      Prostate tumor tissues comprising validation data set were obtained from 79 prostate cancer patients undergoing therapeutic or diagnostic procedures performed as part routine clinical management at MSKCC. Clinical and pathological features of 79 prostate cancer cases comprising validation outcome set are presented in the Table 70. Median follow-up after therapy in this cohort of patients was 70 months. Samples were snap-frozen in liquid nitrogen and stored at −80° C. Each sample was examined histologically using H&amp;E-stained cryostat sections. Care was taken to remove normeoplastic tissues from tumor samples. Cells of interest were manually dissected from the frozen block, trimming away other tissues. All of the studies were conducted under MSKCC Institutional Review Board-approved protocols.  
      Cell Culture. Cell lines used in this study were previously described (19). The LNCap- and PC-3-derived cell lines were developed by consecutive serial orthotopic implantation, either from metastases to the lymph node (for the LN series), or reimplanted from the prostate (Pro series). This procedure generated cell variants with differing tumorigenicity, frequency and latency of regional lymph node metastasis (19). Except where noted, cell lines were grown in RPMI1640 supplemented with 10% FBS and gentamycin (Gibco BRL) to 70-80% confluence and subjected to serum starvation as described (19), or maintained in fresh complete media, supplemented with 10% FBS.  
      Orthotopic Xenografts. Orthotopic xenografts of human prostate PC-3 cells and sublines used in this study were developed by surgical orthotopic implantation as previously described (19). Briefly, 2×10 6  cultured PC3 cells, PC3M or PC3MLN4 sublines were injected subcutaneously into male athymic mice, and allowed to develop into firm palpable and visible tumors over the course of 2-4 weeks. Intact tissue was harvested from a single subcutaneous tumor and surgically implanted in the ventral lateral lobes of the prostate gland in a series of six athymic mice per cell line subtype. The mice were examined periodically for suprapubic masses, which appeared for all subline cell types, in the order PC3MLN4&gt;PC3M&gt;&gt;PC3. Tumor-bearing mice were sacrificed by CO 2  inhalation over dry ice and necropsy was carried out in a 2-4° C. cold room. Typically, bilaterally symmetric prostate gland tumors in the shape of greatly distended prostate glands were apparent. Prostate tumor tissue was excised and snap frozen in liquid nitrogen. The elapsed time from sacrifice to snap freezing was &lt;5 min. A systematic gross and microscopic post mortem examination was carried out.  
      Tissue Processing for mRNA and RNA Isolation. Fresh frozen orthotopic tumor was examined by use of hematoxylin and eosin stained frozen sections. Orthotopic tumors of all sublines exhibited similar morphology consisting of sheets of monotonous closely packed tumor cells with little evidence of differentiation interrupted by only occasional zones of largely stromal components, vascular lakes, or lymphocytic infiltrates. Fragments of tumor judged free of these non-epithelial clusters were used for mRNA preparation. Frozen tissue (1-3 mm×1-3 mm) was submerged in liquid nitrogen in a ceramic mortar and ground to powder. The frozen tissue powder was dissolved and immediately processed for mRNA isolation using a Fast Tract kit for mRNA extraction (Invitrogen, Carlsbad, Calif., see above) according to the manufacturers instructions.  
      RNA and mRNA Extraction. For gene expression analysis, cells were harvested in lysis buffer 2 hrs after the last media change at 70-80% confluence and total RNA or mRNA was extracted using the RNeasy (Qiagen, Chatsworth, Calif.) or FastTract kits (Invitrogen, Carlsbad, Calif.). Cell lines were not split more than 5 times prior to RNA extraction, except where noted.  
      Affymetrix Arrays. The protocol for mRNA quality control and gene expression analysis was that recommended by Affymetrix (http://www.aff metrix.com). In brief, approximately one microgram of mRNA was reverse transcribed with an oligo(dT) primer that has a T7 RNA polymerase promoter at the 5′ end. Second strand synthesis was followed by cRNA production incorporating a biotinylated base. Hybridization to Affymetrix U95Av2 arrays representing 12,625 transcripts overnight for 16 h was followed by washing and labeling using a fluorescently labeled antibody. The arrays were read and data processed using Affymetrix equipment and software as reported previously (18, 19).  
      Data Analysis. Detailed protocols for data analysis and documentation of the sensitivity, reproducibility and other aspects of the quantitative statistical microarray analysis using Affymetrix technology have been reported (18, 19). 40-50% of the surveyed genes were called present by the Affymetrix Microarray Suite 5.0 software in these experiments. The concordance analysis of differential gene expression across the data sets was performed using Affymetrix MicroDB v. 3.0 and DMT v.3.0 software as described earlier (18, 19). We processed the microarray data using the Affymetrix Microarray Suite v.5.0 software and performed statistical analysis of expression data set using the Affymetrix MicroDB and Affymetrix DMT software. This analysis identified a set of 218 genes (91 up-regulated and 127 down-regulated transcripts) differentially regulated in tumors from patients with recurrent versus non-recurrent prostate cancer at the statistically significant level (p&lt;0.05) defined by both T-test and Mann-Whitney test (Table 69). The concordance analysis of differential gene expression across the clinical and experimental data sets was performed using Affymetrix MicroDB v. 3.0 and DMT v.3.0 software as described earlier (19). The Pearson correlation coefficient for individual test samples and appropriate reference standard was determined using the Microsoft Excel software as described in the signature discovery protocol.  
      Survival Analysis. The Kaplan-Meier survival analysis was carried out using the Prism 4.0 software. Statistical significance of the difference between the survival curves for different groups of patients was assessed using Chi square and Logrank tests.  
      Discovery and validation of the prostate cancer recurrence predictor algorithm. According to the present invention, clinically relevant genetic signatures can be found by searching for clusters of co-regulated genes that display highly concordant transcript abundance behavior across multiple experimental models and clinical settings that model or represent malignant phenotypes of interest (Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003; Example 5, supra; Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B. Malignancy-associated regions of transcriptional activation: gene expression profiling identifies common chromosomal regions of a recurrent transcriptional activation in human prostate, breast, ovarian, and colon cancers. Neoplasia, 5: 21-228; Glinsky, G. V., Ivanova, Y. A., Glinskii, A. B. Common malignancy-associated regions of transcriptional activation (MARTA) in human prostate, breast, ovarian, and colon cancers are targets for DNA amplification. Cancer Letters, in press, 2003). Thus, a primary criterion in selecting genes for inclusion within the cluster is the concordance of changes in expression rather than a magnitude of changes (e.g., fold change). Accordingly, transcripts of interest are expected to have a tightly controlled “rank order” of expression within a cluster of co-regulated genes reflecting a balance of up- and down-regulation as a desired regulatory end-point in a cell. A degree of resemblance of the transcript abundance rank order within a gene cluster between a test sample and reference standard is measured by a Pearson correlation coefficient and designated as a phenotype association index (PAI), as described fully in the introduction of the Detailed Description of Preferred Embodiments section. To identify genes with consistently concordant expression patterns across multiple data sets and various experimental conditions, we compared the expression profile of 218 genes (test samples) to the expression profiles of transcripts differentially regulated in multiple experimental models (reference standard) of human prostate cancer (Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003).  
      The transcripts comprising each signature were selected based on Pearson correlation coefficients (r&gt;0.95) reflecting a degree of similarity of expression profiles in clinical tumor samples (recurrent versus non-recurrent tumors) and experimental samples using the following protocol.  
      Step 1. Sets of differentially regulated transcripts were independently identified for each experimental conditions (see below) and clinical samples using the Affymetrix microarray processing and statistical analysis software package as described in this examples&#39;s Materials and Methods section.  
      Step 2. Sub-sets of transcripts exhibiting concordant expression changes in clinical and experimental samples were identified using the Affymetrix MicroDB and DMT software. Sub-sets of transcripts were identified with concordant changes of transcript abundance behavior in recurrent versus non-recurrent clinical tumor samples (218 transcripts) and experimental conditions independently defined for each signature (Signature 1: PC-3MLN4 orthotopic versus s.c. xenografts; Signature 2: PC-3MLN4 versus PC-3M &amp; PC-3 orthotopic xenografts; Signature 3: PC-3/LNCap consensus class, Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003). Thus, from a set of 218 transcripts three concordant sub-sets of transcripts were identified corresponding to each binary comparison of clinical and experimental samples.  
      Step 3. Small gene clusters were selected as sub-sets of genes exhibiting concordant changes of transcript abundance behavior in recurrent versus non-recurrent clinical tumor samples (218 transcripts) and experimental conditions defined for each signature (Signature 1: PC-3MLN4 orthotopic versus s.c. xenografts; Signature 2: PC-3MLN4 versus PC-3M &amp; PC-3 orthotopic xenografts; Signature 3: PC-3/LNCap consensus class, Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003). Expression profiles were presented as log 10  average fold changes for each transcript and processed for visualization and Pearson correlation analysis using Microsoft Excel software. The cut-off criterion for cluster formation was set to exceed a Pearson correlation coefficient 0.95 among the log10 transformed average expression values in the compared groups.  
      Step 4. Small gene clusters exhibiting highly concordant pattern of expression (Pearson correlation coefficient, r&gt;0.95) in clinical and experimental samples (identified in step 3) were evaluated for their ability to discriminate clinical samples with distinct outcomes after the therapy. To assess a potential prognostic relevance of individual gene clusters, we calculated a Pearson correlation coefficient for each of 21 tumor samples (training data set) by comparing the expression profiles of individual samples to the reference expression profiles of relevant experimental samples defined for each signature and an “average” expression profile of recurrent versus non-recurrent tumors. As explained above, we named the corresponding correlation coefficients calculated for individual samples the phenotype association indices (PAIs). We evaluated the prognostic power of identified clusters of co-regulated transcripts based on their ability to segregate the patients with recurrent and non-recurrent prostate tumors into distinct sub-groups and selected a single best performing cluster for each binary condition ( FIG. 57 ; Tables 69 &amp; 70).  
      Step 5. We used Kaplan-Meier survival analysis to assess the prognostic power of each best-performing cluster in predicting the probability that patients would remain disease-free after therapy ( FIG. 58-62 ). We selected the prognosis discrimination cut-off value for each signature based on highest level of statistical significance in patient&#39;s stratification into poor and good prognosis groups as determined by the log-rank test (lowest P value and highest hazard ratio; Table 70 &amp;  FIGS. 58-62 ). Clinical samples having the Pearson correlation coefficient at or higher than the cut-off value were identified as having the poor prognosis signature. Clinical samples with the Pearson correlation coefficient lower the cut-off value were identified as having the good prognosis signature.  
      Step 6. We developed a prostate cancer recurrence predictor algorithm taking into account calls from all three individual signatures. We selected the common prognosis discrimination cut-off value for all three signatures based on highest level of statistical significance in patient&#39;s stratification into poor and good prognosis groups as determined by Kaplan-Meier survival analysis (lowest P value and highest hazard ratio defined by the log-rank test; Table 70 &amp;  FIG. 58-62 ). Clinical samples having the Pearson correlation coefficient at or higher the cut-off value defined by at least two signatures were identified as having the poor prognosis signature. Clinical samples with the Pearson correlation coefficient lower than the cut-off value defined by at least two signatures were identified as having the good prognosis signature. We found that the cut-off value of PAIs&gt;0.2 scored in two of three individual clusters allowed to achieve the 90% recurrence prediction accuracy (Table 70).  
      Step 7. We validated the prognostic power of prostate cancer recurrence predictor algorithm alone and in combination with the established markers of outcome using an independent clinical set of 79 prostate cancer patients ( FIGS. 58-6269  &amp; 71).  
      Results  
      Identification of molecular signatures distinguishing sub-groups of prostate cancer patients with distinct clinical outcomes after therapy. To identify the outcome predictor signatures, we utilized as a training data set the expression analysis of 12,625 transcripts in 21 prostate tumor samples obtained from prostate cancer patients with distinct clinical outcomes after therapy. Using biochemical evidence of relapse after therapy as a criterion of treatment failure, 21 patients were divided into two sub-groups, representing prostate cancer with recurrent (8 patients) and non-recurrent (13 patients) clinical behavior (14). We processed the original U95Av2 GeneChip CEL files using the Affymetrix Microarray Suite 5.0 software and performed statistical analysis of expression data set using the Affymetrix MicroDB and Affymetrix DMT software. This analysis identified a set of 218 genes (91 up-regulated and 127 down-regulated transcripts) differentially regulated in tumors from patients with recurrent versus non-recurrent prostate cancer at the statistically significant level (p&lt;0.05) defined by both T-test and Mann-Whitney test (Table 70).  
      To reduce the number of hits in potential outcome predictor clusters and identify transcripts of potential biological relevance, we compared the expression profile of 218 genes to the expression profiles of transcripts differentially regulated in multiple experimental models of human prostate cancer (Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003, and Example 5, supra) in search for genes with consistently concordant expression patterns across multiple data sets and various experimental conditions. We identified several small gene clusters exhibiting highly concordant pattern of expression (Pearson correlation coefficient, r&gt;0.95) in clinical and experimental samples. We evaluated the prognostic power of each identified cluster of co-regulated transcripts based on ability to segregate the patients with recurrent and non-recurrent prostate tumors into distinct sub-groups. To assess a potential prognostic relevance of individual gene clusters, we calculated a Pearson correlation coefficient for each of 21 tumor samples by comparing the expression profiles of individual samples to the “average” expression profile of recurrent versus non-recurrent tumors and expression profiles of relevant experimental samples (Table 69 and  FIG. 57 ). Based on expected correlation of expression profiles of identified gene clusters with recurrent clinical behavior of prostate cancer, we named the corresponding correlation coefficients calculated for individual samples the phenotype association indices (PAIs).  
      Using this strategy we identified several gene clusters (Tables 69 &amp; 70) discriminating with 86-95% accuracy human prostate tumors exhibiting recurrent or non-recurrent clinical behavior ( FIG. 57  and Tables 69 &amp; 70). The transcripts comprising each signature in Table 69 were selected based on Pearson correlation coefficients (r&gt;0.95) reflecting a degree of similarity of expression profiles in clinical tumor samples (recurrent versus non-recurrent tumors) and experimental samples. Selection of transcripts was performed from sets of genes exhibiting concordant changes of transcript abundance behavior in recurrent versus non-recurrent clinical tumor samples (218 transcripts) and experimental conditions independently defined for each signature (Signature 1: PC-3MLN4 orthotopic versus s.c. xenografts; Signature 2: PC-3MLN4 versus PC-3M &amp; PC-3 orthotopic xenografts; Signature 3: PC-3/LNCap consensus class, Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003, and Example 5, supra). The expression profiles were presented as log10 average fold changes for each transcript.  
               TABLE 69                       Gene expression signatures associated       with recurrent prostate cancer.                  Signature 1                             LocusLink       GenBank   UniGene       Name   Gene Name   ID   ID               MGC5466   Hypothetical protein MGC5466   U90904   Hs.83724       Wnt5A   proto-oncogene Wnt5A   L20861   Hs.152213       KIAA0476   KIAA0476 protein   AB007945   Hs.6684       ITPR1   inositol 1,4,5-trisphosphate   D26070   Hs.198443           receptor, type 1       TCF2   transcription factor 2, hepatic   X58840   Hs.169853                         Signature 2                                     GenBank   UniGene       Gene   Gene Name   ID   ID               MGC5466   Hypothetical protein MGC5466   U90904   Hs.83724       CHAF1A   Chromatin assembly factor 1,   U20979   Hs.79018           subunit A       CDS2   CDP-diacylglycerol synthase 2   Y16521   Hs.24812       IER3   Immediate early response 3   S81914   Hs.76090                         Signature 3                             LocusLink       GenBank   UniGene       Name   Gene Name   ID   ID               PPFIA3   Protein tyrosine phosphatase,   AB014554   Hs.109299           receptor type, f polypeptide       COPEB   Core promoter element binding   AF001461   Hs.285313           protein       FOS   V-fos oncogene homolog   V01512   Hs.25647       JUNB   Jun B proto-oncogene   X51345   Hs.400124       ZFP36   zinc finger protein 36, C3H type   M92843   Hs.343586                  
 
      Table 70 illustrates data from 21 prostate cancer patients who provided tumor samples comprising a signature discovery (training) data set that were classified according to whether they had a good-prognosis signature or poor-prognosis signature based on PAI values defined by either individual recurrence predictor signatures or a recurrence predictor algorithm that takes into account calls from all three signatures. The number of correct predictions in the poor-prognosis and good-prognosis groups is shown as a fraction of patients with the observed clinical outcome after therapy (8 patients developed relapse and 13 patients remained disease-free). Correlation coefficients reflect a degree of similarity of expression profiles in clinical tumor samples (recurrent versus non-recurrent tumors) and experimental samples (Signature 1: PC-3MLN4 orthotopic versus s.c. xenografts; Signature 2: PC-3MLN4 versus PC-3M &amp; PC-3 orthotopic xenografts; Signature 3: PC-3/LNCap consensus class, Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003; and Example 5, supra). P values were calculated with use of the log-rank test and reflect the statistically significant difference in the probability that patients would remain disease-free between poor-prognosis and good-prognosis sub-groups.  
               TABLE 70                          Prostate cancer recurrence prediction accuracy in a good-prognosis       and a poor-prognosis sub-group of patients defined according to whether       they had a good-prognosis or a poor-prognosis signature.                                                 Non-               Recurrence   Correlation   Recurrent   recurrent       P       signature   coefficient   cancer   cancer   Overall   value               Signature 1   r = 0.983   100%    92%   95%   &lt;0.0001               (8 of 8)   (12 of 13)   (20 of 21)       Signature 2   r = 0.963   88%   92%   90%   &lt;0.0001               (7 of 8)   (12 of 13)   (19 of 21)       Signature 3   r = 0.996   75%   92%   86%    0.001               (6 of 8)   (12 of 13)   (18 of 21)       Algorithm   NA   88%   92%   90%   &lt;0.0001               (7 of 8)   (12 of 13)   (19 of 21)                  
 
       FIG. 57  illustrates application of the five-gene cluster (Table 69, signature 1) to characterize clinical prostate cancer samples according to their propensity for recurrence after therapy. The expression pattern of the genes in the recurrence predictor cluster was analyzed in each of twenty-one separate clinical samples. The analysis produces a quantitative phenotype association index (plotted on the Y-axis) for each of the twenty-one clinical prostate cancer samples. Tumors that are likely to recur are expected to have positive phenotype association indices reflecting positive correlation of gene expression with metastasis-promoting orthotopic xenografts, while those that are unlikely to recur are expected to have negative association indices.  
      The figure shows the phenotype association indices for eight samples from patients who later had recurrence as bars 1 through 8, while the association indices for thirteen samples from patients whose tumors did not recur is shown as bars 11 through 23. Eight of the eight samples (or 100%) from patients who later experienced recurrence had positive phenotype association indices and so were properly classified. Twelve of the thirteen samples (or 92.3%) from patients whose tumors did not recur had negative phenotype association indices and so were properly classified as non-recurrent tumors. Thus, overall, twenty of the twenty-one samples (or 95.2%) were properly classified using a five-gene recurrence predictor signature. Two alternative clusters identified using this strategy showed similar sample classification performance (Tables 69 &amp; 70).  
      To further evaluate the prognostic power of the identified gene expression signatures, we performed Kaplan-Meier survival analysis using as a clinical end-point disease-free interval (“DFI”) after therapy in prostate cancer patients with positive and negative PAIs. The Kaplan-Meier survival curves showed a highly significant difference in the probability that prostate cancer patients would remain disease-free after therapy between the groups with positive and negative PAIs defined by the signatures (FIGS.  58 A-C), suggesting that patients with positive PAIs exhibit a poor outcome signature whereas patients with negative PAIs manifest a good outcome signature. The estimated hazard ration for disease recurrence after therapy in the group of patients with positive PAIs as compared with the group of patients with negative PAIs defined by the recurrence predictor signature 3 (Table 69) was 9.046 ( FIG. 58C )(95% confidence interval of ratio, 3.022 to 76.41; P=0.001). 86% of patients with the positive PAIs had a disease recurrence within 5 years after therapy, whereas 85% of patients with the negative PAIs remained relapse-free at least 5 years ( FIG. 58C ). Based on this analysis, we identified the group of prostate cancer patients with positive PAIs as a poor prognosis group and the group of prostate cancer patients with negative PAIs as a good prognosis group.  
      Theoretically, the recurrence predictor algorithm based on a combination of signatures should be more robust than a single predictor signature, particularly during the validation analysis using an independent test cohort of patients. Next we analyzed whether a combination of the three signatures would perform in the patient&#39;s classification test with similar accuracy as the individual signatures. We found that the cut-off value of PAIs&gt;0.2 scored in two of three individual clusters allowed to achieve the 90% recurrence prediction accuracy (Table 70). This recurrence predictor algorithm correctly identified 88% of patients with recurrent and 92% of patients with non-recurrent disease (Table 70). The Kaplan-Meier survival analysis ( FIG. 58D ) showed that the median relapse-free survival after therapy of patients in the poor prognosis group was 26 months. All patients in the poor prognosis group had a disease recurrence within 5 years after therapy, whereas 92% of patients in the good prognosis group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis group of patients as compared with the good prognosis group of patients defined by the recurrence predictor algorithm was 20.32 (95% confidence interval of ratio, 6.047 to 158.1; P&lt;0.0001).  
      Validation of the outcome predictor signatures using independent clinical data set. To validate the potential clinical utility of identified molecular signatures, we evaluated the prognostic power of signatures applied to an independent set of 79 clinical samples obtained from 37 prostate cancer patients who developed recurrence after the therapy and 42 patients who remained disease-free. The Kaplan-Meier survival analysis demonstrated that all three recurrence predictor signatures (Table 69) segregate prostate cancer patients into sub-groups with statistically significant differences in the probability of remaining relapse-free after therapy (Table 71). Interestingly, application of the recurrence predictor algorithm (requiring a cut-off value of PAIs&gt;0.2 scored in two of three individual clusters) appears to perform better than individual signatures in patient&#39;s stratification test using an independent data set (Table 71).  
      Table 71 summarizes classification of 79 prostate cancer patients who provided tumor samples. These samples comprise a signature validation (test) data set and were classified according to whether they had a good-prognosis signature or poor-prognosis signature based on PAI values defined by either individual recurrence predictor signatures or recurrence predictor algorithm that takes into account calls from all three signatures. Kaplan-Meier analysis was performed to evaluate the probability that patients would remain disease free according to whether they had a poor-prognosis or a good-prognosis signature and determine the proportion of patients who would remain disease-free at least 5 years after therapy in a poor-prognosis and a good-prognosis sub-groups. Hazard ratios, 95% confidence intervals, and P values were calculated with use of the log-rank test.  
               TABLE 71                          Stratification of 79 prostate cancer patients into       poor and good prognosis groups at time of diagnosis       based on recurrence predictor signatures.                                         Poor   Good                       prognosis,   prognosis,   Haz-   95% Confidence       Recurrence   5-year   5-year   ard   interval of       signature   survival   survival   ratio   ratio   P value                                             Signature 1   41%   78%   2.858   1.405 to 5.143   0.0028       Signature 2   44%   79%   3.473   1.584 to 5.806   0.0008       Signature 3   41%   76%   3.351   1.810 to 6.907   0.0002       Algorithm   33%   76%   4.224   2.455 to 9.781   &lt;0.0001                  
 
      Kaplan-Meier survival analysis ( FIG. 59A ) showed that the median relapse-free survival after therapy of patients classified within the poor prognosis group (defined by the recurrence predictor algorithm) was 34.6 months. 67% of patients in the poor prognosis group had a disease recurrence within 5 years after therapy, whereas 76% of patients in the good prognosis group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis group as compared with the good prognosis group of patients defined by the recurrence predictor algorithm was 4.224 (95% confidence interval of ratio, 2.455 to 9.781; P&lt;0.0001). Overall, the application of the recurrence predictor algorithm allowed accurate stratification into poor prognosis group 82% of patients who failed the therapy within one year after prostatectomy. The recurrence predictor algorithm seems to demonstrate more accurate performance in patient&#39;s classification compared to the conventional markers of outcome such as preoperative PSA level or RP Gleason sum ( FIGS. 59-60  and Table 72).  
      Recurrence predictor signatures provide additional predictive value over conventional markers of outcome. Next we determined that application of the recurrence predictor signatures provides additional predictive value when combined with conventional markers of outcome such as preoperative PSA level and Gleason score. Both preoperative PSA level and RP Gleason sum were significant predictors of prostate cancer recurrence after therapy in the validation cohort of 79 patients ( FIGS. 59D and 60C ).  
      Kaplan-Meier survival analysis ( FIG. 59D ) showed that the median relapse-free survival after therapy of patients in the poor prognosis group defined by the high preoperative PSA level was 49.0 months. 60% of patients in the poor prognosis group had a disease recurrence within 5 years after therapy, whereas 73% of patients in the good prognosis group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis group as compared with the good prognosis group of patients defined by the preoperative PSA level was 2.551 (95% confidence interval of ratio, 1.344 to 4.895; P=0.0043). However, prediction of the outcome after therapy based on preoperative PSA level accurately stratified into the poor prognosis group only 65% of patients who failed the therapy within one year after prostatectomy (Table 72).  
      Table 72 shows the number of correct predictions in poor-prognosis and good-prognosis groups as a fraction of patients with the observed clinical outcome after therapy (37 patients developed relapse and 42 patients remained disease-free). PSA and Gleason sum cut-off values for segregation of poor-prognosis and good-prognosis sub-groups were defined to achieve the most accurate and statistically significant recurrence prediction in this cohort of patients. Multiparameter nomogram-based prognosis predictor was defined as described in this example&#39;s Materials &amp; Methods using 50% relapse-free survival probability as a cut-off for patient&#39;s stratification into poor and good prognosis subgroups.  
               TABLE 72                          Prostate cancer recurrence prediction accuracy in poor-prognosis and       good-prognosis sub-groups of patients defined by a gene expression-based       recurrence predictor algorithm alone or in combination with established       biochemical and histopathological markers of outcome.                                 Recurrence   Recurrent   Non-recurrent   Year one           predictor   cancer   cancer   recurrence   Overall               Recurrence   68% (25 of 37)   81% (34 of 42)   82% (14 of 17)   75% (59 of 79)       Algorithm       PSA   68% (25 of 37)   67% (28 of 42)   65% (11 of 17)   67% (53 of 79)       PSA &amp; Algorithm   84% (31 of 37)   71% (30 of 42)   88% (15 of 17)   77% (61 of 79)       RP Gleason sum   38% (14 of 37)   90% (38 of 42)   47% (8 of 17)   66% (52 of 79)       RP Gleason sum &amp;   68% (25 of 37)   81% (34 of 42)   82% (14 of 17)   75% (59 of 79)       Algorithm       PSA &amp; RP Gleason   81% (30 of 37)   67% (28 of 42)   82% (14 of 17)   73% (58 of 79)       Nomogram   62% (23 of 37)   79% (33 of 42)   71% (12 of 17)   71% (56 of 79)       Nomogram &amp;   68% (25 of 37)   81% (34 of 42)   82% (14 of 17)   75% (59 of 79)       Algorithm                  
 
      We next determined that application of the recurrence predictor algorithm identifies sub-groups of patients with distinct clinical outcome after therapy in both high and low PSA-expressing groups, thus adding additional predictive value to the therapy outcome classification based on preoperative PSA level alone.  
      In the group of patients with high preoperative PSA level ( FIG. 59B ), the median relapse-free survival after therapy of patients in the poor prognosis sub-group defined by the recurrence predictor algorithm was 36.2 months. 73% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy. Conversely, 73% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 4.315 (95% confidence interval of ratio, 1.338 to 7.025; P=0.0081).  
      In the group of patients with low preoperative PSA level ( FIG. 59C ), the median relapse-free survival after therapy of patients in the poor prognosis sub-group defined by the recurrence predictor algorithm was 42.0 months. 53% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy, whereas 92% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 6.247 (95% confidence interval of ratio, 2.134 to 24.48; P=0.0015). Overall, combining information from the recurrence predictor algorithm with preoperative PSA level measurement allowed 88% of patients who failed the therapy within one year after prostatectomy to be accurately classified within the poor prognosis group (Table 72).  
      Radical prostatectomy (“RP”) Gleason sum is a significant predictor of relapse-free survival in the validation cohort of 79 prostate cancer patients ( FIG. 60C ). Kaplan-Meier survival analysis ( FIG. 60C ) demonstrated that the median relapse-free survival after therapy of patients with the RP Gleason sum 8 &amp; 9 was 21.0 months, thus defining the poor prognosis group based on histopathological criteria. 74% of patients in the poor prognosis group had a disease recurrence within 5 years after therapy, whereas 69% of patients in the good prognosis group (RP Gleason sum 6 &amp; 7) remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis group as compared with the good prognosis group of patients defined by the RP Gleason sum criteria was 3.335 (95% confidence interval of ratio, 2.389 to 13.70; P&lt;0.0001). RP Gleason sum-based outcome classification accurately stratified into poor prognosis group only 47% of patients who failed the therapy within one year after prostatectomy (Table 72).  
      In the group of patients with RP Gleason sum 6 &amp; 7 ( FIG. 60A ), the median relapse-free survival after therapy of patients in the poor prognosis sub-group defined by the recurrence predictor algorithm was 61.0 months. 53% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy, whereas 77% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 3.024 (95% confidence interval of ratio, 1.457 to 8.671; P=0.0055).  
      In the group of patients with RP Gleason sum 8 &amp; 9 ( FIG. 60B ), the median relapse-free survival after therapy in the poor prognosis sub-group defined by the recurrence predictor algorithm was 11.5 months. 100% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy, whereas 67% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 6.143 (95% confidence interval of ratio, 1.573 to 13.49; P=0.0053). Overall, patient&#39;s classification using a combination of the recurrence predictor algorithm and RP Gleason sum allowed 82% of patients who failed the therapy within one year after prostatectomy to be accurately classified as members of the poor prognosis group (Table 72). Based on this analysis we concluded that application of the recurrence predictor algorithm provides an additional predictive value to the therapy outcome classification based on established markers of outcome.  
      Recurrence predictor signatures provide additional predictive value over outcome prediction based on multiparameter nomogram. Classification nomograms are generally recognized most efficient clinically useful models currently available for prediction of the probability of relapse-free survival after therapy of individual prostate cancer patients (Kattan M. W., Eastham J. A., Stapleton A. M., Wheeler T. M., Scardino P. T. A preoperative nomogram for disease recurrence following radical prostatectomy for prostate cancer. J. Natl. Cancer Inst., 90: 766-771, 1998; D&#39;Amico A. V., Whittington R., Malkowicz S. B., Fondurulia J., Chen M-H, Kaplan I., Beard C. J., Tomaszewski J. E., Renshaw A. A., Wein A., Coleman C. N. Pretreatment nomogram for prostate-specific antigen recurrence after radical prostatectomy or external-beam radiation therapy for clinically localised prostate cancer. J. Clin. Oncol., 17: 168-172, 1999; Graefen M., Noldus J., Pichlmeier A., Haese P., Hammerer S., Fernandez S., Conrad R., Henke E., Huland E., Huland H. Early prostate-specific antigen relapse after radical retropubic prostatectomy: prediction on the basis of preoperative and postoperative tumor characteristics. Eur. Urol., 36: 21-30, 1999; Kattan M. W., Wheeler T. M., Scardino P. T. Postoperative nomogram for disease recurrence after radical prostatectomy for prostate cancer. J. Clin. Oncol., 17: 1499-1507, 1999.). We applied the Kattan nomogram utilizing multiple postoperative parameters (Kattan, et al. (1999)) for prognosis prediction classification in the test group of 79 prostate cancer patients.  
      Kaplan-Meier survival analysis ( FIG. 61A ) showed that the median relapse-free survival after therapy of patients in the poor prognosis group defined by the Kattan nomogram was 33.1 months. 72% of patients in the poor prognosis group had a disease recurrence within 5 years after therapy, whereas 81% of patients in the good prognosis group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis group as compared with the good prognosis group of patients defined by the Kattan nomogram was 3.757 (95% confidence interval of ratio, 2.318 to 9.647; P&lt;0.0001). Prediction of the outcome after therapy based on Kattan nomogram accurately stratified into poor prognosis group 71% of patients who failed the therapy within one year after prostatectomy (Table 72).  
      Application of the recurrence predictor algorithm identified sub-groups of patients with distinct clinical outcome after therapy in both poor and good prognosis groups defined by the Kattan nomogram, thus adding additional predictive value to the therapy outcome classification based on nomogram alone.  
      In the poor prognosis group of patients defined by the Kattan nomogram the application of the recurrence predictor algorithm appears to identify two sub-groups of patients with statistically significant difference in the probability to remain relapse-free after therapy ( FIG. 61B ). Median relapse-free survival after therapy of patients in the poor prognosis sub-group defined by the recurrence predictor algorithm was 11.5 months compared to median relapse-free survival of 71.1 months in the good prognosis sub-group ( FIG. 61B ). 89% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy. Conversely, 50% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 3.129 (95% confidence interval of ratio, 1.378 to 7.434; P=0.0068).  
      Similarly, in the good prognosis group of patients identified based on application of the Kattan nomogram, the recurrence predictor algorithm seems to define two sub-groups of patients with statistically significant difference in the probability to remain relapse-free after therapy ( FIG. 61C ). Median relapse-free survival after therapy of patients in the poor prognosis sub-group defined by the recurrence predictor algorithm was 64.8 months. 41% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy. Conversely, 87% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 4.398 (95% confidence interval of ratio, 1.767 to 18.00; P=0.0035). Overall, combination of the recurrence predictor algorithm and Kattan nomogram allowed accurate stratification into poor prognosis group 82% of patients who failed the therapy within one year after prostatectomy (Table 72).  
      Recurrence predictor algorithm defines poor and good prognosis sub-groups of patients diagnosed with the early stage prostate cancer. Identification of sub-groups of patients with distinct clinical outcome after therapy would be particularly desirable in a cohort of patients diagnosed with the early stage prostate cancer. Next we determined that recurrence predictor signatures are useful in defining sub-groups of patients diagnosed with early stage prostate cancer and having a statistically significant difference in the likelihood of disease relapse after therapy.  
      In the group of patients diagnosed with the stage 1C prostate cancer ( FIG. 62A ), the median relapse-free survival after therapy in the poor prognosis sub-group defined by the recurrence predictor algorithm was 12 months. In contrast, the median relapse-free survival after therapy in the good prognosis group was 82.4 months. 77% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy. Conversely, 81% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ration for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 5.559 (95% confidence interval of ratio, 2.685 to 25.18; P=0.0002).  
      In the group of patients diagnosed with the stage 2A prostate cancer ( FIG. 62B ), the median relapse-free survival after therapy in the poor prognosis sub-group defined by the recurrence predictor algorithm was 35.4 months. 86% of patients in the poor prognosis sub-group had a disease recurrence within 5 years after therapy, whereas 78% of patients in the good prognosis sub-group remained relapse-free at least 5 years. The estimated hazard ratio for disease recurrence after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the recurrence predictor algorithm was 7.411 (95% confidence interval of ratio, 2.220 to 40.20; P=0.0024). Based on this analysis we concluded that application of the recurrence predictor algorithm seems to provide potentially useful clinical information in stratification of patients diagnosed with the early stage prostate cancer into sub-groups with statistically significant difference in the likelihood of disease recurrence after therapy.  
      2. Discussion  
      As a result of the broad application of measurements of PSA level in the blood for early detection of prostate cancer in the United States, an increasing proportion of prostate cancer patients are diagnosed with early-stage tumors that apparently confined to the prostate gland and many patients have seemingly indolent disease not affecting individual&#39;s survival (Potosky, A., Feuer, E., Levin, D. Impact of screening on incidence and mortality of prostate cancer in the United States. Epidemiol. Rev., 23: 181-186, 2001). The considerable clinical heterogeneity of the early stage prostate cancer represents a highly significant health care and socioeconomic challenge because prostate cancer is expected to be diagnosed in ˜200,000 individuals every year (Greenlee, R. T., Hill-Hamon, M. B., Murray, T., Thun, M. Cancer statistics, 2001. CA Cancer J. Clin., 51: 15-36, 2001). Consequently, it can be argued that, unlike other types of cancer, development of efficient prognostic tests rather than early detection is critical for improvement of clinical decision-making and management of prostate cancer.  
      We hypothesized that clinically relevant genetic signatures can be found by searching for clusters of co-regulated genes that display highly concordant transcript abundance behavior across multiple experimental models and clinical settings that model or represent malignant phenotypes of interest (Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003; Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B. Malignancy-associated regions of transcriptional activation: gene expression profiling identifies common chromosomal regions of a recurrent transcriptional activation in human prostate, breast, ovarian, and colon cancers. Neoplasia, 5: 21-228; Glinsky, G. V., Ivanova, Y. A., Glinskii, A. B. Common malignancy-associated regions of transcriptional activation (MARTA) in human prostate, breast, ovarian, and colon cancers are targets for DNA amplification. Cancer Letters, in press, 2003). Thus, according to this model the primary criterion in a transcript selection process should be the concordance of changes in expression rather the magnitude of changes (e.g., fold change). One of the predictions of this model is that transcripts of interest are expected to have a tightly controlled “rank order” of expression within a cluster of co-regulated genes reflecting a balance of up- and down-regulated mRNAs as a desired regulatory end-point in a cell. A degree of resemblance of the transcript abundance rank order within a gene cluster between a test sample and reference standard is measured by a Pearson correlation coefficient and designated a phenotype association index (“PAI”).  
      Using this strategy we discovered and validated a prostate cancer recurrence predictor algorithm that is suitable for stratifying patients at the time of diagnosis into poor and good prognosis sub-groups with statistically significant differences in the disease-free survival after therapy. The algorithm is based on application of gene expression signatures associated with biochemical recurrence of prostate cancer. The signatures (Table 69) were defined using clusters of co-regulated genes exhibiting highly concordant expression profiles (r&gt;0.95) in metastatic nude mouse models of human prostate carcinoma and tumor samples from patients with recurrent prostate cancer (see Example 5).  
      A few previous studies have applied oligonucleotide or cDNA microarrays for identification of gene expression signatures associated with biochemical recurrence of human prostate cancer (Dhanasekaran, S. M., Barrette, T. R., Ghosh, D., Shah, R., Varambally, S., Kurachi, K., Pienta, K. J., Rubin, M. A., Chinnalyan, A. M. Delineation of prognostic biomarkers in prostate cancer. Nature, 412:822-826, 2001; Singh, D., Febbo, P. G., Ross, K., Jackson, D. G., Manola, C. L., Tamayo, P., Renshaw, A. A., D&#39;Amico, A. V., Richie, J. P., Lander, E. S., Loda, M., Kantoff, P. W., Golub, T. R., Sellers, W. R. Gene expression correlates of clinical prostate cancer behavior. Cancer Cell, 1: 203-209, 2002; Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, C., Sanda, M. G., Ghosh, D., Pineta, K. J., Sewalt, R. G., Otte, A. P., Rubin, M. A., Chinnalyan, A. M. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature, 419: 624-629, 2002; Henshall, S. M., Afar, D. E., Hiller, J., Horvath, L. G., Quinn, D. I., Rasiah, K. K., Gish, K., Willhite, D., Kench, J. G., Gardiner-Garden, M., Stricker, P. D., Scher, H. I., Grygiel, J. J., Agus, D. B., Mack, D. H., Sutherland, R. L. Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer Res., 63: 4196-4203, 2003). One of the major deficiencies of these studies that somewhat limited their significance was that a single clinical data set was utilized for both signature discovery and validation. To our knowledge, the work reported here is the first genome-wide expression profiling study of human prostate cancer that utilizes one clinical data set for signature discovery and algorithm development, and a second independent data set for validation of the prostate cancer recurrence predictor algorithm.  
      One of the interesting features of described here prostate cancer recurrence predictor algorithm is that it provides additional predictive value over conventional markers of outcome such as pre-operative PSA level and Gleason sum. Another important feature of identified recurrence predictor algorithm is its ability to stratify patients diagnosed with the early stage prostate cancer into sub-groups with statistically-distinct likelihoods of biochemical relapse after therapy. Importantly, the recurrence predictor algorithm segregates into poor prognosis group 88% of patients who subsequently developed disease recurrence within one year after prostatectomy. Based on this analysis we concluded that identified in this study genetic signatures (as well as others that can be determined using the methods of the invention) have a significant potential for developing highly accurate clinical prognostic tests suitable for stratifying prostate cancer patients at the time of diagnosis with respect to likelihood of negative or positive clinical outcome after therapy.  
      The causal genetic, molecular, and biological distinctions between prostate tumors with recurrent and indolent clinical behavior remain largely unknown. The results reported in this example and in Example 5 provide the first experimental evidence of a clinically relevant transcriptional resemblance between metastatic human prostate carcinoma xenografts growing orthotopically in nude mice and primary prostate tumors from patients that subsequently developed a biochemical relapse after therapy. This work provides a model for investigation of the potential functional relevance of identified transcriptional aberrations and suggests that genetically defined metastasis-promoting features of primary tumors seem to be one of the major contributing factors of aggressive clinical behavior and unfavorable prognosis in prostate cancer patients. This conclusion is consistent with results of the several recent studies aimed at definition of metastasis predictor signatures in the primary human tumors representing multiple types of epithelial cancers (van &#39;t Veer, L. J., Dai, H., van de Vijver, M. J., et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 415: 530-536, 2002; van de Vijver, M. J., He, Y. D., van &#39;t Veer, L. J., et al. A gene expression signature as a predictor of survival in breast cancer. N. Engl. J. Med., 347: 1999-2009, 2002; Ramaswamy, S., Ross, K. N., Lander, E. S., Golub, T. R. A molecular signature of metastasis in primary solid tumors. Nature Genetics, 33: 49-54, 2003). Our results indicate that sub-groups of prostate cancer patients with poor and good prognosis gene expression signatures reflect the presence of two genetically defined sub-types of human prostate carcinoma manifesting dramatic statistically significant differences in response to therapy and clinically distinct courses of disease progression.  
      One of the dominant views on prostate cancer pathogenesis is the concept of progression from hormone-dependent early stage prostate cancer to hormone-refractory metastatic late stage disease with the apparent implication of increased proportion of patients with poor prognosis at the advanced stage of progression. However, in our validation data set of 79 samples the actual frequency of recurrence remains relatively constant among the patients with different stages of prostate cancer: 47% (16 of 34) in stage 1C; 56% (9 of 16) in stage 2A; and 41% (12 of 29) in stages 2B/2C/3A. These data suggest that progression of the disease occurs only in a sub-group of patients. Interestingly, in a sub-group of patients with good prognosis signatures the frequency of recurrence appears to increase in the patients with the late-stage prostate cancer: 24% (5 of 21) in stage 1C; 22% (2 of 9) in stage 2A; 33% (3 of 9) in stage 2B; 40% (2 of 5) in stage 2C/3A. These results seem to imply that patients with the good prognosis signatures may represent a sub-group undergoing a classical prostate cancer progression with a gradual increase in malignant potential. The patients with poor prognosis signatures may represent a genetically and biologically distinct sub-type of prostate cancer exhibiting highly malignant behavior at the early stage of disease with the frequency of recurrence 85% (11 of 13) in stage 1C and 100% (7 of 7) in stage 2A patients.  
      In summary, using expression profiles of highly metastatic models of human prostate cancer in nude mice as a predictive reference of expected transcript abundance behavior in recurrent prostate tumors, we identified and validated recurrence predictor signatures of human prostate cancer. Prostate cancer recurrence predictor signatures provide additional predictive value to the conventional markers of outcome and will be clinically useful in stratifying prostate cancer patients into sub-groups with distinct clinical manifestation of disease and different response to therapy.  
     REFERENCES  
     
         
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          10. Welsh, J. B., Sapinoso, L. M., Su, A. I., Kern, S. G., Wang-Rodriguez, J., Moskaluk, C. A., Frierson, H. F., Jr., Hampton, G. M. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res., 61: 5974-5978, 2001.  
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          15. Rhodes, D. R., Barrette, T. R., Rubin, M. A., Ghosh, D., Chinnaiyan, A. M. Meta-analysis of microarrays: interstudy validation of gene expression profiles reveals pathways dysregulation in prostate cancer. Cancer Res., 62: 4427-4433, 2002.  
          16. Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, C., Sanda, M. G., Ghosh, D., Pineta, K. J., Sewalt, R. G., Otte, A. P., Rubin, M. A., Chinnalyan, A. M. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature, 419: 624-629, 2002.  
          17. Henshall, S. M., Afar, D. E., Hiller, J., Horvath, L. G., Quinn, D. I., Rasiah, K. K., Gish, K., Willhite, D., Kench, J. G., Gardiner-Garden, M., Stricker, P. D., Scher, H. I., Grygiel, J. J., Agus, D. B., Mack, D. H., Sutherland, R. L. Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer Res., 63: 4196-4203, 2003.  
          18. LaTulippe, E., Satagopan, J., Smith, A., Scher, H., Scardino, P., Reuter, V., Gerald, W. L. Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastasis. Cancer Res., 62: 4499-4506, 2002.  
          19. Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B., Gebauer, G. Microarray analysis of xenograft-derived cancer cell lines representing multiple experimental models of human prostate cancer. Molecular Carcinogenesis, 37: 209-221, 2003.  
          20. Potosky, A., Feuer, E., Levin, D. Impact of screening on incidence and mortality of prostate cancer in the United States. Epidemiol. Rev., 23: 181-186, 2001.  
          21. Greenlee, R. T., Hill-Hamon, M. B., Murray, T., Thun, M. Cancer statistics, 2001. CA Cancer J. Clin., 51: 15-36, 2001.  
          22. Glinsky, G. V., Krones-Herzig, A., Glinskii, A. B. Malignancy-associated regions of transcriptional activation: gene expression profiling identifies common chromosomal regions of a recurrent transcriptional activation in human prostate, breast, ovarian, and colon cancers. Neoplasia, 5: 21-228.  
          23. Glinsky, G. V., Ivanova, Y. A., Glinskii, A. B. Common malignancy-associated regions of transcriptional activation (MARTA) in human prostate, breast, ovarian, and colon cancers are targets for DNA amplification. Cancer Letters, in press, 2003.  
          24. van &#39;t Veer, L. J., Dai, H., van de Vijver, M. J., et al. Gene expression profiling predicts clinical outcome of breast cancer.  Nature,  415: 530-536, 2002  
          25. van de Vijver, M. J., He, Y. D., van &#39;t Veer, L. J., et al. A gene expression signature as a predictor of survival in breast cancer. N. Engl. J. Med., 347: 1999-2009, 2002.  
          26. Ramaswamy, S., Ross, K. N., Lander, E. S., Golub, T. R. A molecular signature of metastasis in primary solid tumors. Nature Genetics, 33: 49-54, 2003.  
       
    
     EXAMPLE 12  
     Use of Expression Data with Other Metrics to Predict Breast Cancer Patient Survival  
      Introduction  
      Highly accurate prognostic tests are essential for individualized decision-making process during clinical management of cancer patients leading to rational and more efficient selection of appropriate therapeutic interventions and improved outcome after therapy. In breast cancer, patients are classified into broad subgroups with poor and good prognosis reflecting a different probability of disease recurrence and survival after therapy. Distinct prognostic subgroups are identified using a combination of clinical and pathological criteria: age, primary tumor size, status of axillary lymph nodes, histologic type and pathologic grade of tumor, and hormone receptor status (Goldhirsch, A., Glick, J. H., Gelber, R. D., Coates, A. S., Seen, H. J. Meeting highlights: International Consensus Panel on the Treatment of Primary Breast Cancer: Seventh International Conference on Adjuvant Therapy of Primary Breast Cancer. J. Clin. Oncol., 19: 3817-3827, 2001; Eifel, P., Axelson, J. A., Costa, J., et al. National Institute of Health Consensus Development Conference Summary: adjuvant therapy for breast cancer, Nov. 1-3, 2000. J. Natl. Cancer Inst., 93: 979-989, 2001.)  
      One of the most critical treatment decisions during the clinical management of breast cancer patients is the use of adjuvant systemic therapy. Adjuvant systemic therapy significantly improves disease-free and overall survival in breast cancer patients with both lymph-node negative and lymph-node positive disease (Early Breast Cancer Trialists&#39; Collaborative Group. Polychemotherapy for early breast cancer: an overview of the randomized trials. Lancet, 352: 930-942, 1998; Early Breast Cancer Trialists&#39; Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet, 351: 1451-1467, 1998). It is generally accepted that breast cancer patients with poor prognosis would gain the most benefits from the adjuvant systemic therapy (Goldhirsch, et al., 2001; Eifel et al., 2001).  
      Diagnosis of lymph-node status is important in therapeutic decision-making, prediction of disease outcome, and probability of breast cancer recurrence. Invasion into axillary lymph nodes is recognized as one of the most important prognostic factors (Krag, D., Weaver, D., Ashikaga, T., et al. The sentinel node in breast cancer—a multicenter validation study. N. Engl. J. Med., 339: 941-946, 1998; Singletary, S. E., Allred, C., Ashley, P., et al. Revision of the American Joint Committee on cancer staging system for breast cancer. J. Clin. Oncol., 20: 3628-3636, 2002; Jatoli, I., Hilsenbeck, S. G., Clark, G. M., Osborne, C. K. Significance of axillary lymph node metastasis in primary breast cancer. J. Clin. Oncol., 17: 2334-2340, 1999). Most patients diagnosed with lymph-node negative breast cancer can be effectively treated with surgery and local radiation therapy. However, results of several studies show that 22-33% of breast cancer patients with no detectable lymph-node involvement and classified into a good prognosis subgroup develop recurrence of disease after a 10-year follow-up (Early Breast Cancer Trialists&#39; Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet, 351: 1451-1467, 1998). Therefore, accurate identification of breast cancer patients with lymph-node negative tumors who are at high risk of recurrence is critically important for rational treatment decision and improved clinical outcome in the individual patient.  
      Microarray-based gene expression profiling of human cancers rapidly emerged as a new powerful screening technique generating hundreds of novel diagnostic, prognostic, and therapeutic targets (Golub, T. R., Slonim, D. K., Tamayo, P., et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science, 286: 531-537, 1999; Alizadeh, A. A., Eisen, M. B., Davis, R. E., et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature, 403: 503-511, 2000; Alizadeh, A. A., Ross, D. T., Perou, C. M., van de Rijn, M. Towards a novel classification of human malignancies based on gene expression patterns. J. Pathol., 195: 41-52, 2001; Battacharjee, A., Richards, W. G., Staunton, J., et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc. Natl. Acad. Sci. USA, 98: 13790-13795, 2001; Yeoh, E.-J., Ross, M. E., Shurtleff, S. A., et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell, 1: 133-143, 2002; Dyrskot, L., Thykjaer, T., Kruhoffer, M., Jensen, J. L., Marcussen, N., Hamilton-Dutoit, S., Wolf, H., Orntoft, T. Identifying distinct classes of bladder carcinoma using microarrays. Nature Genetics, 33: 90-96, 2003). Recently breast cancer gene expression signatures have been identified that are associated with the estrogen receptor and lymph node status of patients and can aid in classification of breast caner patients into subgroups with different clinical outcome after therapy (Perou, C. M., Sorlie, T., Eisen, M. B., et al. Molecular portrait of human breast tumors. Nature, 406: 747-752, 2000; Gruvberger, S., Ringner, M., Chen, Y., et al. Estrogen receptor status in breast cancer is associated with remarkably distinct gene expression patterns. Cancer Res., 61: 5979-5984, 2001; West, M., Blanchette, C., Dressman, H., et al. Predicting the clinical status of human breast cancer by using gene expression profiles. Proc. Natl. Acad. Sci. USA, 98: 11462-11467, 2001; Ahr, A., Karn, T., Sollbach, C., et al. Identification of high risk breast cancer patients by gene expression profiling. Lancet, 359: 131-132, 2002; van &#39;t Veer, L. J., dai, H., van de Vijver, M. J., et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 415: 530-536, 2002; Sorlie, T., Perou, C. M., Tibshirani, R., et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. USA, 98: 10869-10874, 2001; Heedenfalk, I., Duggan, D., Chen, Y., et al. Gene-expression profiles in hereditary breast cancer. N. Engl. J. Med., 344: 539-548, 2001; van de Vijver, M. J., He, Y. D., van &#39;t Veer, L. J., et al. A gene expression signature as a predictor of survival in breast cancer. N. Engl. J. Med., 347: 1999-2009, 2002; Huang, E., Cheng, S. H., Dressman, H., Pittman, J., Tsou, M. H., Horng, C. F., Bild, A., Iversen, E. S., Liao, M., Chen, C. M., West, M., Nevins, J. R., Huang, A. T. Gene expression predictors of breast cancer outcome. Lancet, 361: 1590-1596, 2003).  
      One of the significant limitations of these array-based studies is that they generated vast data sets comprising many attractive targets with diagnostic and prognostic potential. Design and performance of meaningful follow-up experiments such as translation of the array-generated hits into quantitative RT-PCR-based analytical assays would require a significant data reduction. Furthermore, clinical implementation of novel prognostic tests would require integration of genomic data and best-established conventional markers of the outcome.  
      Here, we translate a large microarray-based breast cancer outcome predictor signature into quantitative RT-PCR-based assays of mRNA abundance levels of small gene clusters performing with similar classification accuracy. We demonstrate that identified molecular signatures provide additional predictive values over well-established conventional prognostic markers for breast cancer such as hormone receptor status and lymph node involvement. These data indicate that quantitative laboratory tests measuring expression profiles of identified small gene clusters are useful for stratifying breast cancer patients into sub-groups with distinct likelihood of positive outcome after therapy and assisting in selection of optimal treatment strategies.  
      Materials and Methods  
      The same general methods as described in Example 11 were used to carry out the work reported in this example.  
      Results and Discussion  
      The 70-gene breast cancer metastasis and survival predictor signature represents a heterogeneous set of small gene clusters independently performing with high therapy outcome prediction accuracy. Recent study on gene expression profiling of breast cancer identifies 70 genes whose expression pattern is strongly predictive of a short post-diagnosis and treatment interval to distant metastases (van &#39;t Veer, et al., 2002). The expression pattern of these 70 genes discriminates with 81% (optimized sensitivity threshold) or 83% (optimal accuracy threshold) accuracy the patient&#39;s prognosis in the group of 78 young women diagnosed with sporadic lymph-node-negative breast cancer (this group comprises of 34 patients who developed distant metastases within 5 years and 44 patients who continued to be disease-free at least 5 years after therapy; they constitute clinically defined poor prognosis and good prognosis groups, correspondingly). We reduced the number of genes whose expression patterns represent genetic signatures of breast cancer with “poor prognosis” or “good prognosis.” Measurements of mRNA expression levels of 70 genes in established human breast carcinoma cell lines (MCF7; MDA-MB-435; MDA-MB-468; MDA-MB-231; MDA-MB-435Br1; MDA-MB-435BL3) and primary cultures of normal human breast epithelial cells were performed utilizing Q-RT-PCR method, which is generally accepted as the most reliable method of gene expression analysis and unambiguous confirmation of a gene identity. For each breast cancer cell line concordant sets of genes were identified exhibiting both positive and negative correlation between fold expression changes in cancer cell lines versus control cell line and poor prognosis group versus good prognosis group patient samples. Minimum segregation sets were selected from corresponding concordance sets and individual phenotype association indices were calculated. The four top-performing breast cancer metastasis predictor gene clusters are listed in Table 73.  
      A breast cancer poor prognosis predictor cluster comprising 6 genes was identified (r=0.981) using MDA-MB-468 cell line gene expression profile as a reference standard. 32 of 34 samples from the poor prognosis group had positive phenotype association indices, whereas 29 of 44 samples from the good prognosis group had negative phenotype association indices yielding 78% overall accuracy in sample classification. Another breast cancer poor prognosis predictor cluster comprising 4 genes was identified (r=0.944) using MDA-MB-435BL3 cell line gene expression profile as a reference standard. Using this 4-gene cluster, 28 of 34 samples from the poor prognosis group had positive phenotype association indices, whereas 28 of 44 samples from the good prognosis group had negative phenotype association indices overall yielding 72% accuracy in sample classification.  
      A breast cancer good prognosis predictor cluster comprising 14 genes was identified (r=−0.952) using MDA-MB-435Br1 cell line gene expression profile as a reference standard. 30 of 34 samples from the poor prognosis group had negative phenotype association indices, whereas 34 of 44 samples from the good prognosis group had positive phenotype association indices yielding 82% overall accuracy in sample classification. Another breast cancer good prognosis predictor cluster comprising 13 genes (r=−0.992) was identified using MCF7 cell line gene expression profile as a reference standard. 30 of 34 samples from the poor prognosis group had negative phenotype association indices, whereas 32 of 44 samples from the good prognosis group had positive phenotype association indices yielding 79% overall accuracy in sample classification.  
      The transcripts comprising each signature listed in Table 73 were selected based on Pearson correlation coefficients (r&gt;0.95) reflecting a degree of similarity of expression profiles in clinical tumor samples (34 recurrent versus 44 non-recurrent tumors) and experimental cell line samples. Selection of transcripts was performed from sets of genes exhibiting concordant changes of transcript abundance behavior in recurrent versus non-recurrent clinical tumor samples (70 transcripts) and experimental conditions independently defined for each signature (6-gene signature: MDA-MB468 cells versus control; 4-gene signature: MDA-MB-435BL3 cells versus control; 13-gene signature: MCF7 cells versus control; 14-gene signature: MDA-MB-435Br1 cells versus control)(see also Example 2). mRNA expression levels of 70 genes comprising parent microarray-defined signature (van&#39;t Veer, L. J., et al., 2002; van de Vijver, M. J., et al., 2002) were measured by standard quantitative RT-PCR method in multiple established human breast cancer cell lines using GAPDH expression for normalization and compared to the expression in a control cell line. Control cells were primary cultures of normal human breast epithelial cells. Expression profiles were presented as log10 average fold changes for each transcript.  
               TABLE 73                       Gene expression signatures predicting       survival of breast cancer patients.                                                        Gene ID (Chip                   identified in               van&#39;t Veer, L.       LocusLink       J., et al.,   UniGene       Name   Description   2002)   ID                         6-gene signature (same as Table 27)                             FLT1   Fms-related tyrosine   NM_002019   Hs.381093           kinase 1       BBC3   BCL2 binding component   U82987   Hs.87246           3       TGFB3   Transforming growth   NM_003239   Hs.2025           factor, beta 3       MS4A7   Membrane-spanning 4-   AF201951   Hs.11090           domains       GSTM3   Glutathione S-transferase   NM_000849   Hs.2006           M3       FGF18   Fibroblast growth factor   NM_003862   Hs.49585           18                 4-gene signature                             HEC   Highly expressed in   NM_006101   Hs.58169           cancer       MCM6   Minichromosome   NM_005915   Hs.155462           maintenance deficient 6       GSTM3   Glutathione S-transferase   NM_000849   Hs.2006           M3       FGF18   Fibroblast growth factor   NM_003862   Hs.49585           18                         13-gene signature (same as Table 29)                                     Gene ID (Chip                   identified in               van&#39;t Veer, L.       LocusLink       J., et al.,       Name   Description   2002)   UniGene               CEGP1   SCUBE2 signal peptide,   NM_020974   Hs.222399           CUB domain       FGF18   Fibroblast growth factor   NM_003862   Hs.49585           18       GSTM3   Glutathione S-transferase   NM_000849   Hs.2006           M3       TGFB3   Transforming growth   NM_003239   Hs.2025           factor, beta 3       MS4A7   Membrane-spanning 4-   AF201951   Hs.11090           domains       EST   Hypothetical protein   Contig55377_RC   Hs.218182       AP2B1   Adaptor-related protein   NM_001282   Hs.74626           complex 2       CCNE2   Cyclin E2   NM_004702   Hs.30464       KIAA0175   Maternal embryonic   NM_014791   Hs.184339           leucine zipper kinase       EXT1   Exostoses (multiple) 1   NM_000127   Hs.184161       LOC341692   Similar to Diap3 protein   Contig46218_RC   Hs.283127       PK428   CDC42 binding protein   NM_003607   Hs.18586           kinase alpha                         14-gene signature (same as Table 28)                                     Gene ID (Chip                   identified in               van&#39;t Veer, L.               J., et al.,       Gene   Description   2002)   UniGene               MS4A7   Membrane-spanning 4-   AF201951   Hs.11090           domains       TGFB3   Transforming growth   NM_003239   Hs.2025           factor, beta 3       BBC3   BCL2 binding component 3   U82987   Hs.87246       AP2B1   Adaptor-related protein   NM_001282   Hs.74626           complex 2       ALDH4A1   Aldehyde dehydrogenase   NM_003748   Hs.77448           4 family, member A1       FLJ11190   Chromosome 20, open   NM_018354   Hs.155071           reading frame 46       DC13   DC13 protein   NM_020188   Hs.6879       GMPS   Guanine monophosphate   NM_003875   Hs.5398           synthetase       AKAP2   A kinase (PRKA) anchor   Contig57258_RC   Hs.42322           protein       DCK   Deoxycytidine kinase   NM_000788   Hs.709       ECT2   Epithelial cell   Contig25991   Hs.122579           transforming sequence 2       EST   ESTs, weakly similar to   Contig38288_RC           quiescin       OXCT   3-oxoacid CoA transferase   NM_000436   Hs.177584       EXT1   Exostoses (multiple) 1   NM_000127   Hs.184161                  
 
      To demonstrate the ability to reduce the number of genes in the cluster, while maintaining predictive power, we selected subsets of genes within a minimum segregation set so as to raise the correlation coefficient, and tested the performance of the cluster as the set was reduced from 9 to 2 genes. Specifically, classification was performed in a cohort of 78 breast cancer patients. The outcome predictor clusters were identified using MDA-MB-435BL3 human breast carcinoma cell line as a reference standard. These results are shown in Tables 73.1 and 73.2.  
               TABLE 73.1                          Classification accuracy of breast cancer outcome predictor       algorithm based on 9-gene parent cluster and smaller gene       clusters derived from the parent 9-gene cluster.                                 Number of genes   Correlation   Poor   Good           in cluster   coefficient   prognosis   prognosis   Overall               9 genes   0.945   31 of 34   27 of 44   58 of 78               (91%)   (61%)   (74%)       5 genes   0.900   20 of 34   36 of 44   56 of 44               (59%)   (82%)   (72%)       4 genes   0.956   28 of 34   28 of 44   56 of 44               (82%)   (64%)   (72%)       2 genes   1.000   27 of 34   30 of 44   57 of 44               (79%)   ((68%)   (73%)                  
 
     
       
         
           
               
             
               
                 TABLE 73.2 
               
             
            
               
                   
               
               
                   
               
               
                 Genes contained within reduced clusters 
               
            
           
           
               
               
               
               
            
               
                 9-gene cluster 
                 5-gene cluster 
                 4-gene cluster 
                 2-gene cluster 
               
               
                   
               
               
                 HEC 
                 HEC 
                 HEC 
                 HEC 
               
               
                 AI377418 
                 MCM6 
                 MCM6 
                 FGF18 
               
               
                 MCM6 
                 BBC3 
                 GSTM3 
               
               
                 BBC3 
                 ALDH4 
                 FGF18 
               
               
                 ALDH4 
                 AP2B1 
               
               
                 AP2B1 
               
               
                 PECI 
               
               
                 GSTM3 
               
               
                 FGF18 
               
               
                   
               
            
           
         
       
     
      As described in Example 2, we validated the classification accuracy using an independent data set, and tested performance of the 13 genes good prognosis predictor cluster on a set of 19 samples obtained from 11 breast cancer patients who developed distant metastases within five years after diagnosis and treatment and 8 patients who remained disease free for at least five years (van &#39;t Veer, L. J., et al., 2002). 9 of 11 samples from the poor prognosis group had negative phenotype association indices, whereas 6 of 8 samples from the good prognosis group had positive phenotype association indices yielding 79% overall accuracy in sample classification.  
      Kaplan-Meier analysis showed that metastasis-free survival after therapy was significantly different in breast cancer patients segregated into good and poor prognosis groups based on relative values of expression signatures defined by all four small gene clusters ( FIG. 65A ). These data indicate that quantitative laboratory tests measuring expression profiles of identified small gene clusters are useful in stratifying breast cancer patients into sub-groups with statistically distinct probabilities of remaining disease-free after therapy.  
      Small gene clusters and a large parent signature perform with similar therapy outcome prediction accuracy in an independent cohort of 295 breast cancer patients. Recently the breast cancer prognosis prediction accuracy of the 70-gene signature was validated in a large cohort of 295 patients with either lymph node-negative or lymph node-positive breast cancer (van de Vijver, M. J., et al., 2002). The expression profile of the 70-gene breast cancer outcome predictor signature was highly informative in forecasting the probability of remaining free of distant metastasis and predicting the overall survival after therapy (id.). We compared the classification accuracy of small gene clusters and a large 70-gene parent signature applied to a cohort of 295 patients.  
      As shown in the Table 74, identified small gene clusters and a large parent signature perform similarly in identifying sub-groups of breast cancer patients with poor and good prognosis defined by differences in the probability of the overall survival after therapy. At the several classification threshold levels small gene clusters fully recapitulate or even outperform the 70-gene parent signature in classification accuracy of the 295 breast cancer patients (Table 74). Taken together these data are consistent with the idea that the 70-gene breast cancer prognosis signature represents a heterogeneous set of small gene clusters with high therapy outcome prediction potential. Consistent with this idea, the application of the 14-gene survival predictor signature was highly informative in classification of breast cancer patients into sub-groups with statistically significant difference in the probability of survival after therapy ( FIG. 68 ). Interestingly, the highly significant difference (p&lt;0.0001) in the survival probability between poor and good prognosis groups defined by the 14-gene signature was achieved using multiple classification threshold levels providing additional flexibility in selection of a desirable 5-or 10-year survival level defining good prognosis group ( FIG. 68B ).  
      To generate the data in Table 74, 295 breast cancer patients were classified according to whether they had a good-prognosis signature or poor-prognosis signature defined by individual therapy outcome predictor signatures. Kaplan-Meier analysis was performed to evaluate the probability that patients would survive according to whether they had a poor-prognosis or a good-prognosis signature and determine the proportion of patients who would survive at least 5 or 10 years after therapy in poor-prognosis and good-prognosis sub-groups. Hazard ratios, 95% confidence intervals, and P values were calculated with use of the log-rank test. The number of correct predictions in poor-prognosis and good-prognosis groups is shown as a fraction of patients with the observed clinical outcome after therapy (79 patients died and 216 patients remained alive). The classification performance of different signatures were evaluated using one common threshold level (0.00) and optimized threshold levels adjusted for each gene cluster to achieve the most statistically significant (highest hazard ratio and lowest P value) discrimination in survival probability between patients assigned to poor and good prognosis groups.  
               TABLE 74                          Stratification of 295 breast cancer patients at the time of diagnosis into poor       and good prognosis groups using different therapy outcome predictor signatures                                                 Poor   Good                           Outcome   prognosis,   prognosis,   Correct   Correct       signature   5-(10)-   5-(10)-   predictions,   predictions,       95%       (cut off   year   year   poor   good   Hazard   Confidence       value)   survival   survival   outcome   outcome   ratio   interval   P value               70-gene   75%   97%   70 of 79   106 of 216   6.327   2.498 to 6.077   &lt;0.0001       (0.45)   (56%)   (92%)   (89%)   (49%)       70-gene   64%   91%   42 of 79   174 of 216   3.867   3.405 to 9.809   &lt;0.0001       (0.00)   (46%)   (80%)   (53%)   (81%)       13-gene   73%   98%   71 of 79   106 of 216   7.005   2.560 to 6.237   &lt;0.0001       (0.12)   (56%)   (93%)   (90%)   (49%)       13-gene   73%   97%   69 of 79   115 of 216   6.519   2.728 to 6.610   &lt;0.0001       (0.04)   (54%)   (92%)   (87%)   (53%)       13-gene   73%   96%   67 of 79   118 of 216   5.698   2.663 to 6.450   &lt;0.0001       (0.00)   (54%)   (90%)   (85%)   (55%)       14-gene   77%   96%   72 of 79    79 of 216   5.220   1.912 to 4.874   &lt;0.0001       (0.37)   (62%)   (91%)   (91%)   (37%)       14-gene   76%   95%   69 of 79    95 of 216   4.701   2.038 to 5.016   &lt;0.0001       (0.28)   (59%)   (89%)   (87%)   (44%)       14-gene   75%   92%   58 of 79   130 of 216   3.637   2.217 to 5.419   &lt;0.0001       (0.00)   (55%)   (85%)   (73%)   (60%)       14-gene   65%   91%   45 of 79   176 of 216   4.171   3.632 to 10.21   &lt;0.0001       (−0.55)    (45%)   (81%)   (57%)   (81%)        6-gene   78%   96%   70 of 79    85 of 216   4.543   1.901 to 4.756   &lt;0.0001       (−0.12)    (62%)   (88%)   (89%)   (39%)        6-gene   78%   92%   64 of 79   101 of 216   3.314   1.757 to 4.282   &lt;0.0001       (0.00)   (60%)   (86%)   (81%)   (47%)        4-gene   73%   93%   60 of 79   136 of 216   4.389   2.723 to 6.735   &lt;0.0001       (0.20)   (53%)   (85%)   (76%)   (63%)        4-gene   75%   93%   60 of 79   119 of 216   3.519   2.050 to 4.983   &lt;0.0001       (0.00)   (58%)   (84%)   (76%)   (55%)                  
 
      The 70-gene signature, in contrast to small gene clusters, is not suitable for breast cancer outcome prediction in patients with estrogen receptor negative tumors. Consistent with well-established prognostic value of the estrogen receptor status of breast tumors (see Introduction), 97 percent of patients in the good prognosis group defined by the 70-gene signature had estrogen receptor positive (ER+) tumors (van de Vijver, M. J., et al., 2002). Conversely, ninety six percent of breast cancer patients with the estrogen receptor negative (ER−) tumors (66 of 69 patients at the cut off level &lt;0.45) had expression profile of the 70 genes predictive of a poor outcome after therapy. Two important conclusions can be drawn from this association. First, breast cancer patients with ER+tumors and poor prognosis expression profile of the 70 genes may have yet unidentified functional defect within an ER-response pathway. Second, a 70-gene signature appears to assign rather uniformly a vast majority of the patients with ER-tumors into poor prognosis category and, therefore, is not suitable for prognosis prediction in this group of breast cancer patients.  
      In agreement with many previous observations, patients with ER− tumors had significantly worst survival after therapy compared to the patients with ER+tumors in the cohort of 295 breast cancer patients ( FIG. 64A ). The Kaplan-Meier survival analysis ( FIG. 64A ) showed that the median relapse-free survival after therapy of patients with the ER− tumors was 9.7 years. Only 47.1% of patients with ER-negative tumors survived 10 years after therapy compared to 77.4% patients with ER+tumors. The estimated hazard ration for survival after therapy in the poor prognosis group as compared with the good prognosis group of patients defined by the ER status was 3.258 (95% confidence interval of ratio, 2.792 to 8.651; P&lt;0.0001).  
      Next we determined that application of a survival predictor algorithm would identify sub-groups of patients with distinct clinical outcome after therapy in breast cancer patients with ER-negative tumors, thus providing additional predictive value to the therapy outcome classification based on ER status alone. We were unable to generate statistically meaningful prognostic stratification of ER-negative breast cancer patients using a parent 70-gene signature (data not shown). However, we were able to identify two small gene clusters comprising 5 and 3 genes (Table 75) that appear highly informative in classifying breast cancer patients with ER-negative tumors into good and poor prognosis sub-groups with statistically distinct probability of survival after therapy ( FIG. 64B ).  
               TABLE 75                          Gene expression signatures predicting survival of breast       cancer patients with estrogen receptor-negative tumors.                                     Gene ID (Chip                   identified in               van&#39;t Veer, L. J.,   UniGene       Gene   Description   et al., 2002)   ID                         5-gene signature                             EST   Unknown   Contig63649_RC           L2DTL   RA-regulated   NM_016448   Hs.126774           nuclear matrix-           associated           protein       DCK   Deoxycytidine   NM_000788   Hs.709           kinase       DKFZP564D0462   G protein-   AL080079   Hs.44197           coupled           receptor 126       LOC286052   Hypothetical   AA555029_RC   Hs.100691           protein           LOC286052                 3-gene signature                             GNAZ   Guanine nucleo-   NM_002073   Hs.92002           tide binding           protein       PK428   CDC42 binding   NM_003607   Hs.18586           protein kinase           alpha       LYRIC   LYRIC/3D3   AK000745   Hs.243901                  
 
      In the group of 69 breast cancer patients with ER-negative tumors ( FIG. 64B ), the median survival after therapy of patients in the poor prognosis sub-group defined by the survival predictor algorithm was 5.2 years. Only 30% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 77% patients in the good prognosis sub-group. The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the survival predictor algorithm was 3.609 (95% confidence interval of ratio, 1.477 to 5.792; P=0.0021).  
      Outcome classification of breast cancer patients with ER-positive tumors using a 14-gene survival predictor signature. To further validate the clinical utility of identified signatures, we determined that application of a 14-gene survival predictor cluster is informative in classifying breast cancer patients with ER-positive tumors. Kaplan-Meier analysis showed that application of the 14-gene survival predictor signature identified three sub-groups of patients with statistically distinct probabilities of survival after therapy in the cohort of 226 breast cancer patients with ER-positive tumors (FIGS.  67 A&amp;B). The median survival after therapy of patients in the poor prognosis sub-group defined by the 14-gene survival predictor signature was 7.2 years ( FIG. 67A ). Only 41% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 100% patients in the good prognosis sub-group (P&lt;0.0001). A large, statistically distinct sub-group of patients with an intermediate expression pattern of the 14-gene signature and an intermediate prognosis was identified by Kaplan-Meier survival analysis ( FIG. 67B ). The patients in the sub-group with an intermediate prognosis had 90% 5-year survival and 76% 10-year survival after therapy ( FIG. 67B ). Thus, the 14-gene survival predictor signature is highly informative in classifying breast cancer patients with ER-positive tumors into good, intermediate, and poor prognosis sub-groups with statistically significant differences in the probability of survival after therapy (FIGS.  67 A&amp;B).  
      Therapy outcome prediction in breast cancer patients with lymph node-negative disease using survival predictor signatures. Invasion into axillary lymph nodes is considered as one of the most important negative prognostic factors in breast cancer and patients with no detectable lymph node involvement are classified as having good prognosis (Krag, et al., 1998; Singletary, et al., 2002; Jatoli, et al., 1999). Breast cancer patients with lymph node negative disease typically would not be selected for adjuvant systemic therapy and usually treated with surgery and radiation. Recent data demonstrated that up to 33% of these patients would fail therapy and develop recurrence of the disease after a 10-year follow-up (Early Breast Cancer Trialists&#39; Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet, 351: 1451-1467, 1998). Therefore, we tested whether application of the 14-gene survival predictor signature would aid in identifying breast cancer patients with lymph-node negative tumors that are at high risk of treatment failure.  
      Kaplan-Meier analysis showed that the 14-gene survival predictor signature (Tables 29 and 73) identified two sub-groups of patients with statistically distinct probability of survival after therapy in the cohort of 151 breast cancer patients with lymph node negative disease ( FIG. 63A ). The median survival after therapy of patients in the poor prognosis sub-group defined by the 14-gene survival predictor signature was 7.7 years ( FIG. 63A ). Only 46% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 82% patients in the good prognosis sub-group (P&lt;0.0001). The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the 14-gene survival predictor signature was 5.067 (95% confidence interval of ratio, 3.174 to 11.57; P&lt;0.0001).  
      Kaplan-Meier analysis also demonstrated that the 14-gene survival predictor signature identified two sub-groups of patients with statistically distinct probability of survival after therapy in the cohort of 109 breast cancer patients with ER-positive tumors and lymph node negative disease ( FIG. 63B ). The median survival after therapy of patients in the poor prognosis sub-group defined by the 14-gene survival predictor signature was 11.0 years ( FIG. 63B ). 10-year survival after therapy in the poor prognosis sub-group was 57% compared to 86% patient&#39;s survival in the good prognosis sub-group (P&lt;0.0001). The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the 14-gene survival predictor signature was 5.314 (95% confidence interval of ratio, 2.775 to 17.79; P&lt;0.0001).  
      Next we determined that application of small gene clusters comprising 5 and 3 genes (Table 75) that appear highly informative in classification of breast cancer patients with ER-negative tumors into good and poor prognosis sub-groups with statistically distinct probability of survival after therapy ( FIG. 64B ), also are informative in classification of sub-group of ER-negative patients with lymph node-negative disease. In the group of 42 breast cancer patients with ER-negative tumors and lymph node-negative disease ( FIG. 63C ), the median survival after therapy of patients in the poor prognosis sub-group defined by the survival predictor algorithm was 5.2 years. Only 34% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 74% patients in the good prognosis sub-group. The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the survival predictor algorithm was 3.237 (95% confidence interval of ratio, 1.139 to 6.476; P=0.0243). Thus, the survival predictor signatures identified in accordance with the methods of the invention are highly informative in classifying breast cancer patients with lymph node-negative disease and either ER-positive or ER-negative tumors into good and poor prognosis sub-groups with statistically significant difference in the probability of survival after therapy (FIGS.  63 B&amp;C).  
      Therapy outcome prediction in breast cancer patients with lymph node-positive disease using survival predictor signatures. Breast cancer patients with invasion into axillary lymph node are considered as having a poor prognosis and usually treated with adjuvant systemic therapy. The patients with poor prognosis are thought to benefit most from adjuvant systemic therapy (see Introduction). In the cohort of 295 breast cancer patients, ten of 151 (6.6%) patients who had lymph node-negative disease and 120 of the 144 (83.3%) patients who had lymph node-positive disease had received adjuvant systemic therapy (van de Vijver, et al. 2002). This treatment strategy was clearly beneficial for patients with lymph node-positive disease, because sub-groups of patients with distinct lymph node status in the cohort of 295 patients had statistically indistinguishable survival after therapy (data not shown). Next we determined therapy outcome prediction using survival predictor signatures identified in accordance with the present invention to be informative in breast cancer patients with lymph node-positive disease.  
      Kaplan-Meier analysis show that application of the 14-gene survival predictor signature identify three sub-groups of patients with statistically distinct probability of survival after therapy in the cohort of 144 breast cancer patients with lymph node positive disease ( FIG. 66A ). The median survival after therapy of patients in the poor prognosis sub-group defined by the 14-gene survival predictor signature was 9.5 years ( FIG. 66A ). Only 43% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 98% patients in the good prognosis sub-group (P&lt;0.0001). Large statistically distinct sub-group of patients with an intermediate expression pattern of the 14-gene signature and an intermediate prognosis was identified by Kaplan-Meier survival analysis ( FIG. 66A ). The patients in the sub-group with an intermediate prognosis had 86% 5-year survival and 73% 10-year survival after therapy ( FIG. 66A ). Thus, 14-gene survival predictor signature appears highly informative in classification of breast cancer patients with lymph node-positive disease into good, intermediate, and poor prognosis sub-groups with statistically significant difference in the probability of survival after therapy ( FIG. 66A ).  
      Using the 14-gene survival predictor signature we identified two sub-groups of patients with statistically distinct probabilities of survival after therapy in the cohort of 117 breast cancer patients with ER-positive tumors and lymph node positive disease ( FIG. 66B ). The median survival after therapy of patients in the poor prognosis sub-group defined by the 14-gene survival predictor signature was 11.0 years ( FIG. 66B ). 10-year survival after therapy in the poor prognosis sub-group was 68% compared to 98% patient&#39;s survival in the good prognosis sub-group (P=0.0026). The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the 14-gene survival predictor signature was 6.810 (95% confidence interval of ratio, 1.566 to 8.358; P=0.0026).  
      Next we determined that the small gene clusters comprising 5 and 3 genes (Table 75) also are informative in classifying sub-groups of ER-negative patients with lymph node-positive disease. In the group of 27 breast cancer patients with ER-negative tumors and lymph node-positive disease ( FIG. 66C ), the median survival after therapy of patients in the poor prognosis sub-group defined by the survival predictor algorithm was 4.4 years. Only 24% of patients in the poor prognosis sub-group survived 10 years after therapy compared to 82% patients in the good prognosis sub-group. The estimated hazard ration for survival after therapy in the poor prognosis sub-group as compared with the good prognosis sub-group of patients defined by the survival predictor algorithm was 3.815 (95% confidence interval of ratio, 0.9857 to 9.660; P=0.0530). Thus, survival predictor signatures identified in accordance with the present invention also is informative in classifying breast cancer patients with lymph node-positive disease into good and poor prognosis sub-groups with statistically significant differences in the probability of survival after therapy ( FIGS. 66A &amp; 66B ).  
      Estimated long-term survival benefits of using gene expression profiling as a component of multiparameter therapy outcome classification of breast cancer patients. Next we estimated the potential clinical benefits of applying gene expression survival predictor signatures identified in accordance with the methods of the present invention for classifying breast cancer patients at the time of diagnosis into sub-groups with distinct probabilities of survival after therapy. In our estimate we used the assignment of the patient into a poor outcome classification sub-group as a criterion of treatment failure and reason for prescription of additional cycle(s) of adjuvant systemic therapy. We have made the estimate of potential therapeutic benefits in the cohort of 295 breast cancer patients (van de Vijver, et al. 2002) and based our estimate on the assumption that the use of additional cycle(s) of adjuvant systemic therapy would be prescribed to patients classified within a poor prognosis sub-group. In the cohort of 295 breast cancer patients, ten of 151 (6.6%) patients who had lymph node-negative disease and 120 of the 144 (83.3%) patients who had lymph node-positive disease had received adjuvant systemic therapy (id.), indicating that a major difference in treatment protocols between LN+ and LN− sub-groups was the application of adjuvant systemic therapy in patients with lymph node positive disease. We accepted the actual 5- and 10-year survival in the corresponding classification categories as the expected therapy outcome for a given sub-group. We assumed that each additional cycle of adjuvant systemic therapy would result in the same therapy outcome as was actually documented in the most relevant sub-groups of the 295 patients. Therapy outcome for patients classified into poor prognosis sub-groups and treated with additional cycle(s) of adjuvant systemic therapy is expected to be in 37% of patients in good therapy outcome category for ER+LN+ and ER+LN-poor signature sub-groups and in 41% of patients in good therapy outcome category for ER-LN+ and ER-LN− poor signature sub-groups (Table 76). Finally, we assumed that patients classified into good prognosis sub-groups would receive the same treatment and would have the same outcome as in the original cohort of 295 patients (van de Vijver, et al., 2002). Based on these assumptions we calculated the number of patients that would be expected to have good and poor survival outcome after therapy and estimated the expected 10-year survival in each classification sub-groups (Table 76).  
      The estimate of potential therapeutic benefits provided in Table 76 is based on the cohort of 295 breast cancer patients (van de Vijver, et al. 2002) and premised on the assumption that additional cycle(s) of adjuvant systemic therapy would be prescribed to patients classified into poor prognosis sub-groups. In the cohort of 295 breast cancer patients, ten of 151 (6.6%) patients who had lymph node-negative disease and 120 of the 144 (83.3%) patients who had lymph node-positive disease had received adjuvant systemic therapy (id.). We accepted the actual 5- and 10-year survival in the corresponding classification categories as the expected therapy outcome for a given sub-group. We assumed that each additional cycle of adjuvant systemic therapy would result in the same therapy outcome as was actually documented in the most relevant sub-groups of the 295 patients. Therapy outcome for patients classified into poor prognosis sub-groups and treated with additional cycle(s) of adjuvant systemic therapy is expected to be in 37% of patients in good therapy outcome category for ER+LN+ and ER+LN− poor signature sub-groups and in 41% of patients in good therapy outcome category for ER-LN+ and ER-LN− poor signature sub-groups.  
               TABLE 76                          Estimated therapeutic benefits of using gene expression survival       predictor signatures for classification of breast cancer patients                                                                 Estimated                   Number   Good   Good   increase in       Classification   5-year   10-year   (%) of   outcome   outcome   10-year       category   survival   survival   patients   (current)   (projected)   survival, %                                                 LN-negative   82%   69%   151/295                                (51%)       LN-positive   85%   72%   144/295                    (49%)       LN− Good   92%   82%   95/151   95   95   0.00       signature           (63%)       LN− Poor   64%   46%   56/151   0   17 (56 × 0.3)     23%       signature           (37%)       LN+ Good   98%   98%   43/144   43   43   0.00       signature           (30%)       LN+   86%   73%   67/144   0   20 (67 × 0.3)     10%       Intermediate           (47%)       LN+ Poor   68%   43%   34/144   0   10 (34 × 0.3)     13%       signature           (24%)       Overall               138/295 (47%)   185/295 (63%)     5%       ER+ tumors   90%   77%   226/295                    (77%)       ER− tumors   62%   47%   69/295                   (23%)       ER+ LN−       Good   97%   86%   69/109   69   69   0.00       signature           (63%)       Poor   76%   57%   40/109   0   15 (40 × 0.37)     17%       signature           (37%)       ER− LN−       Good   74%   74%   16/42    16   16   0.00       signature           (38%)       Poor   50%   34%   26/42    0   11 (25 × 0.41)     44%       signature           (62%       ER+ LN+       Good   98%   98%   43/117   43   43   0.00       signature           (37%)       Poor   86%   68%   74/117   0   27 (74 × 0.37)     16%       signature           (63%)       ER− LN+       Good   82%   82%   11/27    11   11   0.00       signature           (41%)       Poor   47%   24%   16/27    0    7 (16 × 0.41)    100%       signature           (59%)       Overall               139/295 (47%)   199/295 (67%)     6%                  
 
      One of the most interesting end-points of this analysis is the prediction that patients with ER-LN− and ER-LN+breast cancer classified into poor prognosis sub-groups would be expected to show a most dramatic increase in 10-year survival after therapy (Table 76). This prediction is consistent with the generally accepted notion that breast cancer patients with poor prognosis would benefit most from adjuvant systemic therapy (see Introduction). The estimated modest increase in the overall 10-year survival (Table 76) may translate every year into ˜7,000-9,000 more breast cancer survivors after 10-year follow-up. Our ability to accurately segregate at the time of diagnosis breast cancer patients with low probability of survival after therapy should lead to more rapid development of novel efficient therapeutic modalities specifically targeting most aggressive therapy-resistant breast cancers.  
      While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications may be made without departing from the invention, the scope of which is limited only by the appended claims. All references cited, including scientific publications, patent applications, and issued patents, are herein incorporated by reference in their entirety for all purposes.