Patent Publication Number: US-2007099197-A1

Title: Methods of prognosis of prostate cancer

Description:
This application is a continuation of pending U.S. patent application Ser. No. 10/603,505, filed Jun. 24, 2003, which claims the benefit of provisional application 60/391,309, filed Jun. 24, 2002, which is incorporated herein in its entirety. 
    
    
     FIELD OF THE INVENTION  
      The invention relates to the identification of nucleic acid and protein expression profiles and nucleic acids, products, and antibodies thereto that are outcome prognostic in prostate cancer.  
     BACKGROUND OF THE INVENTION  
      Prostate cancer will account for an estimated 30% (189,000) of new cancer cases in men in the United States in 2002 (1). Many of these newly diagnosed cases are a result of the extensive use of prostate-specific antigen (PSA) screening and the subsequent diagnosis of prostate cancer at an early stage and age. However, despite the introduction of PSA screening the mortality from prostate cancer has remained relatively constant. The implications of this are that: (1) there are a large group of men diagnosed with prostate cancer for whom radical treatment is probably unnecessary and who will die with their prostate cancer rather than from it; and (2) there are a group of men for whom early detection offers the possibility of cure that may be denied by delay. Consequently, identifying these groups of men at the time of diagnosis is critical to the optimal management of prostate cancer.  
      While the benefits of PSA screening are widely debated, this serum marker remains one of only a small number of preoperative parameters of prognostic utility. In order to enhance the predictive value of individual parameters with outcomes, nomograms have been developed that incorporate parameters that are measured routinely in clinical practice to predict the probability of PSA relapse free survival of individual patients both prior to and following therapy (2-6). Models such as these currently form the basis of routine clinical decision-making, but such classification systems cannot explore differences in outcomes observed between cancers with similar histopathological features. Hence, there remains a critical need for increased accuracy in the subcategorization of prostate cancers to identify those with an aggressive phenotype.  
      There are a considerable number of publications assessing the ability of biomarkers to predict an earlier time to relapse of prostate cancer following radical prostatectomy (reviewed in ref. (17)). Despite these data, there remain no molecular markers of routine clinical utility which differentiate localized prostate cancers with an aggressive phenotype, and clinicians still rely on conventional preoperative and postoperative prognostic indicators such as pretreatment PSA levels, pathological stage and Gleason grade in routine decision-making. This most likely reflects the fact that studies that have correlated differences in gene expression with patient outcome have assessed candidate genes with limited predictive power that provide no additional prognostic information above the conventional variables. This accentuates the need to discover novel genes with strong predictive ability.  
      One approach is to define patterns of gene expression that correlate with disease phenotype and patient outcome. Here, we undertook a systematic search for novel biomarkers of prostate cancer prognosis by outcome-based analyses of transcript profiles.  
     SUMMARY OF INVENTION  
      The present invention evaluates gene expression profile and identifies prognostic genes of prostate cancers. The present invention provides a method of determining prognosis of prostate cancer and predicting prostate cancer outcome of a patient. The method comprises the steps of first establishing the threshold value of at least one prognostic gene of prostate cancer. Then, the amount of the prognostic gene from a prostate tissue of a patient inflicted of prostate cancer is determined. The amount of the prognostic gene present in that patient is compared with the established threshold value of the prognostic gene, whereby the prognostic outcome of the patient is determined.  
      In certain embodiments, the amount of the prognostic gene is determined by the quantitation of a transcript encoding the sequence of the prognostic gene; or a polypeptide encoded by the transcript. The quantitation of the transcript can be based on hybridization to the transcript. The quantitation of the polypeptide can be based on antibody detection. The method optionally comprises a step of amplifying nucleic acids from the tissue sample before the evaluating. In some embodiments, the evaluating is of a plurality of sequences. The method may further comprises determining prostate-specific antigen (PSA) level. The prognosis contributes to selection of a therapeutic strategy.  
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
      The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.  
       FIG. 1  shows cluster analysis of prostate cancer samples from 72 patients treated for localised prostate cancer by RP. Each column represents a single RP specimen and each row represents one of the 264 genes which demonstrated a strong association with PSA relapse in our model. The dendogram at the top shows the degree to which each prostate cancer is related to the others with respect to gene expression. The 17 patients known to have experienced a PSA relapse are indicated by an “R”. The relative level of expression is indicated by the color scale at the bottom and is indicative of the normalized average intensity units of fluorescence signal detected by microarray analysis.  
       FIG. 2A  shows the expression of trp-p8 mRNA detected by oligonucleotide microarray in prostate cancer samples and in normal body tissues. Samples are: prostate cancer 1-74, adrenal glands 75-77, aorta 78-80, artery 81-83, bladder 84-86, bone marrow 87-89, colonic epithelium 90-92, cerebral cortex 93-95, colon 96-98, colonic muscle 99-101, esophagus 102-104, heart 105-107, kidney 108-110, liver 111-113, lung 114-116, lymph node 117-119, muscle 120-122, oral mucosa 123-125, pharyngeal mucosa 126-128, pancreas 129-131, parathyroid glands 132-133, pituitary 134-136, prostate 137-143, retina 144-146, skin 147-149, small intestine 150-152, spleen 153-155, stomach 156-158, trachea 159-161, tongue 162-164, ureter 165-167, vagus nerve 168-170, vein 171-174.  
       FIG. 2B  shows the expression of trp-p8 mRNA, and  FIG. 2C  shows the PSA mRNA; both detected by oligonucleotide microarray in LuCaP-35 tumors at days 0 to 100 post castration. The expression level of trp-p8 and PSA is shown as normalized average intensity units (Y-axis) of fluorescence signal detected by microarray analysis.  
      units (Y-axis) of fluorescence signal detected by microarray analysis.  
       FIG. 3  shows the Trp-p8 mRNA expression detected by in situ hybridization in radical prostatectomy cases treated with or without neoadjuvant hormone therapy prior to surgery.  
       FIG. 3A : A prostate cancer from a patient treated with RP only showing positive trp-p8 mRNA expression in malignant prostate epithelium.  FIG. 3B : A prostate cancer from a patient treated with RP and NHT showing positive trp-p8 mRNA expression.  FIG. 3C : A prostate cancer from a patient treated with RP only with no detectable trp-p8 mRNA expression in the malignant epithelium, and Figure D: A prostate cancer from a patient treated with preoperative NHT with no evidence of trp-p8 expression.  
       FIG. 4A  shows the trp-p8 protein sequence.  FIG. 4B  shows the trp-p8 mRNA sequence. 
    
    
     DETAILED DESCRIPTION OF INVENTION  
      Current models of prostate cancer classification are poor at distinguishing between tumors that have similar histopathological features but vary in clinical course and outcome. In the present invention, we have applied classical survival analysis to genome-wide gene expression profiles of prostate cancers and preoperative prostate-specific antigen levels from each patient, to identify prognostic markers of disease relapse that provide additional predictive value relative to prostate-specific antigen concentration. The present invention demonstrates that multivariable survival analysis can be applied to gene expression profiles of prostate cancers with censored follow-up data and used to identify molecular markers of prostate cancer relapse with strong predictive power and relevance to the etiology of this disease.  
      Prostate Cancer and Treatments  
      Prostate cancer is found mainly in older men. Prostate cancer is the most commonly diagnosed internal malignancy and second most common cause of cancer death in men in the U.S., resulting in approximately 40,000 deaths each year. Landis et al. (1998) CA Cancer J. Clin. 48:6-29; and Greenlee, et al. (2000) CA Cancer J. Clin 50:7-13. The incidence of prostate cancer has been increasing rapidly over the past 20 years in many parts of the world. Nakata, et al. (2000) Int. J. Urol. 7:254-257; and Majeed, et al. (2000) BJU Int. 85:1058-1062. It develops as the result of a pathologic transformation of normal prostate cells. In tumorigenesis, the cancer cell undergoes initiation, proliferation, and loss of contact inhibition, culminating in invasion of surrounding tissue and, ultimately, metastasis.  
      Prostate cancer is a disease in which malignant (cancer) cells form in the tissues of the prostate. The prostate is a gland in the male reproductive system located just below the bladder (the organ that collects and empties urine) and in front of the rectum (the lower part of the intestine). It is about the size of a walnut and surrounds part of the urethra (the tube that empties urine from the bladder). The prostate gland produces fluid that makes up part of the semen. See generally, Boyle, et al. (2002) Textbook of Prostate Cancer Isis Medical Media, ISBN: 1901865304; Kantoff (ed. 2002) Prostate Cancer: Principles and Practice Lippincott, ISBN: 0781720060; Carroll (2001) Prostate Cancer Decker, ISBN: 1550091301; Belldegrun, et al. (2000) New Perspectives in Prostate Cancer Isis Medical Media, ISBN: 1901865568; Lepor (1999) Prostatic Diseases Saunders, ISBN: 072167416X; Petrovich, et al. (eds. 1996) Carcinoma of the Prostate: Innovations in Management, Springer Verlag, ISBN: 3540587497; and standard prostate cancer medical texts.  
      Four types of standard treatment are used for prostate cancer: watchful waiting, surgery, radiation therapy, or hormone ablation therapy. See, e.g., the National Cancer Institute (NCI) description of prostate cancer, www.cancer.gov.  
      Watchful waiting is closely monitoring a patient&#39;s condition but withholding treatment until symptoms appear or change. This is usually used in older men with other medical problems and early stage disease.  
      Surgery is usually offered to prostate cancer patients in good health who are younger than 70 years old. Main surgery options are pelvic lymphadenectomy, radical protatectomy, perineal prostatectomy, and transurethral resection of the prostate.  
      Pelvic lymphadenectomy is a surgical procedure to take out lymph nodes in the pelvis to see if they contain cancer. If the lymph nodes contain cancer, the doctor will not remove the prostate and may recommend other treatment. Radical prostatectomy (RP) is surgery to remove the entire prostate. Radical prostatectomy is done only if tests show the cancer has not spread outside the prostate. The two types of radical prostatectomy are retropubic prostatectomy, which removes the prostate through an incision made in the abdominal wall, and removal of surrounding lymph nodes (lymphadenectomy) can be done at the same time; and perineal prostatectomy, which is surgery to remove the prostate through an incision made between the scrotum and the anus, and if surrounding lymph nodes are to be removed, this is usually done through a separate incision. Transurethral resection of the prostate is a surgical procedure to remove tissue from the prostate using an instrument inserted through the urethra. This operation is sometimes done to relieve symptoms caused by the tumor before other treatment is given. Transurethral resection of the prostate may also be done in men who cannot have a radical prostatectomy because of age or illness.  
      Impotence and leakage of urine from the bladder or stool from the rectum may occur in men treated with surgery. In some cases, doctors can use a technique known as nerve-sparing surgery. This type of surgery may save the nerves that control erection. However, men with large tumors or tumors that are very close to the nerves may not be able to have this surgery.  
      Radiation therapy is the use of x-rays or other types of radiation to kill cancer cells and shrink tumors. Radiation therapy may use external radiation (using a machine outside the body) or internal radiation. Internal radiation involves putting radioisotopes (materials that produce radiation) through thin plastic tubes into the area where cancer cells are found. Prostate cancer is treated with external and internal (implant) radiation. Radiation therapy may be used alone or in addition to surgery. Impotence and urinary problems may occur in men treated with radiation therapy.  
      Hormone therapy is the fourth of the standard treatments. Hormones are chemicals produced by glands in the body and circulated in the bloodstream. Hormone therapy is the use of hormones to stop cancer cells from growing. Male hormones (especially testosterone) can help prostate cancer grow. To stop the cancer from growing, female hormones or drugs that decrease production of male hormones may be given. Hormone therapy used in the treatment of prostate cancer may include the following: estrogens (hormones that promote female sex characteristics) can prevent the testicles from producing testosterone, however, estrogens are seldom used today in the treatment of prostate cancer because of the risk of serious side effects; luteinizing hormone-releasing hormone agonists also can prevent the testicles from producing testosterone, e.g., leuprolide, goserelin, and buserelin; antiandrogens can block the action of androgens (hormones that promote male sex characteristics), two examples are flutamide and bicalutamide; drugs that can prevent the adrenal glands from making androgens include ketoconazole and aminoglutethimide; and orchiectomy is surgery to remove the testicles, the main source of male hormones, to decrease hormone production. Hot flashes, impaired sexual function, and loss of desire for sex may occur in men treated with hormone therapy.  
      Deaths from prostate cancer are typically a result of metastasis of a prostate tumor. Therefore, early detection of the development of prostate cancer is critical in reducing mortality from this disease. Measuring levels of prostate-specific antigen (PSA) has become a very common method for early detection and screening, and may have contributed to the slight decrease in the mortality rate from prostate cancer in recent years. Nowroozi, et al. (1998) Cancer Control 5:522-531. However, many cases are not diagnosed until the disease has progressed to an advanced stage.  
      Prognosis, Outcome  
      Prognosis is typically recognized as a forecast of the probable course and outcome of a disease. See Dorland&#39;s Medical Dictionary. As such, it involves inputs of both statistical probability, requiring numbers of samples, and outcome data. Herein, outcome data is utilized in the form of prostate cancer recurrence after RP. A patient population of many dozens is included, providing statistical power.  
      The ability to determine which cases of prostate cancer will respond to treatment, and to which type of treatment, would be useful in appropriate allocation of treatment resources. As indicated above, the various standard therapies have significantly different risks and potential side effects. Accurate prognosis would also minimize application of treatment regimens which have low likelihood of success. Such also could avoid delay of the application of alternative treatments which may have higher likelihoods of success for a particular presented case. Thus, the ability to evaluate individual prostate cases for markers which subset into responsive and non-responsive groups for particular treatments are very useful.  
      Current models of prostate cancer classification are poor at distinguishing between tumors that have similar histopathological features but vary in clinical course and outcome. Kattan, et al. (1998) J. Nat&#39;l Cancer Inst. 90:766-771; and Kattan, et al. (1999) J. Clin. Oncol. 17:1499-1507. Identification of novel prognostic molecular markers is a priority if radical treatment is to be offered on a more selective basis to those prostate cancer patients with clinically significant disease. A novel strategy is described to discover molecular markers for prostate cancer prognosis by assessing genome-wide gene expression in many localized prostate cancers and modeling these data based on each patient&#39;s known clinical outcome and preoperative serum prostate-specific antigen concentration. The study herein is directed to molecularly define different forms of prostate cancer which can translate directly into prognosis. And such prognosis allows for application of a treatment regimen having a greater statistical likelihood of cost effective treatments and minimization of negative side effects from the different treatment options.  
      Prostate cancer biopsy samples were collected and analyzed for gene expression across most genes of the human genome. Among genes detected at appropriate levels, correlations with outcome data were evaluated. Genes whose expression levels correlated with statistical significance to outcome data were identified.  
      This approach identified about 270 genes that demonstrated a strong association (P&lt;0.01) with disease outcome, e.g., prostate cancer relapse, and were superior in their predictive ability relative to prostate-specific antigen levels, one of the standard markers. One of these genes, the putative calcium channel protein trp-p8, is androgen-regulated and loss of trp-p8 appears to be associated with aggressive disease. The findings provide a method of survival analysis of gene expression profiles of cancers with censored follow-up data and identify novel molecular markers of prostate cancer progression with strong predictive power that may be used to select prostate cancers with an aggressive phenotype.  
      Thus, the invention herein provides statistical correlations of marker expression in appropriate samples with disease outcome.  
      Survival Analysis  
      The present invention provides the application of classical multivariable survival analysis to a prostate cancer microarray data set incorporating the expression profiles of over 46,000 genes, to identify markers of disease outcome. This technique provides several significant advances over previous methods of analyses that have been used to discover markers of disease outcome from microarray data. In contrast to previously described statistical methods that rely on the classification of tumors based on known outcome (18) or known classifiers of patient outcome (eg. estrogen receptor status) (19, 20), this technique provides for censored data. This enables these analyses to proceed prior to the occurrence of all events, in this case, PSA relapse. Moreover, this survival analysis incorporates the time taken to PSA relapse and may also include covariates (eg. preoperative serum PSA levels) in order to identify genes that provide additional predictive value above conventional markers of outcome. The statistical analyses described herein have also incorporated a stringent method of estimating the pFDR that was recently described (10). This method is designed specifically for the analysis of microarray data where general dependence between hypotheses or “clumpy dependence” exists, where 50 or more genes interact in common pathways to produce some overall process (10). However, this is the first instance that it has been applied to microarray data from a survival analysis.  
      A recently published analysis to discover new markers of prostate cancer outcome utilized microarray analyses of prostate cancers to classify small groups of tumors where the recurrence status was known (21). That study found that no single gene was statistically associated with recurrence at P&lt;0.05 and instead adopted a 5-gene model that most commonly included chromogranin A and inositol triphosphate receptor 3 (IP3R). The significant differences between our study and these previously published data are (1) our adoption of a Cox proportional hazards model, and (2) our observation that 277 individual genes were predictive for prostate cancer relapse, none of which overlapped with the genes in the 5-gene model identified by Singh et al. (2002). There are two prevailing explanations for the latter discrepancy. Firstly, the number of genes interrogated by oligonucleotide microarrays in our study was 4-fold greater; trp-p8 is an example of a gene which was not present in the oligonucleotide array used in the previous study. As a result, the genes identified by Singh et al. (2002), were associated with P values of less significance than those presented in Tables 1 and 2. Secondly, by utilizing a statistical method that applies to censored data, we were able to take into account the varying times to prostate cancer relapse in this model. Therefore, we were able to use our full data set in the analysis, rather than restricting the analysis to those patients with a specified length of follow-up. The larger data set and concomitant increase in statistical power may also contribute to our results differing from those of Singh et al.  
      The TRP channels are made of subunits with six membrane-spanning domains with both carboxy and amino termini located intracellularly that probably form into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into the cell. Trp-p8 or TRPM8 is a member of the TRPM subfamily of TRP ion channels that have potential roles in Ca 2+ -dependent signaling, control of cell cycle proliferation, cell division and cell migration. Ligand binding to some membrane receptors initiates a sequence of events that lead to the activation of phospholipase C, generating inositol-1,4,5-triphosphate which opens the intracellular ion channel IP3R and liberates Ca 2+  from the endoplasmic reticulum. Activation of the TRP channels accompanies this chain of events, allowing the influx of calcium ions into the cells, although their activation is not necessarily directly linked to Ca 2+  depletion from internal stores (22). Calnexin, which is also identified in this analysis as a marker of potential prognostic utility (P=0.004), is believed to be a key chaperone involved in the folding, assembly and oligomerization of newly synthesised IP3R receptors (24). Thus, our study implicates an important role for the phosphatidylinositol signal transduction.  
      Our observation that loss of trp-p8 is associated with a poor prognosis is also reminiscent of the prognostic role of another of the TRPM subfamily, TRPM1 or melastatin, in melanoma. Downregulation of melastatin mRNA in primary cutaneous melanoma is a prognostic marker for metastasis in patients with localized melanoma and is independent of conventional clinicopathological predictors of metastases (25). Recent studies showed that the rat (26) and mouse (27) orthologues of trp-p8 are functional calcium channels that respond to cold stimuli. Although cold is unlikely to be the natural stimulus for trp-p8 in the prostate, the implication that the human trp-p8 protein may be a functional Ca 2+  channel suggests a role in the regulation of intracellular Ca 2+  levels with possible effects on cell motility, cell proliferation and resistance to apoptotic stimuli.  
      In summary, our analyses have identified a group of genes that strongly correlate with prostate cancer relapse and contribute unique information to relapse prediction above preoperative PSA.  
      Prognosis Determination  
      One application of the survival analysis results is to generate a prognostic test for prostate cancer. First, we use TAQMAN® analysis to determine the absolute levels of prognostic genes in 75-150 or more prostate cancer patients. Then we correlate the absolute levels of the prognostic genes with patient outcome by a statistical analysis and determine threshold levels of prognostic genes; from which we establish a profile of the threshold level of each prognostic gene associated with a good outcome. For determining the prognosis of a prostate cancer patient, the absolute level of one or more prognostic genes of this patient is determined. Then the absolute level of one or more prognostic genes of this patient is compared with the above established threshold values. Absolute level higher (or lower depending on the prognostic gene) than the threshold values indicates good outcome.  
      The normalized quantitative level of absolute gene expression of a prognostic gene, from which outcome is predicted, is determined first. Quantitative polymerase chain reaction (PCR)-based methods can be applied. RT-PCR (reverse transcriptase PCR) primers are designed for selected prognostic genes, in order to perform a TaqMan® analysis.  
      TAQMAN® analysis is a real-time quantitative PCR, which is a powerful method used for gene expression analysis, genotyping, pathogen detection/quantitation, mutation screening and DNA quantitation. See, e.g., Bartlett (2003) PCR Protocols (2 d  ed.) Humana Press; and O&#39;Connell (2002) RT-PCR Protocols, Humana Press. The technology uses, e.g., an ABI Prism instrument (TAQMAN®) to detect accumulation of PCR products continuously during the PCR process thus allowing easy and accurate quantitation in the early exponential phase of PCR. The basis for PCR quantitation in the ABI instrument is to continuously measure PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TAQMAN® probe. This probe is composed of a short (ca. 20-25 bases) oligodeoxynucleotide that is labeled with two different flourescent dyes. On the 5′ terminus is a reporter dye and on the 3′ terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR amplicon. When the probe is intact, energy transfer occurs between the two flourophors and emission from the reporter is quenched by the quencher. During the extension phase of PCR, the probe is cleaved by 5′ nuclease activity of Taq polymerase thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity. The laser light source excites each well and a CCD camera measures the fluorescence spectrum and intensity from each well to generate real-time data during PCR amplification. The ABI Prism software examines the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are then plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early log phase of product accumulation. This is accomplished by assigning a fluorescence threshold above background and determining the time point at which each sample&#39;s amplification plot reaches the threshold (defined as the threshold cycle number or CT). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube as described previously.  
      The TAQMAN® primers are designed within the open-reading frame of the prognostic gene of interest so that the amplicon averages 80 bp. Prostate tissue samples from 70-150 or more prostate cancer patients with known histories are collected and RNA is extracted from these samples using standard methods. TAQMAN® analysis is performed on these samples for the appropriate genes. Using the TAQMAN® analysis, the normalized absolute levels of the prognostic genes are then correlated with patient outcome. Using statistical analyses the threshold level of gene expression, which predicts outcome, is then determined. Subsequent patient samples can then be analyzed for potential of relapse and the physician can better define the patient treatment based on whether the patient is predicted to relapse. Subsetting of the data into various outcomes is achieved through statistical analyses. (Snedecor and Cochran (1994) Statistical Methods (8 th  ed.) Iowa State University Press; and Duda, et al. (2001) Pattern Classification (2 d  ed.) Wiley and Sons.)  
      Genes, Markers, Kits  
      The present study provides specific identification of multiple genes whose expression levels in biological samples will serve as markers to evaluate prostate cancer cases. These markers have been selected for statistical correlation to disease outcome data on a large number of prostate cancer patients.  
      The expression levels of these markers in a biological sample may be evaluated by many methods. They may be evaluated for RNA expression levels. Hybridization methods are typically used, and may take the form of a PCR or related amplification method. Alternatively, a number of qualitative or quantitative hybridization methods may be used, typically with some standard of comparison, e.g., actin message. Alternatively, measurement of protein levels may performed by many means. Typically, antibody based methods are used, e.g., ELISA, radioimmunoassay, etc., which may not require isolation of the specific marker from other proteins. Other means for evaluation of expression levels may be applied upon purification of the marker. Antibody purification may be performed, though separation of protein from others, and evaluation of specific bands or peaks on protein separation may provide the same results. Thus, e.g., mass spectroscopy of a protein sample may indicate that quantitation of a particular peak will allow detection of the corresponding marker. Multidimensional protein separations may provide for quantitation of specific purified entities.  
      Tables 1A-C describe markers of the invention useful for the prognosis of prostate cancer.  
      Table 1A shows radical prostatectomy samples that were analyzed using the Eos Hu03 GENECHIP®, which contains 59680 probesets. Each probeset&#39;s intensity measure was entered as a continuous explanatory variable in a Cox proportional hazards regression survival analysis predicting relapse. Pretreatment PSA concentration was entered as a predictor in each analysis. The interquartile range hazard ratio (IQR HR) for each probeset was then calculated. This approach was used since in conventional Cox proportional hazards analyses, the hazards ratios for a covariate are computed by raising e, the base of natural logarithms, to the power of its regression coefficient. However, because the expression data are treated here as continuous covariates, hazards ratios expressed in this manner illustrate only the change in risk of relapse associated with a change of 1 unit on the expression scale, a change too small to be meaningful. To put the hazard ratios and associated confidence limits on a more interpretable scale, presented here is the hazards ratio associated with a change in expression values equivalent to 1 interquartile range (IQR) of the sample data for each probeset. The IQR is simply the 75th percentile minus the 25th percentile, and thus contains the middle 50 percent of observations. From this analysis, 266 probesets were found to be significant predictors of relapse at P&lt;0.01.  
      Table 1B lists the accession numbers for Pkey&#39;s lacking UnigeneID&#39;s for table 1A. For each probeset is listed the gene cluster number from which oligonucleotides were designed. Gene clusters were compiled using sequences derived from Genbank ESTs and mRNAs. These sequences were clustered based on sequence similarity using Clustering and Alignment Tools (DoubleTwist, Oakland Calif.). Genbank accession numbers for sequences comprising each cluster are listed in the “Accession” column.  
      Table 1C shows genomic positioning for those Pkey&#39;s lacking Unigene ID&#39;s and accession numbers in table 1A. For each predicted exon, is listed the genomic sequence source used for prediction. Nucleotide locations of each predicted exon are also listed.  
                               TABLE 1A                       Pkey   ExAccn   UnigeneID   Unigene Title   p value                                                    428664   AK001666   Hs.189095   similar to SALL1 (sal ( Drosophila )-like   3.80177E−05       439785   AA845608   Hs.132860   ESTs   0.000106034       413924   AL119964   Hs.75616   seladin-1   0.000157824       459680   H96982   Hs.42321   ESTs   0.00019382       431542   H63010   Hs.5740   ESTs   0.000250668       404824           C22000161*: gi|2443331|dbj|BAA22375.1| (D     0.000290214       446021   BE389213   Hs.286   ribosomal protein L4   0.000320882       434999   AW975059       gb: EST387164 MAGE resequences, MAGN Homo   0.000341555       458509   AA654650   Hs.282906   ESTs   0.000351184       406722   H27498   Hs.293441     Homo sapiens  SNC73 protein (SNC73) mRNA,   0.000536315       423381   BE250014       gb: 600943007F1 NIH_MGC_15  Homo sapiens  c     0.000602528       419037   R39895   Hs.257391   hypothetical protein DKFZp761J1523   0.00065526       414898   AA157726   Hs.264330   N-acylsphingosine amidohydrolase (acid c     0.000707085       404582           Target Exon   0.00074185       458607   AV656002       ESTs, Moderately similar to unnamed prot     0.000805762       402861   D14661       Wilms&#39; tumour 1-associating protein   0.000870602       441494   AW452344   Hs.129977   ESTs   0.000875883       452753   AA028049   Hs.277728   SEC14 ( S. cerevisiae )-like 2   0.000934337       422516   BE258862   Hs.117950   multifunctional polypeptide similar to S   0.000969694       443675   AI081397       ESTs   0.000984435       425297   AA354685       gb: EST63062 Jurkat T-cells V  Homo sapien     0.001036315       419517   AF052107   Hs.90797     Homo sapiens  clone 23620 mRNA sequence   0.001065289       441345   AW068579   Hs.7780     Homo sapiens  mRNA; cDNA DKFZp564A072 (fr     0.00111943       438611   AW204707   Hs.123387   ESTs   0.001135255       434949   AW976087       ESTs, Highly similar to AF161437 1 HSPC3   0.001142057       430845   AF024690   Hs.248056   G protein-coupled receptor 43   0.001172874       429446   AI547111       gb: PN2.1_A01_G12.r mynorm  Homo sapiens  c     0.001185816       444773   BE156256   Hs.11923   hypothetical protein   0.001200592       446702   R44518   Hs.143496   ESTs   0.001311934       415179   D80630       gb: HUM091D02B Human fetal brain (TFujiwa     0.0013887       448479   H96115   Hs.21293   UDP-N-acteylglucosamine pyrophosphorylas   0.001402576       430799   C19035   Hs.164259   ESTs   0.001404901       454930   AW845987   Hs.68864   ESTs, Weakly similar to phosphatidylseri   0.001417466       407241   M34516       gb: Human omega light chain protein 14.1   0.001504145       421970   AF227156   Hs.110103   RNA polymerase I transcription factor RR   0.001519398       434808   AF155108   Hs.256150     Homo sapiens , Similar to RIKEN cDNA 2810   0.001610938       400207           Eos Control   0.00161581       423318   AW467064   Hs.5740   ESTs   0.001622161       413102   AI199981   Hs.109694   ESTs, Weakly similar to T27691 hypotheti     0.001683835       411630   U42349   Hs.71119   Putative prostate cancer tumor suppresso     0.001688301       419872   AI422951   Hs.146162   ESTs   0.001710345       402812           NM_004930*:  Homo sapiens  capping protein   0.001742994       427418   AA402587       LAT1-3TM protein   0.001743363       416276   U41060   Hs.79136   LIV-1 protein, estrogen regulated   0.001830512       457397   AW969025   Hs.109154   ESTs   0.001994494       403372   AW249152       sirtuin (silent mating type information   0.002012497       415344   T65456       gb: yc73a11.r1 Soares infant brain 1NIB H   0.002025172       422017   NM003877   Hs.110776   STAT induced STAT inhibitor-2   0.002053043       406554           Target Exon   0.002105231       446057   AI420227   Hs.149358   ESTs, Weakly similar to A46010 X-linked   0.002151173       407040   X03689       gb: Human mRNA fragment for elongation fa     0.002199926       419657   AK001043   Hs.92033   integrin-linked kinase-associated serine   0.002290654       457662   AA907734   Hs.124895   ESTs   0.002413693       447308   AI005334   Hs.22015   ESTs, Weakly similar to 138344 titin, ca   0.002472822       420707   BE312807   Hs.143407   ESTs, Weakly similar to A54849 collagen   0.002479439       426429   X73114   Hs.169849   myosin-binding protein C, slow-type   0.00251185       429289   AI400746   Hs.62187   phosphatidylinositol glycan, class K   0.002513019       454275   AW293900   Hs.304842   ESTs, Weakly similar to AMYH_YEAST GLUCO     0.002559888       408603   R25283   Hs.326416     Homo sapiens  mRNA; cDNA DKFZp564H1916 (f     0.002571063       434614   AI249502   Hs.29669   ESTs   0.002629652       406558           C5000893: gi|6226859|P38525|EFG_THEMA   0.002723963       440325   NM003812   Hs.7164   a disintegrin and metalloproteinase doma   0.002768837       440518   AA888046   Hs.233235   ESTs   0.002805131       424099   AF071202   Hs.139336   ATP-binding cassette, sub-family C (CFTR   0.002848507       421655   AA464812       gb: zw63h05.r1 Soares_total_fetus_Nb2HF8 —     0.002855486       445375   AW779857   Hs.166987   ESTs, Weakly similar to B35363 synapsin   0.002861874       456647   AI252640   Hs.110364   peptidylprolyl isomerase C (cyclophilin   0.002867794       433293   AF007835   Hs.32417   hypothetical protein MGC4309   0.002897453       430389   AL117429   Hs.240845   DKFZP434D146 protein   0.002920262       423479   NM014326   Hs.129208   death-associated protein kinase 2   0.00294831       443884   N20617   Hs.194397   leptin receptor   0.002997251       457926   AA452378         Homo sapiens  mRNA; cDNA DKFZp547J125 (fr     0.003054911       459710   AI701596   Hs.121592   ESTs   0.003061123       404560           Target Exon   0.003092402       438657   AI141396   Hs.158741   ESTs   0.003131957       400282           NM_005313:  Homo sapiens  glucose regulated   0.003134356       416144   AA381556   Hs.331803   heat shock 60 kD protein 1 (chaperonin)   0.003162736       430677   Z26317       desmoglein 2   0.003170664       423562   AJ005197   Hs.7984   pleckstrin homology, Sec7 and coiled/coi   0.003217503       401040           C11000425: gi|4507721|ref|NP_003310.1|ti   0.003244184       419733   AW362955         Homo sapiens  cDNA FLJ14415 fis, clone HE   0.003251143       415439   R21114   Hs.21383   ESTs   0.003317352       458054   AW979052   Hs.5734   meningioma expressed antigen 5 (hyaluron     0.003355436       435346   AI248389   Hs.188105   ESTs   0.00337758       410452   AW749026       gb: RC3-BT0319-100100-012-b05 BT0319 Homo   0.003407284       427548   AA813784   Hs.123001   ESTs   0.003456322       438918   AI126484   Hs.127486   ESTs   0.00347913       448076   AJ133123   Hs.20196   adenylate cyclase 9   0.003583335       420339   AW968259   Hs.186647   ESTs   0.003607275       426514   BE616633   Hs.170195   bone morphogenetic protein 7 (osteogenic   0.003628615       452143   N29649   Hs.260855     Homo sapiens  cDNA: FLJ21410 fis, clone C   0.003701377       422813   AV656571   Hs.121068   transmembrane 4 superfamily member 6   0.00379349       401524           Target Exon   0.003793904       453768   BE382670   Hs.198511     Homo sapiens  mRNA; cDNA DKFZp761I177 (fr     0.003810346       424954   NM000546   Hs.1846   tumor protein p53 (Li-Fraumeni syndrome)   0.003826169       440409   AW294316   Hs.125608   ESTs   0.003879241       452286   AI358570   Hs.123933   ESTs, Weakly similar to ZN91_HUMAN ZINC   0.003898535       444756   AA278501   Hs.200332   hypothetical protein FLJ20651   0.003922529       429769   NM004917   Hs.218366   kallikrein 4 (prostase, enamel matrix, p   0.003947007       443403   R01027   Hs.133560   ESTs   0.003959306       400219           Eos Control   0.003966793       448489   AI523875       gb: tg97d04.x1 NCI_CGAP_CLL1  Homo sapiens     0.004120703       428378   AA427571   Hs.98531   ESTs   0.004121896       449909   AA004681   Hs.59432   ESTs   0.004158168       425127   AW841272   Hs.330418     Homo sapiens  cDNA: FLJ22459 fis, clone H   0.004166839       427485   AF039652   Hs.178655   ribonuclease H1   0.004198226       416305   AU076628   Hs.79187   coxsackie virus and adenovirus receptor   0.004214942       415075   L27479   Hs.77889   Friedreich ataxia region gene X123   0.00422178       414091   T83742   Hs.334616   gb: yd67g02.s1 Soares fetal liver spleen   0.004236934       446415   T27097   Hs.22790   ESTs   0.004250994       407218   AA095473   Hs.28505   ubiquitin-conjugating enzyme E2H (homolo     0.004267222       436626   W35362   Hs.103012   ESTs   0.00432651       448519   AW175665   Hs.278695     Homo sapiens  prostein mRNA, complete cds   0.004332167       409841   AW502139       gb: UI-HF-BR0p-ajr-e-05-0-UI.r1 NIH_MGC_5   0.004357117       423022   AA320525   Hs.201076   ESTs   0.004401104       429332   AF030403   Hs.199263   Ste-20 related kinase   0.004405129       417834   BE172058   Hs.82689   tumor rejection antigen (gp96) 1   0.004424022       419808   AW008030   Hs.337536     Homo sapiens  cDNA: FLJ21568 fis, clone C   0.004471786       450088   AW292933   Hs.254110   ESTs   0.004491465       431151   BE207083       gb: ba10d10.y1 NIH-MGC_7  Homo sapiens  cDN   0.00450798       431281   AW970573       gb: EST382654 MAGE resequences, MAGK Homo   0.004657684       420960   Z45662   Hs.90797     Homo sapiens  clone 23620 mRNA sequence   0.004798622       409540   AW409569   Hs.101550   gb: fh01e09.x1 NIH_MGC_17  Homo sapiens  cD   0.004819322       456643   AW751497   Hs.98370   cytochrome P450, subfamily IIS, polypept   0.004821217       449889   AA004613   Hs.168672   ESTs   0.004888264       413074   AI871368   Hs.8417   hypothetical protein DKFZp761M0423   0.004890295       452099   BE612992   Hs.27931   hypothetical protein FLJ10607 similar to   0.004925393       434263   N34895   Hs.44648   ESTs   0.004967084       400296   AA305627   Hs.139336   ATP-binding cassette, sub-family C (CFTR   0.004996569       435981   H74319   Hs.188620   ESTs   0.005005242       409430   R21945   Hs.346735   splicing factor, arginine/serine-rich 5   0.005047202       414916   AA206991       high-mobility group (nonhistone chromoso     0.005130846       434855   AA765019   Hs.191850   ESTs   0.005199586       406651   AI559224       gb: tq32c02.x1 NCI_CCAP_Ut1  Homo sapiens     0.005212356       440675   AW005054   Hs.47883   ESTs, Weakly similar to KCC1_HUMAN CALCI     0.005249269       437412   BE069288   Hs.34744     Homo sapiens  mRNA; cDNA DKFZpS47C136 (fr     0.005270232       400487           ENSP00000238977*: Interferon-induced prot     0.005353963       443366   AI053501   Hs.278869   ESTs, Moderately similar to 2109260A B c   0.005371997       410054   AL120050   Hs.58220     Homo sapiens  cDNA: FLJ23005 fis, clone L   0.005404329       409344   R47279   Hs.285673   hypothetical protein FLJ20950   0.005429984       421215   AI868634   Hs.246358   ESTs, Weakly similar to T32250 hypotheti     0.005442884       450661   AW952160       ESTs   0.005447857       424269   AW137691   Hs.104696   ESTs   0.005483308       412294   AA689219       poly(A)-binding protein, nuclear 1   0.005530138       404511           NM_004349:  Homo sapiens  core-binding fact   0.005558982       437006   AW976322   Hs.291561   ESTs   0.005639929       432989   NM014074       PRO0529 protein   0.00572161       417584   AA252468   Hs.1098   DKFZp434J1813 protein   0.005734515       437992   AW450086   Hs.145989   ESTs, Highly similar to DHHC-domain-cont     0.005769051       447506   R78778   Hs.29808     Homo sapiens  cDNA: FLJ21122 fis, clone C   0.005799441       420929   AI694143   Hs.296251   programmed cell death 4   0.00585145       415121   D60971   Hs.34955     Homo sapiens  cDNA FLJ13485 fis, clone PL   0.005963023       404662           Target Exon   0.006001874       445878   AI262974   Hs.145587   ESTs   0.006055258       421090   BE301870   Hs.101813   solute carrier family 9 (sodium/hydrogen   0.006079413       405155           Target Exon   0.006110052       427379   D79254   Hs.256066   ESTs   0.006133565       412561   NM002286   Hs.74011   lymphocyte-activation gene 3   0.006142277       434257   AF121255   Hs.193053   eukaryotic translation initiation factor   0.006144213       400141           Eos Control   0.006200101       453359   AA448787   Hs.24872   ESTs   0.006315475       433151   AW973735   Hs.17631   hypothetical protein DKFZp434E2135   0.006324267       449791   AI248740   Hs.133323   ESTs   0.006355539       405722   BE410124       NM_021120:  Homo sapiens  discs, large (Dro     0.006388997       427527   AI809057   Hs.293441   immunoglobulin heavy constant mu   0.006397862       411487   AF116666   Hs.70333   hypothetical protein MGC10753   0.006474544       417407   AA923278   Hs.290905   ESTs, Weakly similar to protease [H.sapi     0.00651405       437233   D81448   Hs.339352     Homo sapiens  brother of CDO (BOC) mRNA,   0.006535001       443425   AI056776   Hs.133397   ESTs, Weakly similar to I78885 serine/th   0.006574089       409179   BE062633   Hs.28338   KIAA1546 protein   0.006647277       431947   AL359613   Hs.49933   hypothetical protein DKFZp762D1011   0.006663987       402339           NM_003425*:  Homo sapiens  zinc finger prot     0.006744987       422262   AL022315   Hs.113987   lectin, galactoside-binding, soluble, 2   0.006803463       404458           CX000877*: gi|11877268|emb|CAC18893.1|(A     0.006816499       431693   AI459519       serine (or cysteine) proteinase inhibito     0.006849491       428734   BE303044   Hs.192023   eukaryotic translation initiation factor   0.00696046       444204   AI129194   Hs.143040   ESTs   0.007032748       406837   R70292   Hs.156110   immunoglobulin kappa constant   0.007051544       442482   NM014039   Hs.8360   PTD012 protein   0.007051611       412006   AW451618       ESTs   0.00705506       435354   AA678267   Hs.117115   ESTs   0.007095576       403505   M97639       receptor tyrosine kinase-like orphan rec   0.007139282       451946   AI824901   Hs.281012   ESTs, Highly similar to strong homology   0.007271734       433339   AF019226   Hs.8036   glioblastoma overexpressed   0.007286776       436924   AA741001   Hs.326006   ESTs   0.007312314       431578   AB037759   Hs.261587   GCN2 elF2alpha kinase   0.007346563       419551   AW582256   Hs.91011   anterior gradient 2 ( Xenepus laevis ) hom   0.007352833       434256   AI378817   Hs.191847   ESTs   0.00736484       439778   AL109729   Hs.99364   putative transmembrane protein   0.0073683       423443   AI432601   Hs.168812     Homo sapiens  cDNA FLJ14132 fis, clone MA   0.007425186       405293           Target Exon   0.007457507       426357   AW753757   Hs.12396   gb: RC3-CT0283-271099-021-a08 CT0283 Homo   0.007488395       422921   BE062045         Homo sapiens  cDNA: FLJ23260 fis, clone C   0.007499187       417501   AL041219   Hs.82222   sema domain, immunoglobulin domain (Ig),   0.007512156       426091   BE544541   Hs.249495   heterogeneous nuclear ribonucleoprotein   0.007576069       416974   AF010233   Hs.80667   RALBPI associated Eps domain containing   0.007594318       449787   AA005341   Hs.283559   ESTs   0.007675199       412162   AA100600   Hs.69192   gb: zn63b10.s1 Stratagene HeLa cell s3 93   0.007681586       413522   BE145897       gb: MRO-HT0208-221299-204-b07 HT0208 Homo   0.007824405       426788   U66615   Hs.172280   SWI/SNF related, matrix associated, acti     0.007843962       414586   AA306160   Hs.76506   lymphocyte cytosolic protein 1 (L-plasti     0.007931767       450382   AA397658   Hs.60257     Homo sapiens  cDNA FLJ13598 fis, clone PL   0.007975007       404242           ENSP00000252213*: SODIUM BICARBONATE COTR   0.008032744       400206           Eos Control   0.008161865       441011   AW137447   Hs.126408   ESTs   0.008169197       449223   AB002348   Hs.23263   KIAA0350 protein   0.008169995       451776   W45679   Hs.169854   hypothetical protein SP192   0.008174536       418354   BE386973   Hs.84229   splicing factor, arginine/serine-rich 8   0.00821493       435188   AA669512   Hs.116679   ESTs, Weakly similar to A42826T-cell le     0.00826337       415457   AW081710   Hs.7369   ESTs, Weakly similar to ALU1_HUMAN ALU S   0.008283276       432981   NM002733   Hs.3136   protein kinase, AMP-activated, gamma 1 n   0.008309431       433468   AA832055   Hs.170222   ESTs, Weakly similar to ALU1_HUMAN ALU S   0.008310151       457269   AI338993   Hs.134535   ESTs   0.00834154       431676   AI685464       gb: tt88f04.x1 NCI_CGAP_Pr28  Homo sapiens     0.008414644       426501   AW043782   Hs.293616   ESTs   0.008416828       447623   AA350235   Hs.6127     Homo sapiens  cDNA: FLJ23020 fis, clone L   0.008419744       429678   N70394   Hs.238956   ESTs   0.008452349       444370   NM015344   Hs.11000   leptin receptor overlapping transcript-1   0.00847352       404557           C8001174*: gi|10432400|emb|CAC10290.1|(A     0.008502518       422867   L32137   Hs.1584   cartilage oligomeric matrix protein (pse     0.008537039       441283   AA927670   Hs.131704   ESTs   0.008562466       424640   AA344559   Hs.164428   ESTs   0.008568818       452793   AW138760   Hs.61484   ESTs   0.008570907       420527   AA332287   Hs.175110   ESTs   0.00858412       421515   YI1339   Hs.105352   GalNAc alpha-2, 6-sialyltransferase I, 1   0.008588847       430316   NM000875   Hs.239176   insulin-like growth factor 1 receptor   0.008606329       436524   AA922236   Hs.221037   ESTs   0.008616325       444700   NM003645   Hs.11729   fatty-acid-Coenzyme A ligase, very long-   0.008668985       441222   AI277237   Hs.44208   hypothetical protein FLJ23153   0.008703638       429170   NM001394   Hs.2359   dual specificity phosphatase 4   0.008704913       454393   BE153288       gb: PM0-HT0335-180400-008-c08 HT0335 Homo   0.008716471       456107   AA160000   Hs.137396   ESTs, Weakly similar to JC5238 galactosy   0.008767147       402091           Target Exon   0.008853214       409115   AI223335   Hs.50651   Janus kinase 1 (a protein tyrosine kinas   0.008866852       423250   BE061916   Hs.125849   chromosome 8 open reading frame 2   0.008901601       428944   AA780181   Hs.41182     Homo sapiens  DC47 mRNA, complete cds   0.008970935       419052   T83291   Hs.220624   ESTs   0.008998014       446203   Z47553   Hs.14286   flavin containing monooxygenase 5   0.009023814       428180   AI129767   Hs.182874   guanine nucleotide binding protein (G pr     0.009035339       452264   AU077013   Hs.28757   transmembrane 9 superfamily member 2   0.009036494       446425   AW295364   Hs.255418   ESTs   0.009058296       446547   AI334965   Hs.176976   ESTs   0.009087495       419555   AA244416       gb: nc07d11.s1 NCI_CGAP_Prl  Homo sapiens     0.009114049       422068   AI807519   Hs.104520     Homo sapiens  cDNA FLJ13694 fis, clone PL   0.009119167       434826   AF155661   Hs.22265   pyruvate dehydrogenase phosphatase   0.009188183       411950   T28407   Hs.81564   platelet factor 4   0.009188186       457146   BE271371       biphenyl hydrolase-like (serine hydrolas     0.009228646       454131   AI215902   Hs.88845   ESTs, Highly similar to T50835 hypotheti     0.009282618       404483           C8000657*: gi|1504040|dbj|BAA13219.1|(D8   0.009290064       421351   AU076667   Hs.103755   receptor-interacting serine-threonine ki   0.00929738       417963   AA210718   Hs.104157   ESTs, Weakly similar to KIAA0694 protein   0.009334158       429011   AA443182   Hs.188835   ESTs   0.009370261       425380   AA356389   Hs.32148   AD-015 protein   0.009402223       442315   AA173992   Hs.7956   ESTs, Moderately similar to ZN91_HUMAN Z   0.009446269       424546   BE465173   Hs.194031   ESTs   0.009446339       444524   AI160643   Hs.28332     Homo sapiens  cDNA: FLJ21560 fis, clone C   0.009472535       408446   AW450669   Hs.45068   hypothetical protein DKFZp434I143   0.009508794       422669   H12402   Hs.119122   ribosomal protein L13a   0.00950994       420593   AA280356   Hs.187634   ESTs   0.009517511       447502   AA312531   Hs.26471   Bardet-Biedl syndrome 4   0.0096083       412825   AW167439   Hs.190651     Homo sapiens  cDNA FLJ13625 fis, clone PL   0.009645426       434401   AI864131   Hs.71119   Putative prostate cancer tumor suppresso     0.009778291       432826   X75363   Hs.250770   ACO for serine protease homologue   0.009849589       428840   M15990   Hs.194148   v-yes-1 Yamaguchi sarcoma viral oncogene   0.009881804       413592   AA130654   Hs.302274     Homo sapiens  cDNA FLJ12328 fis, clone MA   0.009899125       443102   AI247472   Hs.132965   ESTs   0.009964996                 Pkey: Unique Eos probeset identifier number            ExAccn: Exemplar Accession number, Genbank accession number            UnigeneID: Unigene number            Unigene Title: Unigene gene title            p value: p value for relapse prediction (see Table 1A description)             
 
     
       
         
           
               
               
               
             
               
                 TABLE 1B 
               
               
                   
               
               
                   
               
               
                 Pkey 
                 CAT Number 
                 Accession 
               
               
                   
               
             
            
               
                 409841 
                 1156088_1 
                 AW502139 AW502432 AW502235 AW501683 AW502647 
               
               
                 410452 
                 1204142_1 
                 AW749026 BE066111 T97135 
               
               
                 412006 
                 127108_1 
                 AW451618 AA846096 AI004201 AI242026 N38791 AI032976 AA099469 N45423 
               
               
                 412294 
                 128797_2 
                 AA689219 AI983045 T16928 Z45040 R20321 
               
               
                 413522 
                 1374614_1 
                 BE145897 BE145816 BE145885 
               
               
                 414916 
                 15071_24 
                 AA206991 BE564126 AA092392 AA090034 AA090545 AA093840 N84434 BE269369 
               
               
                   
                   
                 AI535705 AI535744 AI535682 AF283771 H28296 H27400 BE618821 AI873907 BE622711 
               
               
                   
                   
                 AI471738 AA557452 AA304303 AW794938 AA600212 AW027283 AW938645 AI654646 
               
               
                   
                   
                 AA370554 AA356536 AA715713 N87841 AW575412 AA987424 AA319424 BE084055 
               
               
                   
                   
                 AA827973 AA330422 AW630429 N38949 AA360952 AA045606 BE257213 AW768545 
               
               
                   
                   
                 AA101746 AI335554 N26696 AI630155 AW170282 AA206705 AA357094 AW603120 
               
               
                   
                   
                 AW793181 AI127978 AA639183 AW020136 BE536372 AA093946 AA730118 BE079411 
               
               
                   
                   
                 T90564 D83849 D20752 W07682 BE540914 F22618 AI041775 AA196344 AA366696 
               
               
                   
                   
                 AA083771 AA054783 AA330028 BE544267 AA247271 AW958331 BE073175 AW945457 
               
               
                   
                   
                 AA229491 AW874401 R34185 R81133 W32781 AI191194 BE277231 W79255 AW800102 
               
               
                   
                   
                 AI935842 AA928301 AA230310 AI742195 BE566990 AW673140 AI829489 AA054719 
               
               
                   
                   
                 AW512749 AA782987 AI088142 AW103898 AA714697 AW574795 AI056134 AW162373 
               
               
                   
                   
                 BE148890 AW068721 AW076120 AA563764 AW016252 AW016253 AI338171 AI085967 
               
               
                   
                   
                 AI338788 BE542084 AI186025 AI963188 AW079946 AI034040 AI961313 AI831345 N79755 
               
               
                   
                   
                 AA029435 AA910600 AA618386 AI336429 AA230308 AI346567 AA541647 AW024986 
               
               
                   
                   
                 AI926174 AA878167 AW026237 AA668251 W15170 AA129635 AI363729 AA309687 
               
               
                   
                   
                 AI453176 AI282417 H89557 AW264978 D55190 AA188911 AI471512 AI537126 AW675575 
               
               
                   
                   
                 AI673287 AI476121 AA563901 AA353344 N93269 N80559 L13805 AA564621 AI056119 
               
               
                   
                   
                 AI587020 AW874624 AI803890 AW074286 AA745955 AW152331 AI282228 AI081139 
               
               
                   
                   
                 AI147252 AI126996 AI970694 D55874 AA313759 AW023735 AA999920 AI285652 
               
               
                   
                   
                 AI476553 AI252804 AI096960 AW151090 AA876366 W32423 D57151 AA856637 AI954376 
               
               
                   
                   
                 W73923 AL047978 BE041344 AA861867 AI346526 AL047979 AI348036 AI187244 
               
               
                   
                   
                 AA328683 AA197248 N72984 AA862752 AA747207 AA876587 AA845496 AA890470 
               
               
                   
                   
                 AW170401 AI127224 N99881 AW074379 AA938114 AI197777 AI753834 AI346536 
               
               
                   
                   
                 AA331597 AI367738 AA977063 W93785 AA872167 AI932924 AA614560 AI434283 
               
               
                   
                   
                 AI160153 AW130136 BE542026 AA385117 AA130703 AA778269 AI769329 AI285034 
               
               
                   
                   
                 AW340835 AI224601 AA663430 AA846183 AI362627 AA903448 AW238760 AI283178 
               
               
                   
                   
                 AV662138 AI138363 AA860743 AI368179 AI280190 AI139131 AI359157 H99812 
               
               
                   
                   
                 AA771749 AI539068 AI089843 AI566789 AI281240 AI352354 AI769243 AI092187 
               
               
                   
                   
                 AI073627 AI473623 AW276039 AI798397 AI024587 AA889467 AI683918 AW673268 
               
               
                   
                   
                 AA602941 AA861823 AA668586 AA772542 AI077928 AA594116 AI018648 AI421799 
               
               
                   
                   
                 AA705955 AA586855 AA577106 AI131297 AI355412 AI350882 AW265014 AW043934 
               
               
                   
                   
                 AI127696 AW469864 AI041801 AL048264 AA961777 AI246050 AA566002 AI469308 
               
               
                   
                   
                 AA809086 AW768947 AA507781 AI361342 AI368477 AA133897 AI300444 AI768467 
               
               
                   
                   
                 AA773978 AW062352 AA648130 AA827606 AI094950 T61248 AA101747 AI348251 
               
               
                   
                   
                 AI092294 AA565522 T39158 W33201 C75489 AA670425 AA483085 R48684 T28966 
               
               
                   
                   
                 H96803 AA641999 AA709360 H99805 T19371 AW879059 AA524370 AW338262 N72895 
               
               
                   
                   
                 AW591714 T63777 AL047945 AA150131 AA146973 AW878989 AA877803 T56122 
               
               
                   
                   
                 AA147065 AA342484 AA342236 AW270920 AI913364 AW795486 AI865002 W94286 
               
               
                   
                   
                 AA209325 T40443 AI268918 AI418552 T48135 M62207 AA328164 AW795480 BE169953 
               
               
                   
                   
                 BE169983 AA206888 AA132394 AW149866 T57929 W15510 C75674 R81132 AI423687 
               
               
                   
                   
                 AI193465 H28297 AA994473 F04357 BE243460 AA987347 AI376779 AA927274 T03381 
               
               
                   
                   
                 H99134 T03851 AA384714 AW265058 BE041328 BE541757 AI910675 T64485 N89843 
               
               
                   
                   
                 AA688338 T64628 AI143530 AI026855 F03043 AA865434 AA363018 AA459233 AA664746 
               
               
                   
                   
                 N68567 AW467363 T16030 AW149914 AA994312 BE350136 AA307427 AI658528 L13804 
               
               
                   
                   
                 AA384004 N71219 N22172 AW364964 
               
               
                 415179 
                 1527481_1 
                 D80630 D80896 D80895 
               
               
                 415344 
                 1534510_1 
                 T65456 F11749 Z43023 F06216 R18181 R17246 
               
               
                 419555 
                 185884_1 
                 AA244416 AA244401 
               
               
                 419733 
                 187589_1 
                 AW362955 H59488 AI040666 W60959 W94209 H27231 T84625 H75715 W04957 W63676 
               
               
                   
                   
                 AA659693 AA514302 W63789 BE046412 T91396 AI951970 AW044233 N20018 AW663548 
               
               
                   
                   
                 T90114 AI139947 AA809643 AA846232 AA581966 AA789002 AA295134 AW188870 
               
               
                   
                   
                 H75644 AA526037 AA347970 AW961788 H61476 AL133779 AA449282 H28581 AA249370 
               
               
                 421655 
                 204993_1 
                 AA464812 AA431899 AA295193 AW959241 
               
               
                 422921 
                 222939_1 
                 BE062045 Z43804 W35143 AI761615 N33753 BE062044 BE551229 AI088004 N33865 
               
               
                   
                   
                 AA332473 AA374196 N48481 
               
               
                 423381 
                 227731_1 
                 BE250014 BE293608 BE252781 AA325222 AW904396 
               
               
                 425297 
                 249704_1 
                 AA354685 AW962101 H85269 F11427 R55281 
               
               
                 427418 
                 278594_1 
                 AA402587 AI760178 AI911270 AI184927 AI277654 AA402398 AI633280 AW002589 
               
               
                   
                   
                 AI984968 AI810234 AI671725 AI419580 AA705629 AW138044 AI719961 D45607 
               
               
                   
                   
                 AA455831 
               
               
                 429446 
                 304683_1 
                 AI547111 AW973749 AA558007 
               
               
                 430677 
                 3216_1 
                 Z26317 NM_001943 AW991316 BE018413 AW996800 AW996267 AW996264 W73983 
               
               
                   
                   
                 AA313797 BE513193 AW861416 AW857494 AA488331 BE171045 AW366926 BE002219 
               
               
                   
                   
                 AW996792 AW753487 AW361908 BE003946 AW858751 AW858747 AW858750 
               
               
                   
                   
                 AW858755 AW858749 D58979 AW363740 AW859003 AW363742 AW858999 AW471344 
               
               
                   
                   
                 BE072891 AW753745 BE395396 AI378517 D58730 AW748942 BE395765 BE153312 
               
               
                   
                   
                 BE153169 BE153241 AW371849 AW371853 AW748956 AA506621 AA723159 AI933746 
               
               
                   
                   
                 AW473996 AW572140 
               
               
                 431151 
                 328652_−1 
                 BE207083 
               
               
                 431281 
                 330904_1 
                 AW970573 AA501880 AA501870 
               
               
                 431676 
                 336411_1 
                 AI685464 AW971336 AA513587 AA525142 
               
               
                 431693 
                 33663_3 
                 AI459519 AI366092 AF121175 AL042956 F11899 AI436382 AI133591 AI675879 AA081306 
               
               
                   
                   
                 AI948730 BE243645 AA448957 H09862 AI382265 N92723 AL048959 AI356415 BE245782 
               
               
                   
                   
                 AI288626 AI949306 AI814412 AW207026 AI659678 AI984766 AA741391 AI453490 
               
               
                   
                   
                 AW166423 AI799883 AL045697 AI826075 AI952039 AA167742 BE463776 R01203 
               
               
                   
                   
                 AI972947 AI623819 AW167132 AW337996 AW264027 AA209462 AI863491 T65400 
               
               
                   
                   
                 AI394192 R62397 AW968250 BE464852 AW474624 AI758979 AW474705 BE046016 
               
               
                   
                   
                 AI949348 AI289432 AI620722 AW440580 AI610824 AI458169 AW002172 AI634183 
               
               
                   
                   
                 AA648408 AI289435 C00469 R62398 AI287482 H24845 F09546 AI125609 W93405 
               
               
                   
                   
                 AA150039 AA150203 H09775 AI951377 AI631154 AA258738 AA744971 AA449685 
               
               
                   
                   
                 AI434048 AA167836 R01316 T54772 
               
               
                 432989 
                 35719_1 
                 NM_014074 AF111848 
               
               
                 434949 
                 39603_1 
                 AW976087 AA100561 AF161437 D30850 AA767385 AI990080 AI337209 AA086348 
               
               
                   
                   
                 AW002909 AA747908 AW450816 AW361653 BE145974 BE146300 AW292658 
               
               
                 434999 
                 397353_1 
                 AW975059 AA659177 AA733194 
               
               
                 443675 
                 577019_1 
                 AI081397 N94610 AI633993 AW949183 W23817 AW297357 H17610 F32559 
               
               
                 448489 
                 765247_1 
                 AI523875 R45782 R45781 
               
               
                 450661 
                 84193_1 
                 AW952160 AI819147 AA774089 AA010589 AA319638 AI954753 AI634083 H39119 
               
               
                   
                   
                 AA812766 
               
               
                 454393 
                 115888_1 
                 BE153288 BE153151 BE152925 AA078302 
               
               
                 457146 
                 29193_1 
                 BE271371 NM_004332 X81372 AI167945 AW071802 AI818871 AI017491 AA421820 
               
               
                   
                   
                 AA558952 AA910750 AA973795 R54850 AI672895 AI418120 AI268326 AA911487 
               
               
                   
                   
                 AA167197 N46097 X57653 R10551 T28159 AA167111 AW840204 AW276222 R09405 
               
               
                   
                   
                 N46098 AA284554 AW129121 
               
               
                 457926 
                 43767_1 
                 AA452378 AL390181 H05571 R53363 R55079 R11987 R11919 R84811 R19546 AA046904 
               
               
                   
                   
                 R22842 AL134431 F11225 W79925 H10691 AA354088 AW965695 AI198775 AI803682 
               
               
                   
                   
                 AA040404 AI150653 AA040266 AI436656 AW575893 AI703024 AA446858 AI805847 
               
               
                   
                   
                 AI699312 AW575924 R55051 R53965 R39826 AW772031 AA975258 AW901905 R43388 
               
               
                   
                   
                 BE218163 AI074604 AI148281 AA758256 BE501159 H11032 AW131553 F08888 
               
               
                   
                   
                 AW341569 AI347996 AI952708 AI374835 AI089094 AI284927 W74206 AI027303 AI274177 
               
               
                   
                   
                 AW299757 AI377712 AW300882 AA883979 AI239912 AI346165 AA947211 R46050 
               
               
                   
                   
                 AI698833 AA452150 R43898 AA904733 
               
               
                 458607 
                 65602_1 
                 AV656002 AV655810 
               
               
                   
               
               
                   Pkey: Unique Eos probeset identifier number    
               
               
                   CAT number: Gene cluster number    
               
               
                   Accession: Genbank accession numbers    
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 1C 
               
               
                   
               
               
                   
               
               
                 Pkey 
                 Ref 
                 Strand 
                 Nt_position 
               
               
                   
               
             
            
               
                 400487 
                 8919452 
                 Plus 
                 19369-20782 
               
               
                 401040 
                 7232177 
                 Plus 
                 17623-17919 
               
               
                 401524 
                 7770429 
                 Plus 
                 34644-35263 
               
               
                 402091 
                 8117554 
                 Minus 
                 190-306 
               
               
                 402339 
                 7459859 
                 Minus 
                 24698-26511 
               
               
                 402812 
                 6010110 
                 Plus 
                 25026-25091, 25844-25920 
               
               
                 402861 
                 2814366 
                 Minus 
                 14933-15231, 15387-15627 
               
               
                 403372 
                 9087278 
                 Minus 
                 130002-130131 
               
               
                 403505 
                 7577651 
                 Plus 
                 11059-11541 
               
               
                 404242 
                 5672600 
                 Minus 
                 22722-22897, 23164-23433 
               
               
                 404458 
                 7770571 
                 Minus 
                 35710-36276 
               
               
                 404483 
                 8096904 
                 Minus 
                 162212-163710 
               
               
                 404511 
                 8151864 
                 Minus 
                 148501-148659 
               
               
                 404557 
                 7243881 
                 Minus 
                 88508-88699 
               
               
                 404560 
                 8954219 
                 Plus 
                 29247-29437 
               
               
                 404582 
                 9739220 
                 Plus 
                 53230-53424 
               
               
                 404662 
                 9797105 
                 Minus 
                 99466-99713 
               
               
                 404824 
                 6478944 
                 Plus 
                 209436-209545, 209741-209850 
               
               
                 405155 
                 9966228 
                 Plus 
                 130469-130723 
               
               
                 405293 
                 3845419 
                 Minus 
                 16255-16535, 16665-17340 
               
               
                 405722 
                 9800078 
                 Plus 
                 140732-140892, 141099-141268, 
               
               
                   
                   
                   
                 141434-141714, 142048-142192 
               
               
                 406554 
                 7711566 
                 Plus 
                 106956-107121 
               
               
                 406558 
                 7711569 
                 Minus 
                 14052-14190 
               
               
                   
               
               
                   Pkey: Unique number corresponding to an Eos probeset    
               
               
                   Ref: Sequence source. The 7 digit numbers in this colunm are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.    
               
               
                   Strand: Indicates DNA strand from which exons were predicted.    
               
               
                   Nt_position: Indicates nucleotide positions of predicted exons.    
               
               
                   Note:    
               
               
                   the ExAccn number of NM_is abbreviated to NM in Table 1A-C.    
               
            
           
         
       
     
      Table 2 lists the first 50 genes, ranked by P value, identified by survival analysis to be associated with prostate cancer relapse.  
                                   TABLE 2                           UniGene   Genbank                   Rank   cluster   accession   Gene title   Risk of relapse a     P                                                        1   Hs.189095   NM_020436   Sal-like 4   2.040   0       2   Hs.132860   AA845608   ESTs   0.341   0       3   Hs.75616   NM_014762   24-Dehydrocholesterol reductase (seladin-1)   0.293   0       4   Hs.42321   NM_173605   Hypothetical protein LOC283518   2.133   0       5   Hs.80667   NM_004726   RALBP1 associated Eps domain containing 2 (REPS2)   0.172   0       6   Hs.163543   NM_144704   Hypothetical protein FLJ30473   3.241   0       7   Hs.286   NM_000968   Ribosomal protein L4   0.215   0       8   Hs.114670   D49387   Leukotriene B4 12-hydroxydehydrogenase   2.380   0       9   Hs.366053   NM_024080   Transient receptor potential cation channel, subfamily M, member 8 (trp-p8)   0.260   0       10   Hs.366   AL389978   Immunoglobulin heavy chain variable region   2.436   0.001       11   Not available   BE250014   ESTs   0.295   0.001       12   Hs.257391   NM_032293   Hypothetical protein DKFZp761J1523   3.138   0.001       13   Hs.264330   AK024677   N-acylsphingosine amidohydrolase (acid ceramidase)-like   0.256   0.001       14   Hs.123468   NM_033225   CUB and Sushi multiple domains 1   0.185   0.001       15   Not available   AV656002   EST   0.251   0.001       16   Hs.129977   AW452344   ESTs   0.229   0.001       17   Hs.277728   NM_012429   SEC14-like 2   0.348   0.001       18   Hs.117950   NM_006452   Phosphoribosylaminoimidazole carboxylase   0.321   0.001       19   Hs.424973   BC018081   Clone IMAGE: 4793702   0.225   0.001       20   Not available   AA354685   EST   0.363   0.001       21   Hs.356547   NM_138799   Hypothetical protein BC016005   0.337   0.001       22   Hs.7780   AL049969   cDNA DKFZp564A072   0.186   0.001       23   Hs.123387   AW204707   ESTs   0.375   0.001       24   Hs.377879   AK055649   cDNA FLJ31087 fis   3.112   0.001       25   Hs.248056   NM_005306   G protein-coupled receptor 43   0.211   0.001       26   Hs.301947   NM_014509   Kraken-like serine hydrolase   0.212   0.001       27   Hs.11923   NM_018982   Hypothetical protein DJ167A19.1   0.155   0.001       28   Hs.247423   NM_001617   Adducin 2 (β) (ADD2)   2.044   0.001       29   Not available   D80630   EST   2.753   0.001       30   Hs.21293   NM_003115   UDP-N-acteylglucosamine pyrophosphorylase 1   0.185   0.001       31   Hs.292859   C19035   ESTs, moderately similar to VPP2_HUMAN   2.375   0.001       32   Hs.68864   AW845987   Lipase, member H (LIPH), mRNA   0.273   0.001       33   Hs.405944   X57819   Ig λ chain   2.388   0.002       34   Hs.110103   NM_018427   RNA polymerase I transcription factor RRN3   0.337   0.002       35   Hs.256150   NM_080654   NY-REN-41 antigen   2.718   0.002       36   Hs.76847   NM_014610   α Glucosidase II alpha subunit   0.135   0.002       37   Hs.109694   AI199981   Oxysterol binding protein-like 8 (OSBPL8), mRNA.   4.511   0.002       38   Hs.71119   NM_006765   Putative prostate cancer tumor suppressor (N33)   0.281   0.002       39   Hs.146162   AK075364   ESTs.   2.151   0.002       40   Hs.333417   NM_004930   Cappling protein (actin filament) muscle Z-line, β   0.291   0.002       41   Hs.410998   AA402587   ESTs, Highly similar to MLL septin-like fusion   1.507   0.002       42   Hs.79136   NM_012319   LIV-1 protein, estrogen regulated   0.210   0.002       43   Hs.109154   AW969025   ESTs   0.281   0.002       44   Hs.433622   NM_007085   Follistatin-like 1 (FSTL1)   0.233   0.002       45   Not available   T65456   EST   0.195   0.002       46   Hs.405946   NM_003877   Suppressor of cytokine signaling 2 (SOCS2)   0.448   0.002       47   Hs.127699   NM_001369   Dynein, axonemal, heavy polypeptide 5 (DNAH5)   0.284   0.002       48   Hs.422118   NM_001402   Eukaryotic translation elongation factor 1 alpha 1   0.175   0.002       49   Hs.92033   NM_030768   Integrin-linked kinase-associated serine/threonine phosphatase 2C   5.564   0.002       50   Hs.124895   AA907734   ESTs   3.399   0.002                   a The risk of relapse is the IQR HR calculated for each probeset as described in “Materials and Methods.”            
 
      Sequences described therein, where incomplete, may be extended either by informatics techniques, or by techniques of biochemistry and molecular biology. Many well known methods are available. See, e.g., Mount (2001) Bioinformatics: Sequence and Genome Analysis CSH Press, NY; Baxevanis and Oeullette (eds. 1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins (2d. ed.) Wiley-Liss; Ausubel, et al. (eds. 1999 and supplements) Current Protocols in Molecular Biology Lippincott; and Sambrook, et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Vol. 1-3) CSH Press.  
      Nucleic acid sequences are particularly described. Using linkages to publicly accessible databases, e.g., GenBank accession numbers, sequences are described whose presence or absence in the samples provides prognostic capacity. Correlations are made between the detection of such sequence and the outcomes of the prostate cancers. Thus, detection of physically linked, e.g., adjacent or contiguous, sequence will be equivalent. The correlation between presence of a 5′ segment will be equivalent to such with a 3′ segment of the same physical molecule.  
      The tables also provide protein sequences which correspond to the identified nucleic acid sequences. The amino acid embodiments of the markers will also exhibit similar correlations with outcome. Thus, the use of the protein embodiments can also be used in the invention. Proteins or fragments can be produced, and antibodies generated. See, e.g., Coligan (1991) Current Protocols in Immunology Lippincott; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; and Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press.  
      Kits for use in the prognostic methods are also made available. The kits will include reagents for detecting the markers, e.g., at the nucleic acid or protein level. Thus, for nucleic acid expression level prognosis kits, typically PCR primers or detectable hybridization probes will be included. For protein level prognosis kits, typically antibodies will be used to quantitate or detect the appropriate gene products. Typically instructions will be provided, which may include buffers or instructions for proper disposal of the materials.  
      Diagnostic, Therapeutic Applications  
      After prostate cancer has been identified, tests are performed to find out if cancer cells have spread within the prostate or to other parts of the body. Prostate cancer is typically classified into stages I-IV. The following tests and procedures may be used in the staging process: radionuclide bone scan, pelvic lymphadenectomy, CT scan, and seminal vesicle biopsy.  
      The list of targets may have other diagnostic applications besides outcome prediction. These identified markers may be valuable in such stage subsetting, distinct from outcome subsetting. Typically, after initial diagnosis, tests are performed to determine if cancer cells have spread within the prostate or to other parts of the body. Evaluation of the identified markers, singly or in combinations, may substitute for other tests to assign stage, or add to them for confirmation. Alternatively, the detection of one or more of these markers may be used as early detection screens for prostate cancer. Preferably, if the marker is soluble or released into a readily accessible body fluid, e.g., serum, semen, or urine, a diagnostic test for detection may allow for early detection of prostate cancer.  
      The invention is illustrated further by the following examples that are not to be construed as limiting the invention in scope to the specific procedures described in it.  
     EXAMPLES  
     Example 1  
     Study Design  
      Tissue Collection and Preparation of RNA  
      A cohort of 72 fresh-frozen prostate cancers was collected from patients with localized prostate cancer treated by radical prostatectomy RP at St. Vincent&#39;s Hospital, Sydney. The primary outcome, disease-specific relapse, was measured from the date of RP and was defined as a rise in serum PSA above 0.3 ng/ml with subsequent further rises. Following inking of the external limits of the prostate immediately after removal and prior to formalin-fixation, up to six, 5 mm core biopsies were taken and stored at −80° C. for a later RNA extraction. The proportion of invasive cancer in the biopsy sample was then estimated retrospectively by either frozen sectioning of the biopsy and hematoxylin and eosin staining, or by examination of archival formalin-fixed, paraffin-embedded tissue surrounding the biopsy site. Only those biopsies that contained ≧75% invasive cancer were used for subsequent transcript profiling. Only one biopsy per patient was analyzed.  
      Xenograft Model  
      The androgen-dependent LuCaP-35 (7) prostate cancer xenograft was grown as subcutaneous tumors in nude male mice. To study the androgen-withdrawal process, tumor-bearing mice were castrated and monitored for tumor regression and PSA levels. Tumors were harvested from mice prior to castration, and at various time points (1-100 days) post-castration and were processed for microarray analysis. For data analysis and identification of androgen-regulated genes, the samples were binned in two groups consisting of days 0-2 and days 5-100 post-castration. Genes that showed a significant (P&lt;0.01) difference in the means of each group were identified by a standard Student&#39;s t-Test.  
      RNA Extraction and Microarray Protocols  
      Preparation of total RNA from fresh-frozen prostate and xenograft tissue was performed by extraction with Trizol reagent (Life Technologies Inc., Gaithersburg, Md.) and was reverse transcribed using a primer containing oligo(dT) and a T7 promoter sequence. The resulting cDNAs were then in vitro transcribed in the presence of biotinylated nucleotides (Bio-11-CTP and Bio-16-UTP) using the T7 MEGAscript kit (Ambion, Austin, Tex.).  
      The biotinylated targets were hybridized to the Eos Hu03, a customized Affymetrix GENECHIP® (Affymetrix, Santa Clara, Calif.) oligonucleotide array comprising 59,619 probesets representing 46,000 unique sequences including both known and FGENESH predicted exons that were based on the first draft of the human genome. Hybridization signals were visualised using phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, Oreg.). Normalization of the data was performed as follows. The probe-level intensity data from each array were fitted to a fixed gamma distribution with a mean of 300 and a shape parameter of 0.81. This normalization procedure removes between chip variation attributable to non-biological factors. Then for each probeset, a single measure of average intensity was calculated using Tukey&#39;s trimean of the intensity of the constituent probes (8). Finally, a correction for nonspecific hybridization was applied, in which the average intensity measure of a “null” probeset consisting of probes with scrambled sequence was subtracted from all other probesets on the chip.  
      Statistical Methods  
      Prior to survival analysis, a screen was applied to the expression data to eliminate probesets without meaningful variation. For each probeset, the ratio of the 90 th  percentile to the 15 th  percentile intensity measure was required to be at least 2, and the minimum expression level was required to be at least 150 average intensity units. Separate Cox proportional hazards analyses with pretreatment PSA concentration dichotomised at 20 ng/ml and gene expression modeled as a continuous variable were used to identify gene expression that correlated with PSA recurrence (9). The IQR hazards ratio was computed by multiplying the regression coefficient for each probeset by its own interquartile range prior to exponentiation. The positive false discovery rate (pFDR) was calculated using the method described by Storey and Tibshirani (10). Schoenfeld residuals were used to assess the proportional hazards assumption for the two probesets for trp-p8 and the assumption was found to be upheld in both cases.  
      Variables of clinical relevance were also modeled in univariate analyses for their ability to predict disease-free survival in the 72 prostate cancers using the Cox proportional hazards model. Trp-p8 mRNA expression assessed by ISH, was reported as proportions within histological groups and compared between groups using a Fisher&#39;s Exact test. The expression dataset of 277 selected probesets from 72 samples was reordered according to cluster analysis in both dimensions (probesets and samples). In each analysis, the distance metric was the square root of (1−r), where r is the standard pearson product-moment correlation. The clustering algorithm used was Ward&#39;s minimum variance method (11).  
      In order to evaluate the ability of the 11 genes used by Singh et al., to accurately predict relapse status in aggregate in our dataset, we entered these eleven probesets into a multivariate Cox regression model, and used variable selection methods to choose a subset of predictors. Three different methods were used (forward selection, backward elimination, and stepwise selection), all using P=0.15 as inclusion/exclusion criterion). In each case, the final model using 4 probesets had a significance level of P=0.0029 by the likelihood ratio test.  
      All statistical analyses were performed using SAS (SAS Institute Inc., Cary, N.C.).  
      Tissue Microarray and In Situ Hybridization  
      Tissue microarrays were constructed as described previously (12), and were comprised of prostate cancer samples from 95 patients that are part of a previously published cohort of patients treated for localized prostate cancer by RP alone at St. Vincent&#39;s Hospital, Sydney (13). In addition, 13 prostate cancer specimens were collected from patients treated for localized prostate cancer by RP who had received at least 3 months (range 3-10 months) of preoperative neoadjuvant hormonal treatment (5 with anti-androgens alone, 6 with a combination of a Gn-RH analogue and anti-androgens and 2 with a Gn-RH analogue alone). Trp-p8 expression in these 13 samples was assessed on conventional tissue sections.  
      For ISH, a 424-base pair probe for trp-p8 was derived from the 3′ end of the trp-p8 gene and transcribed to produce a DIG-labeled riboprobe using an RNA DIG-labeling kit (Roche, y™ Mannheim, Germany). ISH was performed on the VENTANA DISCOVERY™ instrument (Ventana Medical Systems, Tucson, Ariz.) using the RIBOMAP™ kit with protease P2 for 2 minutes (Ventana Medical Systems, Tucson, Ariz.) and hybridization for 8 hours at 65° C. Chromogenic detection was achieved with the BLUEMAP™ detection system as described by the manufacturer (Ventana Medical Systems, Tucson, Ariz.).  
     Example 2  
     Expression Profiling of Prostate Cancers  
      In this study, we sought to discover novel biomarkers that might predict for PSA relapse following radical prostatectomy utilizing outcome-based statistical tools to analyze gene expression profiles of 72 prostate cancers. A criteria for selection was the ability to predict recurrence better than preoperative serum PSA concentration alone, since PSA is one of only a handful of markers that provide preoperative prognostic information. The 72 prostate tissues were collected at the time of radical prostatectomy (RP) from patients undergoing treatment for localized prostate cancer at St. Vincent&#39;s Hospital Campus, Sydney, Australia. At last follow-up (median=28.25 months, range 4.9-90.3 months), 17 of the 72 (23.6%) patients had relapsed, of which 14 demonstrated a rise in postoperative PSA levels while 3 patients were diagnosed with a rising PSA and local recurrence of disease. Consistent with published data (5, 6, 13), the significant predictors of prostate cancer relapse in this cohort on univariate analysis were Gleason score (HR=1.88, P=0.027), surgical margins (HR=4.90, P=0.035) and preoperative PSA concentration (HR=4.43, P=0.006) (Table 1). The overall relapse rate of 23.6% and median time to relapse of 14 months in this group of 72 patients was similar to that observed in a cohort of 732 patients treated for localized prostate cancer by RP at the same institution between 1986 and 1999 (13).  
               TABLE 3                          Clinicopathological characteristics of the prostate cancer       cohort (n = 72) that were utilized in the survival analysis.                         Variable   HR (confidence levels)   P               Gleason score a     1.88 (1.08-3.29)   0.027       Preoperative PSA concentration    4.43 (1.53-12.79)   0.006       &lt;20 ng/ml vs. ≧20 ng/ml       Seminal vesicle involvement   2.33 (0.88-6.14)   0.086       positive vs. negative       Surgical margins   4.90 (1.12-21.5)   0.035       positive vs. negative                   a Gleason score was modeled as a continuous variable.             
 
      RNA was extracted from a core biopsy taken at the time of RP for each of the 72 cases that comprised ≧75% cancer tissue. Biotinylated RNA from each sample was then analyzed with a customized GENECHIP® expression array, the Eos Hu03 (14). This single GENECHIP® microarray design is representative of greater than 90% of the expressed human genome based on the first public draft and comprises 59,619 probesets representative of both known and predicted genes (15). An initial screen was applied to the microarray probesets to choose genes expressed with reliable intensity and adequate cross-sample variance. This screen reduced the initial set of 59,619 probesets to a subset of 8,521 probesets for further examination.  
     Example 3  
     Survival Analysis  
      Each probeset&#39;s intensity value was entered as a continuous explanatory variable in a Cox proportional hazards survival analysis predicting relapse. Pretreatment PSA concentration was also entered as a predictor in each analysis. From this analysis, 264 probesets were found to be significant predictors of relapse at P&lt;0.01. To assist interpretation, we next calculated the interquartile range hazard ratio (IQR HR) for each probeset. Because the expression data are treated here as continuous covariates, hazards ratios expressed in their natural scale illustrate only the change in risk of relapse associated with a change of 1 unit on the expression scale, a change too small to be comprehended easily. To put the hazard ratios and associated confidence limits on a more interpretable scale, we present here the hazards ratio associated with a change in expression values equivalent to 1 interquartile range (IQR) of the sample data for each probeset. The IQR is simply the 75th percentile minus the 25th percentile, and thus contains the middle 50 percent of observations.  
      The multiple hypothesis testing problem has been recognized as an important issue to address in microarray research. The large number of tests that are performed simultaneously on thousands of probesets greatly increases the chances of making Type I errors (or false-positive findings). To assess the effect of multiple hypothesis testing, we adapted a method developed by Storey and Tibshirani (2001) for calculating the positive false discovery rate (pFDR), an estimate of the proportion of false-positives present in a set of findings (10). This technique was developed explicitly for use with microarray data, for which the usual assumption of independence among tests is untenable. The procedure can be briefly summarized as follows. First, null data were simulated by randomly permuting the relapse status of subjects and re-performing the survival analyses. In each simulation, the number of relapsers and non-relapsers (17 and 55, respectively) remained constant, but these designations were shuffled and assigned to patients at random. The permutation was performed 500 times, and for each simulation, the number of findings at P&lt;0.01 was noted. The mean number of findings across the 500 permutations was 85.9. This figure, an estimate of the expected number of false positives under null conditions, was then divided by the number of actual findings (n=264) to obtain an estimate of the proportion of false-positive findings. After the application of a correction factor (10), the final estimate for the pFDR was 23%. Thus, we can expect that approximately 61 of the 277 findings are false positives.  
      Identification of the Candidate Marker Genes  
      The 277 probesets (Table 1A-1C) identified by survival analysis included both known genes and hypothetical genes of unknown function, as well as ESTs.  
      Cluster analysis performed in both dimensions on the 72 RP samples and these 277 probesets using the Ward&#39;s minimum variance procedure identified two gene expression subgroups ( FIG. 1 ). Sixteen of the 17 patients known to have experienced a PSA relapse were clustered in one gene expression group characterized by a relative increase in expression of 85 genes (cluster 1) and loss of expression of 179 genes (cluster 2;  FIG. 1 ). An additional 22 patients that were disease-free at the time of censoring were located in this expression cluster, and may suggest that these patients have an increased propensity for relapse in the future. Thirty-two patients who were disease-free at the time of censoring constituted the second expression group which also included one patient who had experienced a PSA relapse.  
      Notably, three of the 277 probesets showing strongest correlation with relapse in our model were identified as the gene for the putative calcium channel protein, trp-p8 (16). For all three probesets, loss of expression of trp-p8 mRNA was associated with a significantly shorter time to PSA relapse free survival with an IQR HR of 0.26 (0.12-0.54; P&lt;0.001), 0.32 (0.16-0.66, P=0.0022) and 0.27 (0.12-0.66, P=0.0045), respectively, when PSA was included in the analysis. Notably, loss of trp-p8 remained a significant predictor of PSA relapse when modeled alone or with Gleason score (data not shown). Subsequent analysis showed that expression of trp-p8 mRNA was primarily restricted to the prostate. Low-level expression was detected in normal liver and no detectable expression was seen in 32 distinct other normal tissues examined by oligonucleotide microarray analysis ( FIG. 2   a ). These data confirm the findings of a recent study that also showed that trp-p8 expression was prostate-specific (16). Analysis of 23 cancer cell lines showed that trp-p8 is only expressed at very low levels in the androgen-dependent prostate cancer cell line LnCaP, but not in the androgen independent prostate cancer cell lines, PC-3 and DU-145, consistent with previous data (16). Since this observation alone is not conclusive evidence that trp-p8 expression is androgen-regulated, we next utilized the androgen-dependent LuCaP-35 prostate cancer xenograft model to assess changes in trp-p8 expression that occur during transition from androgen dependence to androgen independence of prostate cancer (7). Male LuCaP-35 mice were castrated and tumors were harvested at several time points (0-100 days) after castration. High levels of trp-p8 expression were detected on days 0-2 after castration, but not on days 5-100 post castration, and correlated significantly with PSA expression in the same mice (Pearson P=0.080;  FIG. 2 , B and C).  
      To gain further insight into the putative association of trp-p8 with androgen regulation, we examined the levels of trp-p8 expression in the prostate tissue of patients who were treated with androgen deprivation therapy (neoadjuvant hormonal therapy, NHT) prior to RP. In situ hybridization (ISH) for trp-8 mRNA was performed on RP specimens from 13 patients who had received at least 3 months preoperative NHT and the levels compared with tissue from 95 patients treated with RP alone ( FIG. 3 ). These latter patients formed part of a large RP cohort described previously (13). While trp-p8 mRNA was detected in 80 of 95 (84%) prostate cancers from patients treated with RP alone, those patients who underwent NHT prior to RP demonstrated significantly less expression of trp-p8, with only 4 of 13 (31%) samples positive for trp-p8 mRNA (Fisher&#39;s Exact test, P&lt;0.001;  FIG. 3 ).  
      Taken together, these data from cell lines, prostate cancer xenografts and clinical specimens, combined with the original finding that trp-p8 mRNA levels correlated strongly with prostate cancer relapse, strongly support the conclusion that trp-p8 expression is androgen-regulated and may be associated with the transition to androgen-independent disease. A monoclonal antibody to trp-p8 can be produced that will be used to assess protein expression by immunohistochemistry in an independent cohort of formalin-fixed, paraffin-embedded prostate cancer specimens with known prostate cancer outcome (13).  
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      It should be apparent that given the guidance, illustrations and examples provided herein, various alternate embodiments, modifications or manipulations of the present invention would be suggested to a skilled artisan and these are included within the spirit and purview of this application and scope of the expanded claims.