Patent Publication Number: US-2004058325-A1

Title: Gene expression in biological conditions

Description:
TECHNICAL FIELD OF THE INVENTION  
       [0001] The present invention relates to method of determining the presence or absence of a biological condition in animal tissue, wherein the expression of genes in normal tissue and tissue from the biological condition is examined and correlated to standards. The invention further relates to treatment of the biological condition and an assay for determining the condition.  
       BACKGROUND  
       [0002] The building of large databases containing human genome sequences is the basis for studies of gene expressions in various tissues during normal physiological and pathologic conditions. Constantly (constitutively) expressed sequences as well as sequences whose expression is altered during disease processes are important for our understanding of cellular properties, and for the identification of candidate genes for future therapeutic intervention. As the number of known genes and ESTs build up in the databases, array-based simultaneous screening of thousands of genes is necessary to obtain a profile of transcriptional behaviour, and to identify key genes that either alone or in combination with other genes, control various aspects of cellular life. One cellular behaviour that has been a mystery for many years is the malignant behaviour of cancer cells. We now know that for example defects in DNA repair can lead to cancer but the cancer-creating mechanism in heterozygous individuals is still largely unknown as is the malignant cell&#39;s ability to repeat cell cycles to avoid apoptosis to escape the immune system to invade and metastasize and to escape therapy. There are hints and indications in these areas and excellent progress has been made, buth the myriad of genes interacting with each other in a highly complex multidimensional network is making the road to insight long and contorted.  
       [0003] Similar appearing tumors—morphologically, histochemically, microscopically—can be profoundly different. They can hav a different invasive and metastasizing properties, as well as respond differently to therapy. There is thus a need in the art for methods which distinguish tumors and tissues on different bases than are currently in use in the clinic.  
       [0004] The malignant transformation from normal tissue to cancer is believed to be a multistep process, in which tumorsuppressor genes, that normally repress cancer growth show reduced gene expression and in which other genes that encode tumor promoting proteins (oncogenes) show an increased expression level. Several tumor suppressor genes have been identified up till now, as e.g. p16, Rb, p53 (Nesrin Özören and Wafik S. El-Deiry, Introduction to cancer genes and growth control, In: DNA alterations in cancer, genetic and epigenetic changes, Eaton publishing, Melanie Ehrlich (ed) p. 1-43, 2000.; and references therein). They are usually identified by their lack of expression or their mutation in cancer tissue.  
       [0005] Other examinations have shown this downregulation of transcripts to be partly due to loss of genomic material (loss of heterozygosity), partly to methylation of promotorregions, and partly due to unknown factors (Nesrin Özören and Wafik S. El-Deiry, Introduction to cancer genes and growth control, In: DNA alterations in cancer, genetic and epigenetic changes, Eaton publishing, Melanie Ehrlich (ed) p. 1-43, 2000.; and references therein).  
       [0006] Several oncogenes are known, e.g. cyclinD1/PRAD1/BCL1, FGFs, c-MYC, BCL-2 all of which are genes that are amplified in cancer showing an increased level of transcript (Nesrin Özören and Wafik S. El-Deiry, Introduction to cancer genes and growth control, In: DNA alterations in cancer, genetic and epigenetic changes, Eaton publishing, Melanie Ehrlich (ed) p. 1-43, 2000.; and references therein). Many of these genes are related to cell growth and directs the tumor cells to uninhibited growth. Others may be related to tissue degradation as they e.g. encode enzymes that break down the surrounding connective tissue.  
       SUMMARY OF THE INVENTION  
       [0007] In one aspect the present invention relates to a method of determining the presence or absence of a biological condition in animal tissue comprising  
       [0008] collecting a sample comprising cells from the tissue and/or expression products from the cells,  
       [0009] assaying a first expression level of at least one gene from a first gene group, wherein the gene from the first gene group is selected from genes expressed in normal tissue cells in an amount higher than expression in biological condition cells, and/or  
       [0010] assaying a second expression level of at least one gene from a second gene group, wherein the second gene group is selected from genes expressed in a normal tissue cells in an amount lower than expression in biological condition cells,  
       [0011] correlating the first expression level to a standard expression level for normal tissue, and/or the second expression level to a standard expression level for biological condition cells to determine the presence or absence of a biological condition in the animal tissue.  
       [0012] Animal tissue may be tissue from any animal, preferably from a mammal, such as a horse, a cow, a dog, a cat, and more preferably the tissue is human tissue. The biological condition may be any condition exhibiting gene expression different from normal tissue. In particular the biological condition relates to a malignant or premalignant condition, such as a tumor or cancer.  
       [0013] Furthermore, the invention relates to a method of determining the stage of a biological condition in animal tissue,  
       [0014] comprising collecting a sample comprising cells from the tissue,  
       [0015] assaying the expression of at least a first stage gene from a first stage gene group and at least a second stage gene from a second stage gene group, wherein at least one of said genes is expressed in said first stage of the condition in a higher amount than in said second stage, and the other gene is a expressed in said first stage of the condition in a lower amount than in said second stage of the condition,  
       [0016] correlating the expression level of the at least two genes to a standard level of expression determining the stage of the condition.  
       [0017] Thereby, it is possible to determine the biological condition in more details, such as determination of a stage and/or a grade of a tumor.  
       [0018] The methods above may be used for determining single gene expressions, however the invention also relates to a method of determining an expression pattern of a colon cell sample, comprising:  
       [0019] collecting sample comprising colon and/or rectum cells and/or expression products from colon and/or rectum cells,  
       [0020] determining the expression level of two or more genes in the sample, wherein at least one gene belongs to a first group of genes, said gene from the first gene group being expressed in a higher amount in normal tissue than in biological condition cells, and wherein at least one other gene belongs to a second group of genes, said gene from the second gene group being expressed in a lower amount in normal tissue than in biological condition cells, and the difference between the expression level of the first gene group in normal cells and biological condition cells being at least two-fold, obtaining an expression pattern of the colon and/or rectum cell sample.  
       [0021] Gene expression patterns may rely on one or a few genes, but more preferred gene expression patterns relies on expression from multiple genes, whereby a combined information from several genes is obtained.  
       [0022] Further, the invention relates to a method of determining an expression pattern of a colon cell sample independent of the proportion of submucosal, muscle, or connective tissue cells present, comprising:  
       [0023] determining the expression of one or more genes in a sample comprising cells, wherein the one or more genes exclude genes which are expressed in the submucosal, muscle, or connective tissue, whereby a pattern of expression is formed for the sample which is independent of the proportion of submucosal, muscle, or connective tissue cells in the sample.  
       [0024] The expression pattern may be used in a method according to this information, and accordingly, the invention also relates to a method of determining the presence or absence of a biological condition in human colon and/or rectum tissue comprising,  
       [0025] collecting a sample comprising cells from the tissue,  
       [0026] determining an expression pattern of the cells as defined above,  
       [0027] correlating the determined expression pattern to a standard pattern,  
       [0028] determining the presence or absence of the biological condition is said tissue.  
       [0029] as well as a method for determining the stage of a biological condition in animal tissue, comprising  
       [0030] collecting a sample comprising cells from the tissue,  
       [0031] determining an expression pattern of the cells as defined above,  
       [0032] correlating the determined expression pattern to a standard pattern,  
       [0033] determining the stage of the biological condition is said tissue.  
       [0034] The invention further relates to a method for reducing cell tumorigenicity of a cell, said method comprising  
       [0035] contacting a tumor cell with at least one peptide expressed by at least one gene selected from genes being expressed in an amount two-fold higher in normal cells than the amount expressed in said tumor cell, or  
       [0036] comprising  
       [0037] obtaining at least one gene selected from genes being expressed in an amount twofold higher in normal cells than the amount expressed in said tumor cell,  
       [0038] introducing said at least one gene into the tumor cell in a manner allowing expression of said gene(s), or  
       [0039] obtaining at least one nucleotide probe capable of hybridising with at least one gene of a tumor cell, said at least one gene being selected from genes being expressed in an amount one-fold lower in normal cells than the amount expressed in said tumor cell, and  
       [0040] introducing said at least one nucleotide probe into the tumor cell in a manner allowing the probe to hybridise to the at least one gene, thereby inhibiting expression of said at least one gene.  
       [0041] In a further aspect the invention relates to a method for producing antibodies against an expression product of a cell from a biological tissue, said method comprising the steps of  
       [0042] obtaining expression product(s) from at least one gene said gene being expressed as defined above,  
       [0043] immunising a mammal with said expression product(s) obtaining antibodies against the expression product.  
       [0044] The antibodies produced may be used for producing a pharmaceutical composition. Further, the invention relates to a vaccine capable of eliciting an immune response against at least one expression product from at least one gene said gene being expressed as defined above.  
       [0045] The invention furthermore relates to the use of any of the methods discussed above for producing an assay for diagnosing a biological condition in animal tissue.  
       [0046] Also, the invention relates to the use of a peptide as defined above as an expression product and/or the use of a gene as defined above and/or th use of a probe as defined above for preparation of a pharmaceutical composition for the treatment of a biological condition in animal tissue.  
       [0047] In a yet further aspect the invention relates to an assay for determining the presence or absence of a biological condition in animal tissue, comprising  
       [0048] at least one first marker capable of detecting a first expression level of at least one gene from a first gene group, wherein the gene from the first gene group is selected from genes expressed in normal tissue cells in an amount higher than expression in biological condition cells,  
       [0049] at least one second marker capable of detecting a second expression level of at least one gene from a second gene group, wherein the second gene group is selected from genes expressed in normal tissue cells in an amount lower than expression in biological condition cells.  
       [0050] In another aspect the invention relates to an assay for determining an expression pattern of a colon and/or rectum cell, comprising at least a first marker and a second marker, wherein the first marker is capable of detecting a gene from a first gene group as defined above, and the second marker is capable of detecting a gene from a second gene group as defined above.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0051] Samples  
       [0052] The samples according to the present invention may be any tissue sample, it is however often preferred to conduct the methods according to the invention on epithelial tissue, such as epithelial tissue from the gastro-intestinal tract, in particular form colon and/or rectum. In particular the epithelial tissue may be mucosa.  
       [0053] The sample may be obtained by any suitable manner known to the man skilled in the art, such as a biopsy of the tissue, or a superficial sample scraped from the tissue. The sample may be prepared by forming a cell suspension made from the tissue, or by obtaining an extract from the tissue.  
       [0054] In one embodiment it is preferred that the sample comprises substantially only cells from said tissue, such as substantially only cells from mucosa of the colon-rectum.  
       [0055] Biological Condition  
       [0056] The methods according to the invention may be used for determining any biological condition, wherein said condition leads to a change in the expression of at least one gene, and preferably a change in a variety of genes.  
       [0057] Thus, the biological condition may be any malignant or premalignant condition, in particular in colon/rectum, such as an adenocarcinoma, a carcinoma, a teratoma, a sarcoma, and/or a lymphoma.  
       [0058] In relation to the gastrointestinal tract, the biological condition may also be colitis ulcerosa, Mb. Crohn, diverticulitis, adenomas.  
       [0059] Single Gene Expression Contra Expression Pattern  
       [0060] The expression level may be determined as single gene approaches, i.e. wherein the determination of expression from one or two or a few genes is conducted. It is preferred that expression from at least one gene from a first (normal) group is determined, said first gene group representing genes being expressed at a higher level in normal tissue, i.e. so-called suppressors, in combination with determination of expression of at least one gene from a second group, said second group representing genes being expressed at a higher level in tissue from the biological condition than in normal tissue, ie. so-called oncogenes. However, determination of the expression of a single gene whether belonging to the first group or second group is within the scope of the present invention. In this case it is preferred that the single gene is selected among genes having a very high change in expression level from normal cells to biological condition cells.  
       [0061] Another approach is determination of an expression pattern from a variety of genes, wherein the determination of the biological condition in the tissue relies on information from a variety of gene expression, i.e. rather on the combination of expressed genes than on the information from single genes.  
       [0062] Colorectal Tumors  
       [0063] The following data presented herein relates to colorectal tumors, and therefore the description has focused on the gene expression level as one way of identifying genes that lose function in cancer tissue. Genes showing a remarkable downregulation (or complete loss) of the expression level—measured as the mRNA transcript, during the malignant progression in colon from normal mucosa through Dukes A superficial tumors to Dukes B, slightly invasive tumors, to Dukes C that have spread to lymphnodes and finally to Dukes D that have metastasized to other organs, has been examined, as well as genes gaining importance during the differentiation towards malignancy.  
       [0064] Gene Groups  
       [0065] The present invention relates to a variety of genes identified either by an EST identification number and/or by a gene identification number. Both type of identification numbers relates to identification numbers of UniGene database, NCBI, build 18.  
       [0066] The various genes have been identified using Affymetrix arrays of the following product numbers:  
       [0067] Human Gene FL array 900 183  
       [0068] HU35K SubA 900 184  
       [0069] HU35K SubB 900 185  
       [0070] HU35K SubC 900 186  
       [0071] HU35K SubD 900 187  
       [0072] First Gene Group  
       [0073] Th first gene group relates to genes being expressed in normal tissue cells in an amount higher than expression in biological condition cells. The term “normal tissue cells” relates to c lls from the same type of tissue that is examined with respect to the biological condition in question. Thus, with respect to colorectal tumors, the normal tissue relates to colorectal tissue, in particular to colorectal mucosa.  
       [0074] The first gene group therefore relates to genes being downregulated in tumors, such genes being expected to serve as tumor suppressor genes, and they are of importance as predictive markers for the disease as loss of one or more of these may signal a poor outcome or an aggressive disease course. Furthermore, they may be important targets for therapy as restoring their expression level, e.g. by gene therapy, may suppress the malignant growth.  
       [0075] For a colorectal tissue sample a gene from the first gene group is preferably selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_H04768_at   chrom 15 no homology       RC_Z39652_at   Y14593 APM-1 gene adipocyte-specific se-           cretory protein; chrom 1q21.3-q23       RC_H30270_at   chrom 18 PAAAA in colon &amp; bladder no           homology       RC_T47089_s_at   tenascin-X; tenascin-X precursor; unidenti-           fied protein       RC_W31906_at   secretagogin; dJ501N12.8 (putative protein)           chrom 6       RC_AA279803_at   chrom 2 no homology       RC_R01646_at   chrom 13q32.1-33.3; AL159152; homolo-           gy to mouse Pcbp1 - poly(rC)-binding           protein 1       RC_AA099820_at   BAC clone AC016778       AA319615_at   secretory carrier membrane protein; secre-           tory carrier membrane protein 2; chrom 15       H07011_at   tetraspan NET-6 mRNA; transmembrane           4 superfamily; chrom 7       RC_T68873_f_at       RC_T40995_f_at       RC_H81070_f_at       RC_N30796_at       RC_W37778_f_at       RC_R70212_s_at       RC_AA426330_at       RC_N33927_s_at       RC_T90190_s_at       RC_AA447145_at       RC_H75860_at       RC_T71132_s_at                  
 
       [0076] and from genes comprising a sequence as identified below  
                                      “Human chromogranin A” “mRNA,” “complete cds”   J03915       Human adipsin/complement factor D “mRNA,” comple-   M84526       te cds         Homo sapiens  MLC-1V/Sb isoform gene   M24248       Human aminopeptidase N/CD13 mRNA encoding   M22324       aminopeptidase “N,” complete cds         H. sapiens  MT-1I mRNA   X76717         H. sapiens  GCAP-II gene   Z70295       Human somatostatin I gene and flanks   J00306       Human YMP “mRNA,” complete cds   U52101         H. sapiens  mRNA for beta subunit of epithelial amiloride-   X87159       sensitive sodium channel       Human K12 protein precursor “mRNA,” complete cds   U77643       Human sulfate transporter (DTD) “mRNA,” complete cds   U14528       Human transcription factor hGATA-6 “mRNA,” complete   U66075       cds.         H. sapiens  SCAD “gene,” exon 1 and joining features   Z80345       Human S-lac lectin L-14-II (LGALS2) gene   M87860       Human mRNA for protein tyrosine phosphatase   D15049         H. sapiens  mRNA for tetranectin   X64559       Human 11 kd protein “mRNA,” complete cds   U28249       Human anti-mullerian hormone type II receptor precursor   U29700       “gene,” complete cds       Human heparin binding protein (HBp17) “mRNA,” complete   M60047       cds       Human ADP-ribosylation factor (hARF6) “mRNA,” complete   M57763       cds       beta-ADD = adducin beta subunit 63 kda isoform/membrane   S81083       skeleton protein, beta-ADD = adducin beta subunit 63 kda       isoform/membrane skeleton protein {alternatively spliced,       exon 10 to 13 region} [human, Genomic, 1851 nt, segment       3 of 3].       Zinc Finger Protein Znf155   HG4243-           HT4513       Human glucagon “mRNA,” complete cds   J04040         H. sapiens  mRNA for hair “keratin,” hHb5   X99140       Human tubulin-folding cofactor E “mRNA,” complete cds   U61232       Human integrin alpha-3 chain “mRNA,” complete cds   M59911       Human NACP gene   U46901         H. sapiens  mRNA for flavin-containing monooxygenase 5   Z47553       (FMO5)       Human mRNA for ATF-a transcription factor   X52943         H. sapiens  intestinal VIP receptor related protein mRNA   X77777                  
 
       [0077] and and from genes comprising a sequence as id ntified below  
                                        Homo sapiens  chromosome 16 BAC clone CIT987SK-   AF001548       815A9 complete sequence.       Human mRNA for ATP synthase alpha “subunit,” complete   D14710       cds       Human mRNA for IgG Fc binding “protein,” complete   D84239       cds         H. sapiens  mRNA for carcinoembryonic “antigen,”   X98311       CGM2       “ Homo sapiens  (clone lamda-hPEC-3) phosphoenolpy-   L05144       ruvate carboxykinase (PCK1)” “mRNA,” “complete       cds”       Human 11-beta-hydroxysteroid dehydrogenase type 2   U26726       “mRNA,” complete cds       “Human intestinal mucin (MUC2)” “mRNA,” “complete cds”   L21998       Human mRNA for KIAA0106 “gene,” complete cds   D14662       metallothionein   V00594       Human mRNA for IgG Fc binding “protein,” complete   D84239       cds         H. sapiens  mRNA for carcinoembryonic “antigen,”   X98311       CGM2       “ Homo sapiens  (clone lamda-hPEC-3) phosphoenolpy-   L05144       ruvate carboxykinase (PCK1)” “mRNA,” “complete       cds”       metallothionein   V00594                  
 
       [0078] In a preferred embodiment a gene from the first gene group is preferably selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_H04768_at   chrom 15 no homology       RC_Z39652_at   Y14593 APM-1 gene adipocyte-specific se-           cretory protein; chrom 1q21.3-q23       RC_H30270_at   chrom 18 PAAAA in colon &amp; bladder no           homology       RC_AA279803_at   chrom 2 no homology       RC_R01646_at   chrom 13q32.1-33.3; AL159152; homolo-           gy to mouse Pcbp1 - poly(rC)-binding           protein 1       RC_AA099820_at   BAC clone AC016778                  
 
       [0079] and from genes comprising a sequence as identified below  
                                                      “Human chromogranin A” “mRNA,” “complete cds”   J03915           Human adipsin/complement factor D “mRNA,” com-   M84526           plete cds             Homo sapi ns  MLC-1V/Sb isoform gene   M24248           Human aminopeptidase N/CD13 mRNA ncoding   M22324           aminopeptidase “N,” complete cds             H. sapiens  MT-1I mRNA   X76717             H. sapiens  GCAP-II gene   Z70295           Human somatostatin I gene and flanks   J00306                      
 
       [0080] or selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_H04768_at   chrom 15 no homology       RC_Z39652_at   Y14593 APM-1 gene adipocyte-specific se-           cretory protein; chrom 1q21.3-q23       RC_H30270_at   chrom 18 PAAAA in colon &amp; bladder no           homology       RC_T47089_s_at   tenascin-X; tenascin-X precursor; unidenti-           fied protein       RC_W31906_at   secretagogin; dJ501N12.8 (putative protein)           chrom 6       RC_AA279803_at   chrom 2 no homology       RC_R01646_at   chrom 13q32.1-33.3; AL159152; homolo-           gy to mouse Pcbp1 - poly(rC)-binding           protein 1       RC_AA099820_at   BAC clone AC016778       AA319615_at   secretory carrier membrane protein; secre-           tory carrier membrane protein 2; chrom 15       H07011_at   tetraspan NET-6 mRNA; transmembrane           4 superfamily; chrom 7                  
 
       [0081] In a more preferred embodiment a gene from the first gene group is selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_H04768_at   chrom 15 no homology       RC_Z39652_at   Y14593 APM-1 gene adipocyte-specific se-           cretory protein; chrom 1q21.3-q23       RC_H30270_at   chrom 18 PAAAA in colon &amp; bladder no           homology       RC_T47089_s_at   tenascin-X; tenascin-X precursor; unidenti-           fied protein       RC_W31906_at   secretagogin; dJ501N12.8 (putative protein)           chrom 6       RC_AA279803_at   chrom 2 no homology       RC_R01646_at   chrom 13q32.1-33.3; AL159152; homolo-           gy to mouse Pcbp1 - poly(rC)-binding           protein 1       AA319615_at   secretory carrier membrane protein; secre-           tory carrier membrane protein 2; chrom 15                  
 
       [0082] In a most preferred embodiment a gene from the first gene group is selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_T47089_s_at   tenascin-X; tenascin-X precursor; unidenti-           fied protein       RC_W31906_at   secretagogin; dJ501N12.8 (putative protein)           chrom 6       RC_AA279803_at   chrom 2 no homology       AA319615_at   secretory carrier membrane protein; secre-           tory carrier membrane protein 2; chrom 15                  
 
       [0083] Second Gene Group  
       [0084] We have determined genes that are up-regulated (or gained de novo) during the malignant progression of colorectal cancer from normal tissue through Dukes A,B,C and to Dukes D. These genes are potential oncogenes and may be those genes that create or enhance the malignant growth of the cells. The expression level of these genes may serve as predictive markers for the disease course, as a high level may signal an aggressive disease course, and they may serve as targets for therapy, as blocking these genes by e.g. anti-sense therapy, or by biochemical means could inhibit, or slow, the tumor growth. Such up-regulated (or gained de novo) genes, oncogenes, may be classified according to the present invention as genes belonging to second genes group.  
       [0085] With respect to colorectal tumors genes belonging to the second gene group are preferably selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_AA609013_s_at   microsomal dipeptidase (also on 6.8 k);           chrom 16       RC_AA232508_at   CGI-89 protein; unnamed protein product;           hypothetical protein       RC_AA428964_at   serine protease-like protease; serine pro-           tease homolog = NES1; normal epithelial cell-           specific 1       RC_T52813_s_at   dJ28O10.2 (G0S2 (PUTATIVE LYMPHO-           CYTE G0/G1 SWITCH PROTEIN 2; chrom           1       RC_AA075642_at   gp-340 variant protein; DMBT1/8kb.2 protein       RC_AA007218_at   chrom 13 no homology       RC_N33920_at   ubiquitin-like protein FAT10; diubiquitin;           dJ271M21.6 (Diubiquitin); chrom 6       RC_N71781_at   KIAA1199 protein, chrom 15       RC_R67275_s_at   alpha-1 (type XI) collagen precursor; colla-           gen, type XI, alpha 1; collagen type XI al-           pha-1 isoform A; chrom 1       RC_W80763_at   hypothetical protein; chrom 17       RC_AA443793_at   chrom 7p22 AC006028 BAC clone       RC_AA034499_s_at   ZNF198 protein; zinc finger protein; FIM           protein; Cys-rich protein; zinc finger protein           198; chrom 13       RC_AA035482_at   chrom 5; AK022505 clone; CalcineurinB           (weakly similar)       RC_AA024482_at   hypothetical protein; unnamed protein pro-           duct; chrom 17       RC_H93021_at   chrom 2; XM_004890 peptidylprolyl isomer-           ase A (cyclophilin A)       RC_AA427737_at   no homology       RC_AA417078_at   chrom 7q31; AF017104 clone       M29873_s_at   cytochrome P450-IIB (hIIB3); 19q13.1-           q13.2       RC_H27498_f_at       RC_T92363_s_at       RC_N89910_at       RC_W60516_at       RC_AA219699_at       RC_AA449450_at                  
 
       [0086] and from genes comprising a sequence as identified below  
                                        Homo sapiens  (clones “MDP4,” MDP7) microsomal   J05257       dipeptidase (MDP) “mRNA,” complete cds       “ Homo sapiens  reg gene” “homologue,” “complete   L08010       cds”         H. sapiens  mRNA for prepro-alpha2(I) collagen   Z74616       “Human S-adenosylhomocysteine hydrolase (AHCY)”   M61832       “mRNA,” “complete cds”       Transcription Factor Iiia   HG4312-           HT4582       Human gene for melanoma growth stimulatory activity   X54489       (MGSA)       Human strom lysin-3 mRNA   X57766       CDC25Hu2 = cdc25 + homolog “[human,” “mRNA,” 3118   S78187       nt]       Human mRNA for cripto protein   X14253       Human transformation-sensitive protein (IEF SSP 3521)   M86752       “mRNA,” complet cds       Human complement component 2 (C2) gene allele b   L09708         H. sapiens  mRNA for ITBA2 protein   X92896         H. sapiens  encoding CLA-1 mRNA   Z22555       “Human fibroblast growth factor receptor 4 (FGFR4)”   L03840       “mRNA,” “complete cds“       “”“Fibronectin,” “Alt. Splice 1”   HG3044-           HT3742       tyk2   X54667       Human mRNA for B-myb gene   X13293       “Human phosphofructokinase (PFKM)” “mRNA,” “complete   U24183       cds”       Human pre-B cell enhancing factor (PBEF) “mRNA,” com-   U02020       plete cds       Human SH2-containing inositol 5-phosphatase (hSHIP)   U57650       “mRNA,” complete cds       Human interleukin 8 (IL8) “gene,” complete cds   M28130       “Human lamin B receptor (LBR)” “mRNA,” “complete cds”   L25931         H. sapiens  mRNA for protein tyrosine phosphatase   Z48541       Human mRNA for unc-18 “homologue,” complete cds   D63851         H. sapiens  mRNA for Zn-alpha2-glycoprotein   X59766           Z25521       “Human asparagine synthetase” “mRNA,” “complete cds”   M27396       Human hepatitis delta antigen interacting protein A (dipA)   U63825       “mRNA,” complete cds       Human splicesomal protein (SAP 61) “mRNA,” complete   U08815       cds       Human protein kinase C-binding protein RACK7 “mRNA,”   U48251       partial cds       Human MAC30 “mRNA,” 3′ end   L19183       Human thrombospondin 2 (THBS2) “mRNA,” complete cds   L12350       “Human nicotinamide N-methyltransferase (NNMT)”   U08021       “mRNA,” “complete cds”         H. sapiens  mRNA for type I interstitial collagenase   X54925       Human cytochrome b561 gene   U29463       Human H19 RNA “gene,” complete cds (spliced in sili-   M32053       co)       Human collagen type XVIII alpha 1 (COL18A1) “mRNA,”   L22548       partial cds       Human clone 23733 “mRNA,” complete cds.   U79274       Human transforming growth factor-beta induced gene pro-   M77349       duct (BIGH3) “mRNA,” complete cds       “Human breast epithelial antigen BA46” “mRNA,” “com-   U58516       plete cds”           X57351         H. sapiens  NGAL gene   X99133       Human mRNA for MDNCF (monocyte-derived neutro-   Y00787       phil chemotactic factor)         H. sapiens  EF-1 delta gene encoding human elongation   Z21507       factor-1-delta         H. sapiens  mRNA for prepro-alpha1(I) collagen   Z74615       Nuclear Factor Nf-II6   HG3494-           HT3688           U29175       “HNL = neutrophil lipocalin” “[human,” “ovarian   S75256       cancer cell line” “OC6,” “mRNA” “Partial,” “534       nt]. /qb = S75256 /ntype = RNA”                  
 
       [0087] In a preferred embodiment the genes belonging to the second gene group are preferably selected individually from genes comprising a sequence as identified below by EST  
                                   UniGene number   Homologous to                  RC_AA007218_at   chrom 13 no homology       RC_AA443793_at   chrom 7p22 AC006028 BAC clone       RC_AA035482_at   chrom 5; AK022505 clone; CalcineurinB           (weakly similar)       RC_H93021_at   chrom 2; XM_004890 peptidylprolyl isomer-           ase A (cyclophilin A)       RC_AA427737_at   no homology       RC_AA417078_at   chrom 7q31; AF017104 clone                  
 
       [0088] and from genes comprising a sequence as identified below  
       [0089] In another preferred embodiment genes from the second gene group are selected individually from genes comprising a sequence as identified below  
                                   UniGene number   Homologous to                  RC_AA609013_s_at   microsomal dipeptidase (also on 6.8 k);           chrom 16       RC_AA232508_at   CGI-89 protein; unnamed protein product;           hypothetical protein       RC_AA428964_at   serine protease-like protease; serine pro-           tease homolog = NES1; normal epithelial cell-           specific 1       RC_T52813_s_at   dJ28O10.2 (G0S2 (PUTATIVE LYMPHO-           CYTE G0/G1 SWITCH PROTEIN 2; chrom           1       RC_AA075642_at   gp-340 variant protein; DMBT1/8kb.2 protein       RC_AA007218_at   chrom 13 no homology       RC_N33920_at   ubiquitin-like protein FAT10; diubiquitin;           dJ271M21.6 (Diubiquitin); chrom 6       RC_N71781_at   KIAA1199 protein, chrom 15       RC_R67275_s_at   alpha-1 (type XI) collagen precursor; colla-           gen, type XI, alpha 1; collagen type XI al-           pha-1 isoform A; chrom 1       RC_W80763_at   hypothetical protein; chrom 17       RC_AA443793_at   chrom 7p22 AC006028 BAC clone       RC_AA034499_s_at   ZNF198 protein; zinc finger protein; FIM           protein; Cys-rich protein; zinc finger protein           198; chrom 13       RC_AA035482_at   chrom 5; AK022505 clone; CalcineurinB           (weakly similar)       RC_AA024482_at   hypothetical protein; unnamed protein pro-           duct; chrom 17       RC_H93021_at   chrom 2; XM_004890 peptidylprolyl isomer-           ase A (cyclophilin A)       RC_AA427737_at   no homology       RC_AA417078_at   chrom 7q31; AF017104 clone       M29873_s_at   Cytochrome P450-IIB (hIIB3); 19q13.1-           q13.2                  
 
       [0090] In a more preferred embodiment genes from the second gene group are selected individually from genes comprising a sequence as identified below  
                                   UniGene number   Homologous to                  RC_AA609013_s_at   microsomal dipeptidase (also on 6.8 k);           chrom 16       RC_AA232508_at   CGI-89 protein; unnamed protein product;           hypothetical protein       RC_AA428964_at   serine protease-like protease; serine pro-           tease homolog = NES1; normal epithelial cell-           specific 1       RC_AA075642_at   gp-340 variant protein; DMBT1/8kb.2 protein       RC_AA007218_at   chrom 13 no homology       RC_N33920_at   ubiquitin-like protein FAT10; diubiquitin;           dJ271M21.6 (Diubiquitin); chrom 6       RC_N71781_at   KIAA1199 protein, chrom 15       RC_R67275_s_at   alpha-1 (type XI) collagen precursor; colla-           gen, type XI, alpha 1; collagen type XI al-           pha-1 isoform A; chrom 1       RC_W80763_at   hypothetical protein; chrom 17       RC_AA034499_s_at   ZNF198 protein; zinc finger protein; FIM           protein; Cys-rich protein; zinc finger protein           198; chrom 13       RC_AA035482_at   chrom 5; AK022505 clone; CalcineurinB           (weakly similar)       RC_AA024482_at   hypothetical protein; unnamed protein pro-           duct; chrom 17       RC_H93021_at   chrom 2; XM_004890 peptidylprolyl isomer-           ase A (cyclophilin A)       RC_AA427737_at   no homology       RC_AA417078_at   chrom 7q31; AF017104 clone       M29873_s_at   cytochrome P450-IIB (hIIB3); 19q13.1-           q13.2                  
 
       [0091] In an even more preferred embodiment genes from the second gene group are selected individually from genes comprising a sequence as identified below  
                                   UniGene number   Homologous to                  RC_AA609013_s_at   microsomal dipeptidase (also on 6.8 k);           chrom 16       RC_AA007218_at   chrom 13 no homology       RC_AA035482_at   chrom 5; AK022505 clone; CalcineurinB           (weakly similar)       RC_H93021_at   chrom 2; XM_004890 peptidylprolyl isomer-           ase A (cyclophilin A)       RC_AA427737_at   no homology       RC_AA417078_at   chrom 7q31; AF017104 clone                  
 
       [0092] such as a sequence as identified below  
                                                   UniGene number   Homologous to                          RC_W80763_at   hypothetical protein; chrom 17                      
 
       [0093] The genes from the second gene group discussed above are preferably genes being expressed in all stages of the biological condition, such as all Dukes stages of a colorectal tumor, to be used for determining the biological condition.  
       [0094] Number of Genes  
       [0095] As discussed above, it is possible to use a single gene approach determining the expression of one of the genes only, in order to determine th biological condition of the tissue. This is particularly relevant for genes mentioned in the tables in Experiments, since these genes have been determined as having a strong indicativity per gene. It is however preferred that expression from at least one gene from the first group as well as expression from one gene from the second group is determined to obtain a more statistically significant result, that is more independent of the expression level of the individual gene. In a preferred embodiment expression from more genes from both groups are determined, such as determination of expression from at least two genes from either of the gene groups, such as determination of expression from at least three genes from either of the gene groups, such as determination of expression from at least four genes from either of the gene groups, such as determination of expression from at least five genes from either of the gene groups, such as determination of expression from at least six genes from either of the gene groups, such as determination of expression from at least seven genes from either of the gene groups.  
       [0096] A pattern of characteristic expression of one gene can be useful in characterizing a cell type source or a stage of disease. However, more genes may be usefully analyzed. Useful patterns include expression of at least one, two, three, five, ten, fifteen, twenty, twenty-five, fifty, seventy-five, one hundred or several hundred informative genes.  
       [0097] Expression Level  
       [0098] Using the results provided in the accompanying figures and tables, a gene is indicated as being expressed if an intensity value of greater than or equal to 20 is shown. Conversely, an intensity value of less than 20 indicates that the gene is not expressed above background levels. Comparison of an expression pattern to another may score a change from expressed to non-expressed, or the reverse. Alternatively, changes in intensity of expression may be scored, either increases or decreases. Any statistically significant change can be used. Typically changes which are greater than 2-fold are suitable. Changes which are greater than 5-fold are highly significant.  
       [0099] The present invention in particular relates to methods using genes wherein the ratio of the expression level in normal tissue to biological condition tissue for suppressor genes or vice versa of the expression level in biological condition tissue to normal tissue for condition genes is as high as possible, such as at least two-fold change in expression, such as at least three-fold, such as at least four fold, such as at least five fold, such as at least six fold, such as at least ten fold, such as at least fifteen fold, such as at least twenty fold.  
       [0100] Stages and Grades  
       [0101] Stage of a colorectal tumor indicates how deep the tumor has penetrated. Superficial tumors are termed Dukes A and Dukes B and Dukes C are used to describe increasing degrees of penetration into the muscle. The grade of a colorectal tumor is expressed on a scale of I-IV (1-4). The grade reflects the cytological appearance of the cells. Grade I cells are almost normal. Grade II cells are slightly deviant. Grade III cells are clearly abnormal. And Grade IV cells are highly abnormal.  
       [0102] It is important to classify the stage of a cancer disease, as superficial tumors may require a less intensive treatment than invasive tumors. We have therefore used the expression level of genes to identify genes whose expression can be used to identify a certain stage of the disease. We have divided these “Classifiers” into those which can be used to identify Dukes A, B, C, and D stages. We expect that measuring the transcript level of one or more of these genes will lead to a classifier that can add supplementary information to the information obtained from the pathological Dukes classification. For example we believe that gene expression levels that signify a Dukes C will be unfavourable to detect in a Dukes A tumor, as they may signal that the Dukes A tumor has the potential to become a Dukes C tumor. The opposite is probably also true, that an expression level that signify Dukes A will be favorable to have in a Dukes C tumor. In that way independent information may be obtained from Dukes pathological classification and a classification based on gene expression levels is made.  
       [0103] Thus, in one embodiment the invention relates to a method as described above further comprising the steps of determining the stage of a biological condition in the animal tissue, comprising assaying a third expression level of at least one gene from a third gene group, wherein a gene from said second gene group, in one stage, is expressed differently from a gene from said third gene group.  
       [0104] In another aspect the invention relates to method of determining the stage of a biological condition in animal tissue,  
       [0105] comprising collecting a sample comprising cells from the tissue,  
       [0106] assaying the expression of at least a first stage gene from a first stage gene group and/or at least a second stage gene from a second stage gene group, wherein at least one of said genes is expressed in said first stage of the condition in a higher amount than in said second stage, and the other gene is a expressed in said first stage of the condition in a lower amount than in said second stage of the condition,  
       [0107] correlating the expression level of the assessed genes to a standard level of expression determining the stage of the condition.  
       [0108] The method of determining the stage of a tumor may be combined with determination of the biological condition or may be an independent method as such. The difference in expression level of a gene from one stage to the expression level of the gene in another group is preferably at least two-fold, such as at least three-fold.  
       [0109] Thus, the invention relates to a method of determining the stage of a colorectal tumor, wherein the stage is selected from colon cancer stages Dukes A, Dukes B, Dukes C, and Dukes D, comprising assaying at least the expression of Dukes A stage gene from a Dukes A stage gene group, at least one Dukes B stage gene from a Dukes B stage gene group, at least the expression of Dukes C stage gene from a Dukes C stage gene group, and/or at least one Dukes D stage gene from a Dukes D stage gene group, wherein at least one gene from each gene group is expressed in a significantly different amount in that stage than in one of the other stages.  
       [0110] The genes selected may be a gene from each gene group being expressed in a significantly higher amount in that stage than in one of the other stages, such as:  
       [0111] a Dukes A stage gene selected individually from any gene comprising a sequence as identified below as EST  
                                                      RC_AA599199_at   ALU seq.           RC_R12694_at   unnamed protein product               BAA91641, chrom 10           RC_H91325_s_at   aldolase B; aldolase B (aa 1-               364); chrom 9           RC_N51709_at   chrom X           RC_N72610_at   —           RC_N69263_at   chrom 10; AK026414 clone               (only 108 nt hom)           RC_T15817_f_at   iNOS, inducible nitric oxide               synthase                      
 
       [0112]                                                      RC_F03077_f   chromosome 17, clone               hRPC.15           RC_AA599199   Alu seq           RC_AA207015   clone RP4-733M16 on chromo-               some 1p36.11-36.23           RC_AA234916   chromosome 19 clone CTC-               461 H2           RC_N92239_a   Wnt inhibitory factor-1 (WIF-1),               chromosome 12           RC_N93958_s   phospholipase A2, group X               (PLA2G10),           U95301_at   phospholipase A2, group X               (PLA2G10),           RC_AA426330   chromosome 17, clone               hRPC.1110_E_20           RC_AA024658   clone SCb-254N2               (UWGC:rg254N02) from 6p21           RC_H88540_a   heat shock protein 90, 1q21.2-               q22                        
       [0113] or any gene comprising a sequence as identified below  
                                      D87444_at   Human mRNA for KIAA0255 “gene,” complete cds       U18291_at   Human CDC16Hs “mRNA,” complete cds       L76568_xpt3_f_at   S26 from  Homo sapiens  excision and cross link repair protein           (ERCC4) “gene,” complete genomic sequence. /gb = L76568           /ntype = DNA /annot = exon       U45328_s_at   “Human ubiquitin-conjugating enzyme (UBE2I)” “mRNA,” “complete           cds”       Z14982_rna1_at     H. sapiens  gene for major histocompatibility complex encoded protea-           some subunit LMP7.       AD000092_cds7_s   RAD23A gene (human RAD23A homolog) extracted from  Homo         _at     sapiens  DNA from chromosome 19p13.2 cosmids “R31240,” R30272           and R28549 containing the “EKLF,” “GCDH,” “CRTC,” and RAD23A           “genes,” genomic sequence       D86973_at   Human mRNA for KIAA0219 “gene,” partial cds       X81636_at     H. sapiens  clathrin light chain a gene       M59916_at   Human acid sphingomyelinase (ASM) “mRNA,” complete cds       X85781_s_at   “ H. sapiens  NOS2” “gene,” “exon 27 /gb = X85781 /ntype = DNA           /annot = exon”       M57731_s_at   “Human gro-beta” “mRNA,” “complete cds”       U49188_at   Human placenta (Diff33) “mRNA,” complete cds       X53800_s_at   Human mRNA for macrophage inflammatory protein-2beta (MIP2beta)       U56816_at   Human kinase Myt1 (Myt1) “mRNA,” complete cds.       HG1067-   Mucin (Gb:M22406)       HT1067_r_at                  
 
       [0114]                                      Human migration inhibitory factor-related protein 8 (MRP8)   M21005       “gene,” complete cds       Human acyloxyacyl hydrolase “mRNA,” complete cds   M62840       Human PEP19 (PCP4) “mRNA,” complete cds   U52969         H. sapiens  Humig mRNA   X72755         H. sapiens  PISSLRE mRNA   X78342         H. sapiens  mRNA for twist “protein,” partial.   Y11180       /gb = Y11180 /ntype = RNA       Human mRNA for TGF-beta superfamily “protein,” com-   AB000584       plete cds       Human mRNA for “MSS1,” complete cds   D11094       Human complement factor B “mRNA,” complete cds   L15702       “ Homo sapiens  GTP-binding protein   M28213       (RAB2)” “mRNA,” “complete cds”       Human translational initiation factor 2 beta subunit (eIF-2-   M29536       beta) “mRNA,” complete cds       Human E16 “mRNA,” complete cds   M80244       IEX-1 = radiation-inducible immediate-early   S81914       gene “[human,” “placenta,”       mRNA “Partial,” 1223 nt]       Human CDC16Hs “mRNA,” complete cds   U18291       Human DD96 “mRNA,” complete cds   U21049       Human (memc) “mRNA,” 3′UTR. /gb = U30999   U30999       /ntype = RNA       “Human ubiquitin-conjugating enzyme   U45328       (UBE2I)” “mRNA,” “complete cds”       “Human fetal brain glycogen phosphorylase   U47025       B” “mRNA,” “complete cds”       “Human BTG2 (BTG2)” “mRNA,” “complete cds”   U72649       Human jun-B mRNA for JUN-B protein   X51345       Human chaperonin 10 “mRNA,” complete cds   U07550         H. sapiens  RING4 cDNA   X57522         H. sapiens  genes TAP1, TAP2, LMP2, LMP7 and DOB.   X66401         H. sapiens  mRNA for alpha 4 protein   Y08915         Homo sapiens  interleukin-1 receptor-associated kinase   L76191       (IRAK) “mRNA,” complete cds       “Human von Willebrand factor” “mRNA,” “3′   M10321       end”       Human chromosome segregation gene homolog CAS   U33286       “mRNA,” complete cds       Human Bruton&#39;s tyrosine kinase-associated protein-135   U77948       “mRNA,” complete cds.       “Human KH type splicing regulatory protein KSRP”   U94832       “mRNA,” “complete cds.”         H. sapiens  ADE2H1 mRNA showing homologies to SAICAR   X53793       synthetase and AIR carboxylase of the purine pathway (EC       “6.3.2.6,” EC 4.1.1.21)                    
       [0115] a Dukes B stage gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_T67463_s_at       cathepsin O2; X; K       RC_W94688_at       perilipin       RC_AA126743_at       Z97200 PAC chrom 1q24;               PMX1 homeobox gene       RC_AA236547_at       no homology       RC_AA255567_at       angiopoietin-related protein-2;               angiopoietin-like 2       RC_AA421256_at       —       RC_AA386386_s   PPPPP   —       _at       RC_AA452549_at   PPPPP   PRO1659; hypothetical protein               chrom 11                  
 
       [0116]                                                      M63262_at   5-lipoxygenase activating protein (FLAP),               13q12           R67290_at   Interleukine 14           N36619_at           L19161_at   translation initiation factor 2, subunit 3″,               Xp22.2-22.1           RC_AA496035   Chromosome 1? (TIGR)           L29217_s_at   CDC-like kinase 3 (CLK3), 15q24           RC_W73194_a   Dermatoponin, 1q12-q23           RC_N69507_a   hypothetical protein PRO1847 (Alu accord-               ing to TIGR)           RC_H15814_s   adipose most abundant gene transcript 1           M84526_at   D component of complement (adipsin)                        
       [0117] or any gene comprising a sequence as identified below  
                                      U57316_at   Human GCN5 (hGCNS) “gene,” complete cds       X66839_at     H. sapiens  MaTu MN mRNA for p54/58N protein       J04599_at   Human hPGI mRNA encoding bone small proteoglycan I “(biglycan),” com-           plete cds       X57579_s_at     H. sapiens  activin beta-A subunit (exon 2)       J02874_at   Human adipocyte lipid-binding “protein,” complete cds       M11749_at   Human Thy-1 glycoprotein “gene,” complete cds       U06863_at   Human follistatin-related protein precursor “mRNA,” complete cds       U51010_s_at   “Human nicotinamide N-methyltransferase” “gene,” “exon 1 and 5′ flanking           region. /gb = U51010 /ntype = DNA /annot = exon”       U08021_at   “Human nicotinamide N-methyltransferase (NNMT)” “mRNA,” “complete           cds”       HG3044-   “”“Fibronectin,” “Alt. Splice 1”       HT3742_s_at       X02761_s_at   Human mRNA for fibronectin (FN precursor)       X02544_at   Human mRNA for alpha1-acid glycoprotein (orosomucoid)       M62505_at   Human C5a anaphylatoxin receptor “mRNA,” complete cds       J05070_at   Human type IV collagenase “mRNA,” complete cds       U16306_at   Human chondroitin sulfate proteoglycan versican V0 splice-variant precursor           peptide “mRNA,” complete cds       M14218_at   Human argininosuccinate lyase “mRNA,” complete cds       L77567_s_at   “ Homo sapiens  mitochondrial citrate transport protein (CTP)” “mRNA,” “3′           end”       M63391_rna1   Human desmin gene, complete cds.       _at       D13643_at   Human mRNA for KIAA0018 “gene,” complete cds       D79985_at   Human mRNA for KIAA0163 “gene,” complete cds                  
 
       [0118]                                      Human adipocyte lipid-binding “protein,” complete cds   J02874       Human A1 protein “mRNA,” complete cds   U29680       Human LGN protein “mRNA,” complete cds   U54999       Human skeletal muscle LIM-protein SLIM2 “mRNA,” partial   U60116       cds       Human mRNA for alpha1-acid glycoprotein (orosomucoid)   X02544       Human mRNA for fibronectin receptor alpha subunit   X06256         H. sapiens  P1-Cdc21 mRNA   X74794         H. sapiens  mRNA for fibulin-2   X82494         H. sapiens  5T4 gene for 5T4 Oncofetal antigen   Z29083         Homo sapiens  mRNA for osteoblast specific factor 2 (OSF-   D13666       2os)       Mac25   HG987-           HT987       “Human lysozyme” “mRNA,” “complete cds with   J03801       an Alu repeat in the 3′ flank”       Human metalloproteinase (HME) “mRNA,” complete cds   L23808       Human alpha-1 collagen type IV gene, exon 52.   M26576       Human lumican “mRNA,” complete cds   U21128       Human mRNA for fibronectin (FN precursor)   X02761       Human mRNA fragment for elongation factor TU (N-   X03689       terminus). /gb = X03689 /ntype = RNA       Human mRNA for type IV collagen alpha-2 chain   X05610       Human mRNA for collagen VI alpha-1 C-terminal globular   X15880       domain       “ H. sapiens ,” gene for Membrane cofactor protein   X59405         H. sapiens  SOD-2 gene for manganese superoxide dismu-   X65965       tase. /gb = X65965 /ntype = DNA /annot = exon         H. sapiens  NMB mRNA   X76534         H. sapiens  vimentin gene   Z19554       Human chaperonin 10 “mRNA,” complete cds   U07550         H. sapiens  RING4 cDNA   X57522         H. sapiens  genes TAP1, TAP2, LMP2, LMP7 and DOB.   X66401         H. sapiens  mRNA for alpha 4 protein   Y08915         Homo sapiens  interleukin-1 receptor-associated kinase   L76191       (IRAK) “mRNA,” complete cds       “Human von Willebrand factor” “mRNA,” “3′   M10321       end”       Human chromosome segregation gene homolog CAS   U33286       “mRNA,” complete cds       Human Bruton&#39;s tyrosine kinase-associated protein-135   U77948       “mRNA,” complete cds.       “Human KH type splicing regulatory protein KSRP”   U94832       “mRNA,” “complete cds.”         H. sapiens  ADE2H1 mRNA showing homologies to SAICAR   X53793       synthetase and AIR carboxylase of the purine pathway (EC       “6.3.2.6,” EC 4.1.1.21)       “”“Globin,” “Beta”   HG1428-           HT1428       “Human alpha-1 collagen type I” “gene,” “3′   M55998       end”         H. sapiens  mRNA for SOX-4 protein   X70683       “Human mRNA for collagen binding   D83174       protein” “2,” “complete cds”       Human SPARC/osteonectin “mRNA,” complete cds   J03040       Human PRAD1 mRNA for cyclin   X59798                    
       [0119] a Dukes C stage gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_D45556_at       chrom 15; AL390085 clone       RC_W86214_at       RC_AA039439_s       novel gene KIAA0134 protein       _at       19q13.3       RC_AA128935_at       RC_AA134158_s       class I homeodomain; homeo-       _at       box protein, chrom 7       RC_AA232646_at       chrom 17, AF266756 sphingo-               sine kinase (SPHK1       RC_AA401184_at       no homology       RC_AA436840_at       RC_AA488655_at       RC_AA181902_at   PPPPP   AC007201 on chrom 19 (only               80 nt hom)                  
 
       [0120]                                                      RC_AA122350   chromosome 8           AA374109_at   spondin 2, extracellular matrix               protein, chromosome 4           RC_AA621755   transcription factor Dp-2, 3q23           RC_AA442069   sodium channel 2, 12q12           RC_T40767_a   chromosome 19           RC_AA488655   Mus?           RC_AA398908           RC_AA447764   hypothetical protein, chromosome 4           RC_N69136_a                        
       [0121] or any gene comprising a sequence as identified below  
                                      M20681_at   Human glucose transporter-like protein-III “(GLUT3),”           complete cds       D50914_at   Human mRNA for KIAA0124 “gene,” partial cds       L37362_at     Homo sapiens  (clone d2-115) kappa opioid receptor           (OPRK1) “mRNA,” complete cds       X66114_rna1     H. sapiens  gene for 2-oxoglutarate carrier protein.       _at       M32053_at   Human H19 RNA “gene,” complete cds (spliced in           silico)       Y00787_s_at   Human mRNA for MDNCF (monocyte-derived           neutrophil chemotactic factor)       U64444_at   Human ubiquitin fusion-degradation protein (UFD1L)           “mRNA,” complete cds       X95325_s_at     H. sapiens  mRNA for DNA binding protein A variant       X02419_rna1     H. sapiens  uPA gene       _s_at       X57522_at     H. sapiens  RING4 cDNA       AB001325_at   Human AQP3 gene for aquaporine 3 (water “channel),”           partail cds       AB002315_at   Human mRNA for KIAA0317 “gene,” complete cds.           /gb = AB002315 /ntype = RNA       L12760_s_at   “Human phosphoenolpyruvate carboxykinase (PCK1)”           “gene,” “complete cds with repeats”       M80899_at   Human novel protein AHNAK “mRNA,” partial           sequence                  
 
       [0122]                                      Ribosomal Protein L39 Homolog   HG2874-           HT3018         Homo sapiens  (clone d2-115) kappa opioid receptor   L37362       (OPRK1) “mRNA,” complete cds       Human kell blood group protein mRNA   M64934           U73167       Human cancellous bone osteoblast mRNA for serin pro-   D87258       tease with IGF-binding “motif,” complete cds       Human interferon-inducible protein 27-Sep “mRNA,” com-   J04164       plete cds       “Human sickle cell beta-globin” “mRNA,” “complete cds”   M25079           M29277       “Human spermidine synthase” “mRNA,” “complete cds”   M34338       Human copine I “mRNA,” complete cds   U83246       “”“Globin,” “Beta”   HG1428-           HT1428       “Human alpha-1 collagen type I” “gene,” “3′ end”   M55998         H. sapiens  mRNA for SOX-4 protein   X70683       “Human mRNA for collagen binding protein” “2,” “complete   D83174       cds”       Human SPARC/osteonectin “mRNA,” complete cds   J03040       Human PRAD1 mRNA for cyclin   X59798                    
       [0123] a Dukes D stage gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_N91920_at   AAAAP   chrom 16p12-p11.2;               XN_007994 retinoblastoma               binding protein       RC_AA621601_at   AAAAP   chrom 17 XM_009868 RAB36               ARS oncogene family                  
 
       [0124]                                                      RC_AA121433   Axin, chromosome 16           RC_N91920_a   RB protein binding protein,               chromosome 16           RC_AA621601   GTP-binding protein Rab36,               chromosome 17           RC_AA454020   NADPH quinone oxidoreduc-               tase homolog; p53 induced,               chromosome 2           RC_Z39652_a   APM-1 gene, chromosome 18                        
       [0125] or any gene comprising a sequence as identified below  
                                      X17644_s —     Human GST1-Hs mRNA for GTP-binding protein       at       Y12812_at     H. sapiens  RFXAP mRNA       X60486_at     H. sapiens  H4/g gene for H4 histone       X52221_at     H. sapiens  ERCC2 “gene,” exons 1 &amp; 2           (partial)       L06175_at     Homo sapiens  P5-1 “mRNA,” complete cds       Z48481_at     H. sapiens  mRNA for membrane-type matrix           metalloproteinase 1       X54232_at   Human mRNA for heparan sulfate proteaglycan           (glypican)       L08010_at   “ Homo sapiens  reg gene” “homologue,”           “complete cds”       L27706_at   Human chaperonin protein (Tcp20) gene           complete cds       L15533_rna1     Homo sapiens  pancreatits-associated protein       _at   (PAP) gene, complete cds.       X51408_at   Human mRNA for n-chimaerin       K02765_at   Human complement component C3 “mRNA,”           alpha and beta “subunits,” complete cds       U38904_at   Human zinc finger protein C2H2-25 “mRNA,”           complete cds                  
 
       [0126]                                        Homo sapiens  FRG1 “mRNA,” complete cds   L76159       Human cyclin protein “gene,” complete cds   M15796       Human U2 small nuclear RNA-associated B″ antigen   M15841       “mRNA,” complete cds       Human mRNA export protein Rae1 (RAE1) “mRNA,” com-   U84720       plete cds.       Human protease-activated receptor 3 (PAR3) “mRNA,”   U92971       complete cds.         H. sapiens  mRNA for mediator of receptor-induced toxicity   X84709         H. sapiens  RFXAP mRNA   Y12812       Human mRNA for “Qip1,“ complete cds   AB002533       Human mRNA for transferrin receptor   X01060       “metastasis-associated gene ”“[human,”“ highly metastatic   S79219       lung cell subline ”“Anip[937],”“ mRNA ”“Partial,”“ 978 nt]”                    
       [0127] The genes selected may b a gene from each gene group being expressed in a significantly lower amount in that stage than in one of the other stages, such as: a Dukes A stage gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_N32411_f_at   PAPPP   Myc-associated zinc-finger               protein of human islet; chrom               16       RC_AA243858_at   PAPPP   KIAA0882 protein       RC_AA486283_at   PAPPP   ras-like protein; ras-related C3               botulinum toxin substrate;               dJ20J23       RC_AA490930_at   PAPPP   chrom 18; KIAA1468 protein       RC_H54088_s_at   PPPPP   ribosomal protein L41       RC_H59052_f_at   PPPPP   fungal sterol-C5-desaturase               homolog; ORF; thymosin beta-4       RC_R49198_s_at   PPPPP   —       RC_T73572_f_at   PPPPP   ferritin L-chain; L apoferritin       RC_AA477483_at   PPPPP   no matching est                  
 
       [0128] or any gene comprising a sequence as identified below  
                                        Homo sapiens  SKB1Hs “mRNA,” complete cds.   AF015913       /gb = AF015913 /ntype = RNA       Mucin (Gb:M22406)   HG1067-           HT1067       Human platelet activating factor “acetylhydrolase,” brain   U72342       “isoform,” 45 kDa subunit (LIS1) gene         Homo sapiens  ERK activator kinase (MEK2) mRNA   L11285       Human 20-kDa myosin light chain (MLC-2) “mRNA,”   J02854       complete cds         H. sapiens  lysosomal acid phosphatase gene (EC 3.1.3.2)   X15525       Exon 1 (and joined CDS).       Human mRNA for matrix Gla protein   X53331         H. sapiens  mRNA for diacylglycerol kinase   X62535       Human heat shock protein (hsp 70) gene, complete cds.   M11717       Human TRPM-2 protein gene   M63379                  
 
       [0129] a Dukes B stag gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_D59847_at   PPAPP   proSAAS; granin-like neuroen-               docrine peptide precursor       RC_F05038_at   PPAPP   polyamine modulated factor-1;               polyamine modulated factor 1       RC_N41059_at   PPAPP   chrom 3       RC_T23460_at   PPAPP   chrom 3; IFNAR2 21q22.11       RC_W42789_at   PPAPP   chrom 8 AF268037 C8ORF4               protein (C8ORF4) chrom 8               ORF       RC_AA460017_i —     PPAPP   BAC clone chrom 16       at       RC_AA482127_at   PPAPP   KIAA1142 protein       RC_AA504806_at   PPAPP   chrom 2 AF052107 clone               23620 mRNA sequence       RC_T90037_at   PPPPP   unnamed protein product,               chrom 4       RC_AA432130_at   PPPPP   KIAA0867 protein, chrom 12                  
 
       [0130] or any gene comprising a sequence as identified below  
                                      Human gene for mitochondrial acetoacetyl-CoA thiolase   D10511       Human mRNA for transcription factor “AREB6,” complete   D15050       cds       Human mRNA for KIAA0248 “gene,” partial cds   D87435         Homo sapiens  (clone CC6) NADH-ubiquinone oxidoreduc-   L04490       tase subunit “mRNA,” 3′ end cds       Human phosphoglucomutase 1 (PGM1) “mRNA,” com-   M83088       plete cds         Homo sapiens  guanylin “mRNA,” complete cds   M97496       “Human trans-Golgi p230 ”“mRNA,”“ complete cds”   U41740         H. sapiens  mRNA for vacuolar proton “ATPase,” subunit D   X71490         H. sapiens  mRNA for 3-hydroxy-3-methylglutaryl coen-   X83618       zyme A synthase       Human mRNA for KIAA0018 “gene,” complete cds   D13643       “Mucin ”“1,”“ ”“Epithelial,”“ Alt. Splice 9”   HG371-           HT26388         H. sapiens  mRNA for L-3-hydroxyacyl-CoA dehydrogenase   X96752                  
 
       [0131] a Dukes C stage gene is selected individually from any gene comprising a sequence as identified below  
                                                          RC_N30231_at   PPPAP   Lsm4 protein; U6 snRNA-                   associated Sm-like protein                   LSm4; glycine-rich protein           RC_W73790_f_at   PPPAP   immunoglobulin-related pro-                   tein 14.1; lambda L-chain C                   region; omega protein, chrom                   22           RC_AA412184_at   PPPAP   chrom 1p36; d89060 dolichyl-                   diphosphooligosaccharide-                   protein glycosyltransferase           RC_AA521303_at   PPPAP   methionine adenosyltransfer-                   ase regulatory beta subunit;                   dTDP-4-keto-6-deoxy-D-                   glucose 4-reductase, chrom 5           RC_AA461174_at   PPPPP   8p21.3-p22 AB020860 anti-                   oncogene           AA393432_s_at   PPPPP   chrom 2, Unknown; unnamed                   protein product AAD20029                      
 
       [0132] or any gene comprising a sequence as identified below  
                                        Homo sapiens  colon mucosa-associated (DRA)   L02785       “mRNA,” complete cds       Human Ig J chain gene   M12759       Human selenium-binding protein (hSBP) “mRNA,”   U29091       complete cds. /gb = U29091 /ntype = RNA         H. sapiens  mRNA for sigma 3B protein   X99459       Human ERK1 mRNA for protein serine/threonine kin-   X60188       ase       Human mRNA for mitochondrial 3-oxoacyl-CoA “thio-   D16294       lase,” complete cds       “Biliary ”“Glycoprotein,”“ Alt. Splice ”“5,”“ A”   HG2850-           HT4814       Human AQP3 gene for aquaporine 3 (water “channel),”   AB001325       partail cds       Human CD14 mRNA for myelid cell-specific leucine-rich   X13334       glycoprotein       Human thioredoxin “mRNA,” nuclear gene encoding mito-   U78678       chondrial “protein,” complete cds       Human mitochondrial ATPase coupling factor 6 subunit   M37104       (ATP5A) “mRNA,” complete cds       “Human MHC class II HLA-DP light chain ”“mRNA,”“ com-   M57466       plete cds”       Human mRNA for early growth response protein 1   X52541       (hEGR1)       Human mRNA for mitochondrial 3-ketoacyl-CoA thiolase   D16481       beta-subunit of trifunctional “protein,” complete cds         Homo sapiens  laminin-related protein (LamA3) “mRNA,”   L34155       complete cds         H. sapiens  mRNA for selenoprotein P   Z11793       Human hkf-1 “mRNA,” complete cds   D76444         Homo sapiens  nuclear domain 10 protein (ndp52) “mRNA,”   U22897       complete cds       Human X104 “mRNA,” complete cds   L27476         H. sapiens  cDNA for RFG   X77548         H. sapiens  mRNA for Progression Associated Protein   Y07909       Human liver “2,4-dienoyl-CoA” reductase “mRNA,” com-   U49352       plete cds       Human A33 antigen precursor “mRNA,” complete cds   U79725         H. sapiens  pS2 protein gene   X52003       Human RASF-A PLA2 “mRNA,” complete cds   M22430         Homo sapiens  pstl mRNA for pancreatic secretory inhibitor   Y00705       (expressed in neoplastic tissue).       Human CO-029   M35252                  
 
       [0133] a Dukes D stage gene is selected individually from any gene comprising a sequence as identified below  
                                          RC_R72886_s_at   PPPPA   KIAA0422; adenylyl cyclase               type VI, chrom 12       RC_AA026030_at   PPPPA   chrom 1       RC_Z39006_at   PPPPA   hypothetical protein, chrom 17       RC_AA435908_at   PPPPA   chrom 19; ac011491 clone and               20 nt hom. RAB2, RAS onco-               gene family       RC_AA057829_s   PPPPA   growth-arrest-specific protein;       _at       growth arrest-specific 6; AXL               stimulatory factor, chrom 13       RC_R72087_at   PPPPA   chrom 5 EST; hom to chrom               20 AL356652 clone       RC_H04242_at   PPPPA   ras related protein Rab5b;               RAB5B, member RAS onco-               gene family       RC_R97304_f_at   PPPPA   HLA-drb5; cell surface gly-               coprotein; MHC HLA-DR-beta               chain precursor chrom 6       RC_N48609_at   PPPPA   chrom 11; AC004584 chrom               17       RC_W86850_f_at   PPPPA   chrom 22 ? X96924 mito-               chondrial citrate tranbsport               region       RC_AA130603_at   PPPPA   ak024908 clone       RC_AA479610_at   PPPPA   singleton ak025344 clone       RC_AA490593_i —     PPPPA   chrom 17 ? Synaptobrevin2       at       (VAMP2) AF135372       RC_AA054321_s   PPPPA   6p21 HLA class i region;       _at       AC004202 clone       RC_D60328_at   PPPPP   chrom 6, unknown; ring finger               protein 5       RC_H96850_at   PPPPP   oligosaccharyltransferase               d89060 1p36.1 (also C-class)       RC_AA127444_at   PPPPP   chrom 1 no homology       RC_AA242824_at   PPPPP   chrom 11; ac005233 PAC               clone chrom 22       AA405775_s_at   PPPPP   similar to CAA16821               (PID: g3255952)                  
 
       [0134] or any gene comprising a sequence as identified below  
                                      Human complement component C3 “mRNA,” alpha and   K02765       beta “subunits,” complete cds         H. sapiens  mRNA for adenosine “triphosphatase,” cal-   Z69881       cium       Human skeletal muscle LIM-protein SLIM1 “mRNA,” com-   U60115       plete cds       Human platelet-derived growth factor receptor alpha   M21574       (PDGFRA) “mRNA,” complete cds       Human mRNA for KIAA0247 “gene,” complete cds   D87434       Human mRNA for KIAA0171 “gene,” complete cds   D79993       Human Down syndrome critical region protein (DSCR1)   U28833       “mRNA,” complete cds       Human Ki nuclear autoantigen “mRNA,” complete cds   U11292                  
 
       [0135] Expression Patterns  
       [0136] The objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided of determining an expression pattern of a cell sample preferably independent of the proportion of submucosal, muscle and connective tissue cells present. Expression is determined of one or more genes in a sample comprising cells, said genes being selected from the same genes as discussed above and shown in the tables of the Examples.  
       [0137] It is an object of the present that characteristic patterns of expression of genes can be used to characterize different types of tissue. Thus, for example gene expression patterns can be used to characterize stages and grades of colorectal tumors. Similarly, gene expression patterns can be sued to distinguish cells having a colorectal origin from other c lls. Moreover, gene expression of cells which routinely contaminate colorectal tumor biopsies has been identified, and such gene expression can be removed or subtracted from patterns obtained from colorectal biopsies. Further, the gene expression patterns of single-cell solutions of colorectal tumor cells have been found to be far freer of interfering expression of contaminating muscle, submucosal, and connective tissue cells that biopsy samples.  
       [0138] The one or more genes exclude genes which are expressed in the submucosal, muscle, and connective tissue. A pattern of expression is formed for the sample which is independent of the proportion of submucosal, muscle, and connective tissue cells in the sample.  
       [0139] In another aspect of the invention a method of determining an expression pattern of a cell sample is provided. Expression is determined of one or more genes in a sample comprising cells. A first pattern of expression is thereby formed for the sample. Genes which are expressed in submucosal, muscle, and connective tissue cells are removed from the first pattern of expression, forming a second pattern of expression which is independent of the proportion of submucosal, muscle, and connective tissue cells in the sample.  
       [0140] Another embodiment of the invention provides a method for determining an expression pattern of a colorectal mucosa or colorectal cancer cell. Expression is determined of one or more genes in a sample comprising colorectal mucosa or colorectal cancer cells; the expression determined forms a first pattern of expression. A second pattern of expression which was formed using the one or more genes and a sample comprising predominantly submucosal, muscle, and connective tissue cells, is subtracted from the first pattern of expression, forming a third pattern of expression. The third pattern of expression reflects expression of the colorectal mucosa or colorectal cancer cells independent of the proportion of submucosal, muscle, and connective tissue cells present in the sample.  
       [0141] Diagnosing  
       [0142] In another embodiment of the invention a method is provided of detecting an invasive tumor in a patient. A marker is detected in a sample of a body fluid. The body fluid is selected from the group consisting of blood, plasma, serum, faeces, mucus, sputum, cerebrospinal fluid and/or urine. The marker is an mRNA or protein expression product of a gene which is more prevalent in submucosal, muscle, and connective tissue than in the body fluid. An increased amount of the marker in the body fluid indicates a tumor which has become invasive in the patient.  
       [0143] In another aspect of the invention a method is provided for diagnosing a colorectal cancer. A first pattern of expression is determined of one or more genes in a colonic tissue sample suspected of being neoplastic. The first pattern of expression is compared to a second and third reference pattern of expression. The second pattern is of the one or more genes in normal colorectal mucosa and the third pattern is of the one or more genes in colorectal cancer. A first pattern of expression which is found to be more similar to the third pattern than the second indicates neoplasia of the colorectal tissue sample.  
       [0144] According to yet another aspect of the invention a method is provided for predicting outcome or prescribing treatment of a colorectal tumor. A first pattern of expression is determined of one or more genes in a colorectal tumor sample. The first pattern is compared to one or more reference patterns of expression determined for colorectal tumors at a grade between I and IV. The reference pattern which shares maximum similarity with the first pattern is identified. The outcome or treatment appropriate for the grade of tumor of the reference pattern with the maximum similarity is assigned to the colorecteal tumor sample.  
       [0145] In another embodiment of the invention a method is provided for determining grade of a colorecteal tumor. A first pattern of expression is determined of one or more genes in a colorectal tumor sample. The first pattern is compared to one or more reference patterns of expression determined for colorectal tumors at a grade between I and IV. The grade of the reference pattern with the maximum similarity is assigned to the colorecteal tumor sample.  
       [0146] Yet another embodiment of the invention provides a method to determine stage of a colorectal tumor as described above. A first pattern of expression is determined of one or more genes in a colorectal tumor sampl. The first pattern is compared to one or more reference patterns of expression determined for colorectal tumors at different stages. The reference pattern which shares maximum similarity with the first pattern is identified. The stage of the reference pattern with the maximum similarity is assigned to the colorecteal tumor sample.  
       [0147] In still another embodiment of the invention a method is provided for identifying a tissue sample as colo-rectal. A first pattern of expression is determined of one or more genes in a tissue sample. The first pattern is compared to a second pattern of expression determined obtained for normal mucosa cells. Similarity between the first and the second patterns suggests that the tissue sample is mucosa in its origin. This method being particularly useful when diagnosing metastasis possibly distant from its origin.  
       [0148] Another aspect of the invention is a method to aid in diagnosing, predicting outcome, or prescribing treatment of a colorectal cancer. A first pattern of expression is determined of one or more genes in a first colorectal tissue sample. A second pattern of expression is determined of the one or more genes in a second colorectal tissue sample. The first colorectal tissue sample is a normal colorectal mucosa sample or an earlier stage or lover grade of colorectal tumor than the second colorectal tissue sample. The first pattern of expression is compared to the second pattern of expression to identify a first set of genes which are increased in the second colorectal tissue sample relative to the first colorectal tissue sample and a second set of genes which are decreased in the second colorectal tissue sample relative to the first colorectal tissue sample. Those genes which are expressed in submucosal, muscle or connective tissue are removed from the first set of genes. Those genes which are not expressed in submucosal, muscle, or connective tissue are removed from the second set of genes.  
       [0149] Independence of Submucosal, Muscle and Connective Tissue  
       [0150] Since a biopsy of the tissue often contains more tissue material, than the tissue to be examined, such as connective tissue, when the tissue to be examined is epithelial or mucosa, the invention also relates to methods, wherein the expression pattern of the tissue is independent of the amount of connective tissue in the sample.  
       [0151] Biopsies contain epithelial cells that most often are the targets for the studies, and in addition many other cells that contaminate the epithelial cell fraction to a varying extent. The contaminants include histiocytes, endothelial cells, leukocytes, nerve cells, muscle cells etc. Micro dissection is the method of choice for DNA examination, but in case of expression studies this procedure is difficult due to RNA degradation during the procedure. The epithelium may be gently removed and the expression in the remaining submucosa and underlying connective tissue (the colon wall) monitored. Genes expressed at high or low levels in the colon wall should be interrogated when performing expression monitoring of the mucosa and tumors. A similar approach could be used for studies of epithelia in other organs.  
       [0152] Normal mucosa lining the colon lumen from colons for colon cancer was scraped off. Then biopsies were taken from the denuded submucosa and connective tissue, reaching approximately 5 mm into the colon wall, and immediately disintegrated in guanidinium isothiocyanate. Total RNA may be extracted, pooled, and poly(A) +  mRNA may be prepared from the pool followed by conversion to double-stranded cDNA and in vitro transcription into cRNA containing biotin-labeled CTP and UTP.  
       [0153] Genes that are expressed and genes that are not expressed in colon wall can both interfere with the interpretation of the expression in a biopsy, and should be interrogated when interpreting expression intensities in tumor biopsies, as the colon wall component of a biopsy varies in amount from biopsy to biopsy.  
       [0154] When having determined the pattern of genes expressed in colon wall components said pattern may be subtracted from a pattern obtained from the sample resulting in a third pattern related to the mucosa (epithelial) cells.  
       [0155] In another aspect of the invention a method is provided for determining an expression pattern of a colorectal tissue sample independent of the proportion of submucosal, muscle and connective tissue cells present. A single-cell suspension of disaggregated colorectal tumor cells is isolated from a colorectal tissue sample comprising colorectal tumor cells is isolated form a coloretal tissue sample comprising colorectal cells, submucosal cells, muscle cells, and connective tissue cells. A pattern of expression is thus formed for the sample which is independent of the proportion of submucosal, muscle, and connective tissue cells in the colorectal tissue sample.  
       [0156] Yet another method relates to elimination mRNA from colon wall components before determining the pattern, e.g. by filtration and/or affinity chromatography to remove mRNA related to the colon wall.  
       [0157] Detection  
       [0158] Working with human tumor material requires biopsies, and working with RNA requires freshly frozen or immediately processed biopsies. Apart from the cancer tissue, biopsies do inevitably contain many different cell types, such as cells present in the blood, connective and muscle tissue, endothelium etc. In the case of DNA studies, microdissection or laser capture are method of choice, however the time dependent degradation of RNA makes it difficult to perform manipulation of the tissue for more than a few minutes. Furthermore, studies of expressed sequences may be difficult on the few cells obtained via microdissection or laser capture, as these may have an expression pattern that deviates from the predominant pattern in a tumor due to large intratumoral heterogeneity.  
       [0159] In the present context high density expression arrays may be used to evaluate the impact of colorectal wall components in colorectal tumor biopsies, and tested preparation of single cell solutions as a means of eliminating the contaminants. The results of these evaluations permit us to design methods of evaluating colorectal samples without the interfering background noise caused by ubiquitous contaminating submucosal, muscle, and connective tissue cells. The evaluating assays of the invention may be of any type.  
       [0160] While high density expression arrays can be used, other techniques are also contemplated. These include other techniques for assaying for specific mRNA species, including RT-PCR and Northern Blotting, as well as techniques for assaying for particular protein products, such as ELISA, Western blotting, and enzyme assays. Gene expression patterns according to the present invention are determined by measuring any gene product of a particular gene, including mRNA and protein. A pattern may be for one or more gene.  
       [0161] RNA or protein can be isolated and assayed from a test sample using any techniques known in the art. They can for example be isolated from fresh or frozen biopsy, from formalin-fixed tissue, from body fluids, such as blood, plasma, serum, urine, or sputum.  
       [0162] The data provided of expression for submucosal, muscle, and connective tissue can be used in at least three ways to improve the quality of data for a tested sample. The genes identified in the data as expressed can be excluded from the testing or from the analysis. Alternatively, the intensity of expression of the genes expressed in the submucosal, muscle, and connective tissue can be subtracted from the intensity of expression determined for the tests tissue.  
       [0163] The data collected and disclosed here as “connective tissue” is presumed to contain both muscle and submucosal gene expression as well. Thus it represents the composite expression of these cell types which can typically contaminate a colorectal biopsy.  
       [0164] Detection of Expression  
       [0165] Expression of genes may in general be detected by either detecting mRNA from the cells and/or detecting expression products, such as peptides and proteins.  
       [0166] mRNA Detection  
       [0167] The detection of mRNA of the invention may be a tool for determining the developmental stage of a cell type may be definable by its pattern of expression of messenger RNA. For example, in particular stages of cells, high levels of ribosomal RNA are found whereas relatively low levels of other types of messenger RNAs may be found. Where a pattern is shown to be characteristic of a stage, a stage may be defined by that particular pattern of messenger RNA expression. The mRNA population is a good determinant of developmental stage, will be correlated with other structural features of the cell. In this manner, cells at specific developmental stages will be characterized by the intracellular environment, as well as the extracellular environment. The present invention also allows the combination of definitions based, in part, upon antigens and, in part, upon mRNA expression. In one embodiment, the two may be combined in a single incubation step. A particular incubation condition may be found which is compatible with both hybridization recognition and non-hybridization recognition molecules. Thus, e.g., an incubation condition may be selected which allows both specificity of antibody binding and specificity of nucleic acid hybridization. This allows simultaneous performance of both types of interactions on a single matrix. Again, where developmental mRNA patterns are correlated with structural features, or with probes which are able to hybridize to intracellular mRNA populations, a cell sorter may be used to sort specifically those cells having desired mRNA population patterns.  
       [0168] It is within the general scope of the present invention to provide methods for the detection of mRNA. Such methods often involve sample extraction, PCR amplification, nucleic acid fragmentation and labeling, extension reactions, transcription reactions and the like.  
       [0169] Sample Preparation  
       [0170] The nucleic acid (either genomic DNA or mRNA) may be isolated from the sample according to any of a number of methods well known to those of skill in the art. One of skill will appreciate that where alterations in the copy number of a gene are to be detected genomic DNA is preferably isolated. Conversely, where expression levels of a gene or genes are to be detected, preferably RNA (mRNA) is isolated.  
       [0171] Methods of isolating total mRNA are well known to those of skill in the art. In one embodiment, the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and polyA.sup.+mRNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987)).  
       [0172] The sample may be from tissue and/or body fluids, as defined elsewhere herein. Before analyzing the sample, e.g., on an oligonucleotide array, it will often be desirable to perform one or more sample preparation operations upon the sample. Typically, these sample preparation operations will include such manipulations as extraction of intracellular material, e.g., nucleic acids from whole cell samples, viruses and the like, amplification of nucleic acids, fragmentation, transcription, labeling and/or extension reactions. One or more of these various operations may be readily incorporated into the device of the present invention.  
       [0173] DNA Extraction  
       [0174] DNA extraction may be relevant in case possible mutations in the genes are to be dtermined in addition to the determination of expression of the genes.  
       [0175] For those embodiments where whole cells, or other tissue samples are being analyzed, it will typically be necessary to extract the nucleic acids from the cells or viruses, prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatments to prepare the sample for subsequent operations, e.g., denaturation of contaminating (DNA binding) proteins, purification, filtration, desalting, and the like.  
       [0176] Liberation of nucleic acids from the sample cells, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods. For example, chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins.  
       [0177] Alternatively, physical methods may be used to extract the nucleic acids and denature DNA binding proteins, such as physical protrusions within microchannels or sharp edged particles piercing cell membranes and extract their contents. Combinations of such structures with piezoelectric elements for agitation can provide suitable shear forces for lysis.  
       [0178] More traditional methods of cell extraction may also be used, e.g., employing a channel with restricted cross-sectional dimension which causes cell lysis when the sample is passed through the channel with sufficient flow pressure. Alternatively, cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current to the sample. More specifically, the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow. Subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture are also possible extraction methods.  
       [0179] Filtration  
       [0180] Following extraction, it will often be desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, salts, and the like. Removal of particulate matter is generally accomplished by filtration, flocculation or the like. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample, passing salts through dialysis membranes, and the like. Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica (i.e., glass wool), or the like. Suitable gel exclusion media, also well known in the art, may also be readily incorporated into the devices of the present invention, and is commercially available from, e.g., Pharmacia and Sigma Chemical.  
       [0181] Alternatively, desalting methods may generally take advantage of the high electrophoretic mobility and negative of DNA compared to other elements. Electrophoretic methods may also be utilized in the purification of nucleic acids from other cell contaminants and debris. Upon application of an appropriate electric field, the nucleic acids present in the sample will migrate toward the positive electrode and become trapped on the capture membrane. Sample impurities remaining free of the membrane are then washed away by applying an appropriate fluid flow. Upon reversal of the voltage, the nucleic acids are released from the membrane in a substantially purer form. Further, coarse filters may also be overlaid on the barriers to avoid any fouling of the barriers by particulate matter, proteins or nucleic acids, thereby permitting repeated use.  
       [0182] Separation of Contaminants by Chromatography  
       [0183] In a similar aspect, the high electrophoretic mobility of nucleic acids with their negative charges, may be utilized to separate nucleic acids from contaminants by utilizing a short column of a gel or other appropriate matrix or gel which will slow or retard the flow of other contaminants while allowing the faster nucleic acids to pass.  
       [0184] This invention provides nucleic acid affinity matrices that bear a large number of different nucleic acid affinity ligands allowing the simultaneous selection and removal of a large number of preselected nucleic acids from the sample. Methods of producing such affinity matrices are also provided. In general the methods involve the steps of a) providing a nucleic acid amplification template array comprising a surface to which are attached at least 50 oligonucleotides having different nucleic acid sequences, and wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said oligonucleotides is greater than about 60 different oligonucleotides per 1 cm.sup.2, and all of said different oligonucleotides have an identical terminal 3′ nucleic acid sequence and an identical terminal 5′ nucleic acid sequence. b) amplifying said multiplicity of oligonucleotides to provide a pool of amplified nucleic acids; and c) attaching the pool of nucleic acids to a solid support.  
       [0185] For example, nucleic acid affinity chromatography is based on the tendency of complementary, single-stranded nucleic acids to form a double-stranded or duplex structure through complementary base pairing. A nucleic acid (either DNA or RNA) can easily be attached to a solid substrate (matrix) where it acts as an immobilized ligand that interacts with and forms duplexes with complementary nucleic acids present in a solution contacted to the immobilized ligand. Unbound components can be washed away from the bound complex to either provide a solution lacking the target molecules bound to the affinity column, or to provide the isolated target molecules themselves. The nucleic acids captured in a hybrid duplex can be separated and released from the affinity matrix by denaturation either through heat, adjustment of salt concentration, or the use of a destabilizing agent such as formamide, TWEEN.TM.-20 denaturing agent, or sodium dodecyl sulfate (SDS). Affinity columns (matrices) are typically used either to isolate a single nucleic acid typically by providing a single species of affinity ligand. Alternatively, affinity columns bearing a single affinity ligand (e.g. oligo dt columns) have been used to isolate a multiplicity of nucleic acids where the nucleic acids all share a common sequence (e.g. a polyA).  
       [0186] Affinity Matrices  
       [0187] The type of affinity matrix used depends on the purpose of the analysis. For example, where it is desired to analyze mRNA expression levels of particular genes in a complex nucleic acid sample (e.g., total mRNA) it is often desirable to eliminate nucleic acids produced by genes that are constitutively overexpressed and thereby tend to mask gene products expressed at characteristically lower levels. Thus, in one embodiment, the affinity matrix can be used to remove a number of preselected gene products (e.g., actin, GAPDH, etc.). This is accomplished by providing an affinity matrix bearing nucleic acid affinity ligands complementary to the gene products (e.g., mRNAs or nucleic acids derived therefrom) or to subsequences thereof. Hybridization of the nucleic acid sample to the affinity matrix will result in duplex formation between the affinity ligands and their target nucleic acids. Upon elution of the sample from the affinity matrix, the matrix will retain the duplexes nucleic acids leaving a sample depleted of the overexpressed target nucleic acids.  
       [0188] The affinity matrix can also be used to identify unknown mRNAs or cDNAs in a sample. Where the affinity matrix contains nucleic acids complementary to every known gene (e.g., in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template) in a sample, capture of the known nucleic acids by the affinity matrix leaves a sample enriched for those nucleic acid sequences that are unknown. In effect, the affinity matrix is used to perform a subtractive hybridization to isolate unknown nucleic acid sequences. The remaining “unknown” sequences can then be purified and sequenced according to standard methods.  
       [0189] The affinity matrix can also be used to capture (isolate) and thereby purify unknown nucleic acid sequences. For example, an affinity matrix can be prepared that contains nucleic acid (affinity ligands) that are complementary to sequences not previously identified, or not previously known to be expressed in a particular nucleic acid sample. The sample is then hybridized to the affinity matrix and those sequences that are retained on the affinity matrix are “unknown” nucleic acids. The retained nucleic acids can be eluted from the matrix (e.g. at increased temperature, increased destabilizing agent concentration, or decreased salt) and the nucleic acids can then be sequenced according to standard methods.  
       [0190] Similarly, the affinity matrix can be used to efficiently capture (isolate) a number of known nucleic acid sequences. Again, the matrix is prepared bearing nucleic acids complementary to those nucleic acids it is desired to isolate. The sample is contacted to the matrix under conditions where the complementary nucleic acid sequences hybridize to the affinity ligands in the matrix. The non-hybridized material is washed off the matrix leaving the desired sequences bound. The hybrid duplexes are then denatured providing a pool of the isolated nucleic acids. The different nucleic acids in the pool can be subsequently separated according to standard methods (e.g. gel electrophoresis).  
       [0191] As indicated above the affinity matrices can be used to selectively remove nucleic acids from virtually any sample containing nucleic acids (e.g., in a cDNA library, DNA reverse transcribed from an mRNA, mRNA used directly or amplified, or polymerized from a DNA template, and so forth). The nucleic acids adhering to the column can be removed by washing with a low salt concentration buffer, a buffer containing a destabilizing agent such as formamide, or by elevating the column temperature.  
       [0192] In one particularly preferred embodiment, the affinity matrix can be used in a method to enrich a sample for unknown RNA sequences (e.g. expressed sequence tags (ESTs)). The method involves first providing an affinity matrix bearing a library of oligonucleotide probes specific to known RNA (e.g., EST) sequences. Then, RNA from undifferentiated and/or unactivated cells and RNA from differentiated or activated or pathological (e.g., transformed) or otherwise having a different metabolic state are separately hybridized against the affinity matrices to provide two pools of RNAs lacking the known RNA sequences.  
       [0193] In a preferred embodiment, the affinity matrix is packed into a columnar casing. The sample is then applied to the affinity matrix (e.g. injected onto a column or applied to a column by a pump such as a sampling pump driven by an autosampler). The affinity matrix (e.g. affinity column) bearing the sample is subjected to conditions under which the nucleic acid probes comprising the affinity matrix hybridize specifically with complementary target nucleic acids. Such conditions are accomplished by maintaining appropriate pH, salt and temperature conditions to facilitate hybridization as discussed above.  
       [0194] For a number of applications, it may be desirable to extract and separate messenger RNA from cells, cellular debris, and other contaminants. As such, the device of the present invention may, in some cases, include an mRNA purification chamber or channel. In general, such purification takes advantage of the poly-A tails on mRNA. In particular and as noted above, poly-T oligonucleotides may be immobilized within a chamber or channel of the device to serve as affinity ligands for mRNA. Poly-T oligonucleotides may be immobilized upon a solid support incorporated within the chamber or channel, or alternatively, may be immobilized upon th surface(s) of the chamber or channel itself. Immobilization of oligonucleotides on the surface of the chambers or channels may be carried out by methods described herein including, e.g., oxidation and silanation of the surface followed by standard DMT synthesis of the oligonucleotides.  
       [0195] In operation, the lysed sample is introduced to a high salt solution to increase the ionic strength for hybridization, whereupon the mRNA will hybridize to th immobilized poly-T. The mRNA bound to the immobilized poly-T oligonucleotides is then washed free in a low ionic strength buffer. The poy-T oligonucleotides may be immobiliized upon poroussurfaces, e.g., porous silicon, zeolites silica xerogels, scintered particles, or other solid supports.  
       [0196] Hybridization  
       [0197] Following sample preparation, the sample can be subjected to one or more different analysis operations. A variety of analysis operations may generally be performed, including size based analysis using, e.g., microcapillary electrophoresis, and/or sequence based analysis using, e.g., hybridization to an oligonucleotide array.  
       [0198] In the latter case, the nucleic acid sample may be probed using an array of oligonucleotide probes. Oligonucleotide arrays generally include a substrate having a large number of positionally distinct oligonucleotide probes attached to the substrate. These arrays may be produced using mechanical or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods.  
       [0199] Light Directed Synthesis of Oligonucleotide Arrays  
       [0200] The basic strategy for light directed synthesis of oligonucleotide arrays is as follows. The surface of a solid support, modified with photosensitive protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A selected nucleotide, typically in the form of a 3′-O-phosphoramidite-activated deoxynucleoside (protected at the 5′ hydroxyl with a photosensitive protecting group), is then presented to the surface and coupling occurs at the sites that were exposed to light. Following capping and oxidation, the substrate is rinsed and the surface is illuminated through a second mask, to expose additional hydroxyl groups for coupling. A second selected nucleotide (e.g., 5′-protected, 3′-O-phosphoramidite-activated deoxynucleoside) is presented to the surface. The selective deprotection and coupling cycles are repeated until the desired set of products is obtained. Since photolithography is used, the process can be readily miniaturized to generate high density arrays of oligonucleotide probes. Furthermore, the sequence of the oligonucleotides at each site is known. See, Pease, et al. Mechanical synthesis methods are similar to the light directed methods except involving mechanical direction of fluids for deprotection and addition in the synthesis steps.  
       [0201] For some embodiments, oligonucleotide arrays may be prepared having all possible probes of a given length. The hybridization pattern of the target sequence on the array may be used to reconstruct the target DNA sequence. Hybridization analysis of large numbers of probes can be used to sequence long stretches of DNA or provide an oligonucleotide array which is specific and complementary to a particular nucleic acid sequence. For example, in particularly preferred aspects, the oligonucleotide array will contain oligonucleotide probes which are complementary to specific target sequences, and individual or multiple mutations of these. Such arrays are particularly useful in the diagnosis of specific disorders which are characterized by the presence of a particular nucleic acid sequence.  
       [0202] Following sample collection and nucleic acid extraction, the nucleic acid portion of the sample is typically subjected to one or more preparative reactions. These preparative reactions include in vitro transcription, labeling, fragmentation, amplification and other reactions. Nucleic acid amplification increases the number of copies of the target nucleic acid sequence of interest. A variety of amplification methods are suitable for use in the methods and device of the present invention, including for example, the polymerase chain reaction method or (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), and nucleic acid based sequence amplification (NASBA).  
       [0203] The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of approximately 30 or 100 to 1, respectively. As a result, where these latter methods are employed, sequence analysis may be carried out using either type of substrate, i.e., complementary to either DNA or RNA.  
       [0204] Frequently, it is desirable to amplify the nucleic acid sample prior to hybridization. One of skill in the art will appreciate that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids.  
       [0205] PCR  
       [0206] Methods of “quantitative” amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. The high density array may then include probes sp cific to the internal standard for quantification of the amplified nucleic acid.  
       [0207] Thus, in one embodiment, this invention provides for a method of optimizing a probe set for detection of a particular gene. Generally, this method involves providing a high density array containing a multiplicity of probes of one or more particular length(s) that are complementary to subsequences of the mRNA transcribed by the target gene. In one embodiment the high density array may contain every probe of a particular length that is complementary to a particular mRNA. The probes of the high density array are then hybridized with their target nucleic acid alone and then hybridized with a high complexity, high concentration nucleic acid sample that does not contain the targets complementary to the probes. Thus, for example, where the target nucleic acid is an RNA, the probes are first hybridized with their target nucleic acid alone and then hybridized with RNA made from a cDNA library (e.g., reverse transcribed polyA.sup.+mRNA) where the sense of the hybridized RNA is opposite that of the target nucleic acid (to insure that the high complexity sample does not contain targets for the probes). Those probes that show a strong hybridization signal with their target and little or no cross-hybridization with the high complexity sample are preferred probes for use in the high density arrays of this invention.  
       [0208] PCR amplification generally involves the use of one strand of the target nucleic acid sequence as a template for producing a large number of complements to that sequence. Generally, two primer sequences complementary to different ends of a segment of the complementary strands of the target sequence hybridize with their respective strands of the target sequence, and in the presence of polymerase enzymes and nucleoside triphosphates, the primers are extended along the target sequence. The extensions are melted from the target sequence and the process is repeated, this time with the additional copies of the target sequence synthesized in the preceding steps. PCR amplification typically involves repeated cycles of denaturation, hybridization and extension reactions to produce sufficient amounts of the target nucleic acid. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which ach primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.  
       [0209] In PCR methods, strand separation is normally achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase. Typical heat denaturation involves temperatures ranging from about 80.degree. C. to 105.degree. C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity.  
       [0210] In addition to PCR and IVT reactions, the methods and devices of the present invention are also applicable to a number of other reaction types, e.g., reverse transcription, nick translation, and the like.  
       [0211] Labelling Before Hybridization  
       [0212] The nucleic acids in a sample will generally be labeled to facilitate detection in subsequent steps. Labeling may be carried out during the amplification, in vitro transcription or nick translation processes. In particular, amplification, in vitro transcription or nick translation may incorporate a label into the amplified or transcribed sequence, either through the use of labeled primers or the incorporation of labeled dNTPs into the amplified sequence.  
       [0213] Hybridization between the sample nucleic acid and the oligonucleotide probes upon the array is then detected, using, e.g., epifluorescence confocal microscopy. Typically, sample is mixed during hybridization to enhance hybridization of nucleic acids in the sample to nucleoc acid probes on the array.  
       [0214] Labelling After Hybridization  
       [0215] In some cases, hybridized oligonucleotides may be labeled following hybridization. For example, where biotin labeled dNTPs are used in, e.g., amplification or transcription, streptavidin linked reporter groups may be used to label hybridized complexes. Such operations are readily integratable into the systems of the present invention. Alternatively, the nucleic acids in the sample may be labeled following amplification. Post amplification labeling typically involves the covalent attachment of a particular detectable group upon the amplified sequences. Suitable labels or detectable groups include a variety of fluorescent or radioactive labeling groups well known in the art. These labels may also be coupled to the sequences using methods that are well known in the art.  
       [0216] Methods for detection depend upon the label selected. A fluorescent label is preferred because of its extreme sensitivity and simplicity. Standard labeling procedures are used to determine the positions where interactions between a sequence and a reagent take place. For example, if a target sequence is labeled and exposed to a matrix of different probes, only those locations where probes do interact with the target will exhibit any signal. Alternatively, other methods may be used to scan the matrix to determine where interaction takes place. Of course, the spectrum of interactions may be determined in a temporal manner by repeated scans of interactions which occur at each of a multiplicity of conditions. However, instead of testing each individual interaction separately, a multiplicity of sequence interactions may be simultaneously determined on a matrix.  
       [0217] Means of detecting labeled target (sample) nucleic acids hybridized to the probes of the high density array are known to those of skill in the art. Thus, for example, where a colorimetric label is used, simple visualization of the label is sufficient. Where a radioactive labeled probe is used, detection of the radiation (e.g with photographic film or a solid state detector) is sufficient.  
       [0218] In a preferred embodiment, however, the target nucleic acids are labeled with a fluorescent label and the localization of the label on the probe array is accomplished with fluorescent microscopy. The hybridized array is excited with a light source at the excitation wavelength of the particular fluorescent label and the resulting fluorescence at the emission wavelength is detected. In a particularly preferred embodiment, the excitation light source is a laser appropriate for the excitation of the fluorescent label.  
       [0219] The target polynucleotide may be labeled by any of a number of convenient detectable markers. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. Other potential labeling moieties include, radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes. Another method for labeling may bypass any label of the target sequence. The target may be exposed to the probes, and a double strand hybrid is formed at those positions only. Addition of a double strand specific reagent will detect where hybridization takes place. An intercalative dye such as ethidium bromide may be used as long as the probes themselves do not fold back on themselves to a significant extent forming hairpin loops. However, the length of the hairpin loops in short oligonucleotide probes would typically be insufficient to form a stable duplex.  
       [0220] Suitable chromogens will include molecules and compounds which absorb light in a distinctive range of wavelengths so that a color may be observed, or emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluorescers. Biliproteins, e.g., phycoerythrin, may also serve as labels.  
       [0221] A wide variety of suitable dyes are available, being primarily chosen to provide an intense color with minimal absorption by their surroundings. Illustrative dye types include quinoline dyes, triarylmethane dyes, acridine dyes, alizarine dyes, phthaleins, insect dyes, azo dyes, anthraquinoid dyes, cyanine dyes, phenazathionium dyes, and phenazoxonium dyes.  
       [0222] A wide variety of fluorescers may be employed either by themselves or in conjunction with quencher molecules. Fluorescers of interest fall into a variety of categories having certain primary functionalities. These primary functionalities include 1- and 2-aminonaphthalene, p,p′-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidzaolylphenylamine, 2-oxo-3-chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins, triarylmethanes and flavin. Individual fluorescent compounds which have functionalities for linking or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6-sulfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid; pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl, N-methyl 2-aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamine; N,N′-dioctadecyl oxacarbocyanine; N,N′-dihexyl oxacarbocyanine; merocyanine, 4-(3′pyrenyl)butyrate; d-3-aminodesoxy-equilenin; 12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene; 2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis&gt;2-(4-methyl-5-phenyl-oxazolyl)!benzene; 6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium) 1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin; chlorotetracycline; N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide; N-&gt;p-(2-benzimidazolyl)-phenyl!maleimide; N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin; 4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resomufin; rose bengal; and 2,4-diphenyl-3(2H)-furanone.  
       [0223] Desirably, fluorescers should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound dye may differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent.  
       [0224] Fluorescers are generally preferred because by irradiating a fluorescer with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.  
       [0225] Detectable signal may also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. One family of compounds is 2,3-dihydro-1,-4-phthalazinedione. The most popular compound is luminol, which is the 5-amino compound. Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylamino&gt;ca!benz analog. These compounds can be made to luminesce with alkaline hydrogen peroxide or calcium hypochlorite and base. Another family of compounds is the 2,4,5-triphenylimidazoles, with lophine as the common name for the parent product. Chemiluminescent analogs include para-dimethylamino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.  
       [0226] Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like. Exemplary spin labels include nitroxide free radicals.  
       [0227] Fragmentation  
       [0228] In addition, amplified sequences may be subjected to other post amplification treatments. For example, in some cases, it may be desirable to fragment the sequence prior to hybridization with an oligonucleotide array, in order to provide segments which are more readily accessible to the probes, which avoid looping and/or hybridization to multiple probes. Fragmentation of the nucleic acids may generally be carried out by physical, chemical or enzymatic methods that are known in the art.  
       [0229] Sample Analysis  
       [0230] Following the various sample preparation operations, the sample will generally be subjected to one or more analysis operations. Particularly preferred analysis operations include, e.g., sequence based analyses using an oligonucleotide array and/or size based analyses using, e.g., microcapillary array electrophoresis.  
       [0231] Capillary Electrophoresis  
       [0232] In some embodiments, it may be desirable to provide an additional, or alternative means for analyzing the nucleic acids from the sample  
       [0233] Microcapillary array electrophoresis generally involves the use of a thin capillary or channel which may or may not be filled with a particular separation medium. Electrophoresis of a sample through the capillary provides a size based separation profile for the sample. Microcapillary array electrophoresis generally provides a rapid method for size based sequencing, PCR product analysis and restriction fragment sizing. The high surface to volume ratio of these capillaries allows for the application of higher electric fields across the capillary without substantial thermal variation across the capillary, consequently allowing for more rapid separations. Furthermore, when combined with confocal imaging methods, these methods provide sensitivity in the range of attomoles, which is comparable to the sensitivity of radioactive sequencing methods.  
       [0234] In many capillary electrophoresis methods, the capillaries, e.g., fused silica capillaries or channels etched, machined or molded into planar substrates, are filled with an appropriate separation/sieving matrix. Typically, a variety of sieving matrices are known in the art may be used in the microcapillary arrays. Examples of such matrices include, e.g., hydroxyethyl cellulose, polyacrylamide, agarose and the like. Gel matrices may be introduced and polymerized within the capillary channel. However, in some cases, this may result in entrapment of bubbles within the channels which can interfere with sample separations. Accordingly, it is often desirable to place a preformed separation matrix within the capillary channel(s), prior to mating the planar elements of the capillary portion. Fixing the two parts, e.g., through sonic welding, permanently fixes the matrix within the channel. Polymerization outside of the channels helps to ensure that no bubbles are formed. Further, the pressure of the welding process helps to ensure a void-free system.  
       [0235] In addition to its use in nucleic acid “fingerprinting” and other sized based analyses, the capillary arrays may also be used in sequencing applications. In particular, gel based sequencing techniques may be readily adapted for capillary array electrophoresis.  
       [0236] Expression Products  
       [0237] In addition to detection of mRNA or as the sole detection method expression products from the genes discussed above may be detected as indications of the biological condition of the tissue. Expression products may be detected in either the tissue sample as such, or in a body fluid sample, such as blood, serum, plasma, faeces, mucus, sputum, cerebrospinal fluid, and/or urine of the individual.  
       [0238] The expression products, peptides and proteins, may be detected by any suitable technique known to the person skilled in the art.  
       [0239] In a preferred embodiment the expression products are detected by means of specific antibodies directed to the various expression products, such as immunofluorescent and/or immunohistochemical staining of the tissue.  
       [0240] Immunohistochemical localization of expressed proteins may be carried out by immunostaining of tissue sections from the single tumors to determine which cells expressed the protein encoded by the transcript in question. The transcript levels were used to select a group of proteins supposed to show variation from sample to sample, making possible a rough correlation between level of protein detected and intensity of the transcript on the microarray.  
       [0241] For example sections were cut from paraffin-embedded tissue blocks, mounted, and deparaffinized by incubation at 80 C° for 10 min, followed by immersion in heated oil at 60 C for 10 min (Estisol 312, Estichem A/S, Denmark) and rehydration. Antigen retrieval is achieved in TEG (TrisEDTA-Glycerol) buffer using microwaves at 900 W. The tissue sections cooled in the buffer for 15 min before a brief rinse in tap water. Endogenous peroxidase activity is blocked by incubating the sections with 1% H202 for 20 min, followed by three rinses in tap water, 1 min each. The sections are then soaked in PBS buffer for 2 min. The next steps are modifi d from the descriptions given by Oncogene Science Inc., in the Mouse Immunohistochemistry Detection System, XHCO1 (UniTect, Uniondale, N.Y., USA). Briefly, the tissue sections are incubated overnight at 4 C with primary antibody (against beta-2 microglobulin (Dako), cytokeratin 8, cystatin-C (both from Europa, US), junB, CD59, E-cadherin, apo-E, cathepsin E, vimentin, IGFII (all from Santa Cruz), followed by three rinses in PBS buffer for 5 min each. Afterwards, the sections are incubated with biotinylated secondary antibody for 30 min, rinsed three times with PBS buffer and subsequently incubated with ABC (avidin-biotinlylated horseradish peroxidase complex) for 30 min, followed by three rinses in PBS buffer.  
       [0242] Staining is performed by incubation with AEC (3-amino-ethylcarbazole) for 10 min. The tissue sections are counter stained with Mayers hematoxylin, washed in tap water for 5 min. and mounted with glycerol-gelatin. Positive and negative controls may be included in each staining round with all antibodies.  
       [0243] In yet another embodiment the expression products may be detected by means of conventional enzyme assays, such as ELISA methods.  
       [0244] Furthermore, the expression products may be detected by means of peptide/protein chips capable of specifically binding the peptides and/or proteins assessed. Thereby an expression pattern may be obtained.  
       [0245] Assay  
       [0246] Thus, in a further aspect the invention relates to an assay for determining an expression pattern of a colon and/or rectum cell, comprising at least a first marker and/or a second marker, wherein the first marker is capable of detecting a gene from a first gene group as defined above, and the second marker is capable of detecting a gene from a second gene group as defined above.  
       [0247] In a preferred embodiment the assay comprises at least two markers for each gene group.  
       [0248] correlating the first expression level and the second expression level to a standard level of the assessed genes to determine the presence or absence of a biological condition in the animal tissue.  
       [0249] The marker (s) are preferably specifically detecting a gene as identified herein, in particular the genes of the tables in the examples and as discussed above.  
       [0250] As discussed above the marker may be any nucleotide probe, such as a DNA, RNA, PNA, or LNA probe capable of hybridising to mRNA indicative of the expression level. The hybridisation conditions are preferably as described below for probes.  
       [0251] In another embodiment the marker is an antibody capable of specifically binding the expression product in question.  
       [0252] Detection  
       [0253] Patterns can be compared manually by a person or by a computer or other machine. An algorithm can be used to detect similarities and differences. The algorithm may score and compare, for example, the genes which are expressed and the genes which are not expressed. Alternatively, the algorithm may look for changes in intensity of expression of a particular gene and score changes in intensity between two samples. Similarities may be determined on the basis of genes which are expressed in both samples and genes which are not expressed in both samples or on the basis of genes whose intensity of expression are numerically similar.  
       [0254] Generally, the detection operation will be performed using a reader device external to the diagnostic device. However, it may be desirable in some cases, to incorporate the data gathering operation into the diagnostic device itself.  
       [0255] The detection apparatus may be a fluorescence detector, or a spectroscopic detector, or another detector.  
       [0256] Although hybridization is one type of specific interaction which is clearly useful for use in this mapping embodiment, antibody reagents may also be very useful.  
       [0257] Data Gathering and Analysis  
       [0258] Gathering data from the various analysis operations, e.g., oligonucleotide and/or microcapillary arrays, will typically b carried out using methods known in the art. For example, the arrays may be scanned using lasers to excite fluorescently labeled targets that have hybridized to regions of probe arrays mentioned above, which can then be imaged using charged coupled devices (“CCDs”) for a wide field scanning of the array. Alternatively, another particularly useful method for gathering data from the arrays is through the use of laser confocal microscopy which combines the ease and speed of a readily automated process with high resolution detection.  
       [0259] Following the data gathering operation, the data will typically be reported to a data analysis operation. To facilitate the sample analysis operation, the data obtained by the reader from the device will typically be analyzed using a digital computer. Typically, the computer will be appropriately programmed for receipt and storage of the data from the device, as well as for analysis and reporting of the data gathered, i.e., interpreting fluorescence data to determine the sequence of hybridizing probes, normalization of background and single base mismatch hybridizations, ordering of sequence data in SBH applications, and the like.  
       [0260] It is an object of the present invention to provide a biological sample which may be classified or characterized by analyzing the pattern of specific interactions mentioned above. This may be applicable to a cell or tissue type, to the messenger RNA population expressed by a cell to the genetic content of a cell, or to virtually any sample which can be classified and/or identified by its combination of specific molecular properties.  
       [0261] Pharmaceutical Composition  
       [0262] The invention also relates to a pharmaceutical composition for treating the bioligical condition, such as colorectal tumors.  
       [0263] In one embodiment the pharmaceutical composition comprises one or more of the peptides being expression products as defined above. In a preferred embodiment, the peptides are bound to carriers. The peptides may suitably be coupled to a polymer carrier, for example a protein carrier, such as BSA. Such formulations are well-known to the person skilled in the art.  
       [0264] The peptides may be suppressor peptides normally lost or decreased in tumor tissue administered in order to stabilise tumors towards a less malignant stage. In another embodiment the peptides are onco-peptides capable of eliciting an immune response towards the tumor cells.  
       [0265] In another embodiment the pharmaceutical composition comprises genetic material, either genetic material for substitution therapy, or for suppressing therapy as discussed below.  
       [0266] In a third embodiment the pharmaceutical composition comprises at least one antibody produced as described above.  
       [0267] In the present context the term pharmaceutical composition is used synonymously with the term medicament. The medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition as defined above in combination with pharmaceutically acceptable additives. Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration. For most indications a localised or substantially localised application is preferred.  
       [0268] Strategies in formulation development of medicaments and compositions based on the compounds of the present invention generally correspond to formulation strategies for any other protein-based drug product. Potential problems and the guidance required to overcome these problems are dealt with in several textbooks, e.g. “Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga, Technomic Publishing AG, Basel, 1995.  
       [0269] Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. The preparation may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or which nhance the effectiven ss or transportation of the preparation.  
       [0270] Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art. The formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.  
       [0271] The preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect. Additional formulations which are suitable for other modes of administration include suppositories, and, in some cases, oral formulations. For suppositories, traditional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.  
       [0272] The preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosag ranges are of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 1000 μg, such as in the range of from about 1 μg to 300 μg, and especially in the range of from about 10 μg to 50 μg. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosis would be in the interval 30 mg to 70 mg per 70 kg body weight.  
       [0273] Some of the compounds of the present invention are sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous to include a compound, which promote delivery of the active substance to its target.  
       [0274] In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc.  
       [0275] Vaccines  
       [0276] In a further embodiment the present invention relates to a vaccine for the prophylaxis or treatment of a biological condition comprising at least one expression product from at least one gene said gene being expressed as defined above.  
       [0277] The term vaccines is used with its normal meaning, i.e preparations of immunogenic material for administration to induce in the recipient an immunity to infection or intoxication by a given infecting agent. Vaccines may be administered by intravenous injection or through oral, nasal and/or mucosal administration. Vaccines may be either simple vaccines prepared from one species of expression products, such as proteins or peptides, or a variety of expression products, or they may be mixed vaccines containing two or more simple vaccines. They are prepared in such a manner as not to destroy the immunogenic material, although the methods of preparation vary, depending on the vaccine.  
       [0278] The enhanced immune response achieved according to the invention can be attributable to e.g. an enhanced increase in the level of immunoglobulins or in the level of T-cells including cytotoxic T-cells will result in immunisation of at least 50% of individuals exposed to said immunogenic composition or vaccine, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 92%, such as at least 94%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 98.5%, for example at least 99%, for example at least 99.5% of the individuals exposed to said immunogenic composition or vaccine are immunised.  
       [0279] Compositions according to the invention may also comprise any carrier and/or adjuvant known in the art including functional equivalents thereof. Functionally equivalent carriers are capable of presenting the same immunogenic determinant in essentially the same steric conformation when used under similar conditions. Functionally equivalent adjuvants are capable of providing similar increases in the efficacy of the composition when used under similar conditions.  
       [0280] Therapy  
       [0281] The invention further relates to a method of treating individuals suffering from the biological condition in question, in particular for treating a colorectal tumor.  
       [0282] In one embodiment the invention relates to a method of substitution therapy, ie. administration of genetic material generally expressed in normal cells, but lost or decreased in biological condition cells (tumor suppressors). Thus, the invention relates to a method for reducing cell tumorigenicity of a cell, said method comprising  
       [0283] obtaining at least one gene selected from genes being expressed in an amount twofold higher in normal cells than the amount expressed in said tumor cell (tumor suppressors),  
       [0284] introducing said at least one gene into the tumor cell in a manner allowing expression of said gene(s).  
       [0285] The at least one gene is preferably selected individually from genes comprising a sequence as identified below  
                                      RC_H04768_at   chrom 15 no homology       RC_Z39652_at   Y14593 APM-1 gene adipocyte-specific se-           cretory protein; chrom 1q21.3-q23       RC_H30270_at   chrom 18 PAAAA in colon &amp; bladder no           homology       RC_T47089_s_at   tenascin-X; tenascin-X precursor; unidenti-           fied protein       RC_W31906_at   secretagogin; dJ501N12.8 (putative protein)           chrom 6       RC_AA279803_at   chrom 2 no homology       RC_R01646_at   chrom 13q32.1-33.3; AL159152; homolo-           gy to mouse Pcbp1 - poly(rC)-binding           protein 1       AA319615_at   secretory carrier membrane protein; secre-           tory carrier membrane protein 2; chrom 15                  
 
       [0286] and from  
                                      “Human chromogranin A” “mRNA,” “complete cds”   J03915       Human adipsin/complement factor D “mRNA,” com-   M84526       plete cds         Homo sapiens  MLC-1V/Sb isoform gene   M24248       Human aminopeptidase N/CD13 mRNA encoding   M22324       aminopeptidase “N,” complete cds         H. sapiens  MT-1I mRNA   X76717         H. sapiens  GCAP-II gene   Z70295       Human somatostatin I gene and flanks   J00306       Human YMP “mRNA,” complete cds   U52101         H. sapiens  mRNA for beta subunit of epithelial amiloride-   X87159       sensitive sodium channel       Human K12 protein precursor “mRNA,” complete cds   U77643       Human sulfate transporter (DTD) “mRNA,” complete cds   U14528       Human transcription factor hGATA-6 “mRNA,” complete   U66075       cds.         H. sapiens  SCAD “gene,” exon 1 and joining features   Z80345       Human S-lac lectin L-14-II (LGALS2) gene   M87860       Human mRNA for protein tyrosine phosphatase   D15049         H. sapiens  mRNA for tetranectin   X64559       Human 11 kd protein “mRNA,” complete cds   U28249       Human anti-mullerian hormone type II receptor precursor   U29700       “gene,” complete cds       Human heparin binding protein (HBp17) “mRNA,” complete   M60047       cds       Human ADP-ribosylation factor (hARF6) “mRNA,” complete   M57763       cds       beta -ADD = adducin beta subunit 63 kda isoform/membrane   S81083       skeleton protein, beta -ADD = adducin beta subunit 63 kda       isoform/membrane skeleton protein {alternatively spliced,       exon 10 to 13 region} [human, Genomic, 1851 nt, segment       3 of 3].       Zinc Finger Protein Znf155   HG4243-           HT4513       Human glucagon “mRNA,” complete cds   J04040         H. sapiens  mRNA for hair “keratin,” hHb5   X99140       Human tubulin-folding cofactor E “mRNA,” complete cds   U61232       Human integrin alpha-3 chain “mRNA,” complete cds   M59911       Human NACP gene   U46901         H. sapiens  mRNA for flavin-containing monooxygenase 5   Z47553       (FMO5)       Human mRNA for ATF-a transcription factor   X52943         H. sapiens  intestinal VIP receptor related protein mRNA   X77777                  
 
       [0287] In a preferred embodiment at least two different genes are introduced into the tumor cell.  
       [0288] In another aspect the invention relates to a therapy whereby genes generally correlated to disease are inhibited by one or more of the following methods:  
       [0289] A method for reducing cell tumorigenicity of a cell, said method comprising  
       [0290] obtaining at least one nucleotide probe capable of hybridising with at least one gene of a tumor cell, said at least one gene being selected from genes being expressed in an amount at least one-fold lower in normal cells than the amount expressed in said tumor cell, and  
       [0291] introducing said at least one nucleotide probe into the tumor cell in a manner allowing the probe to hybridise to the at least one gene, thereby inhibiting expression of said at least one gene. This method is preferably based on anti-sense technology, whereby the hybridisation of said probe to the gene leads to a down-regulation of said gene.  
       [0292] The down-regulation may of course also be based on a probe capable of hybridising to regulatory components of the genes in question, such as promoters.  
       [0293] The probes are preferably selected from probes capable of hybridising to a nucleotide sequence comprising a sequence as identified below  
                                          RC_AA609013_s_at   APPPP   microsomal dipeptidase (also               on 6.8 k); chrom 16       RC_AA232508_at   APPPP   CGI-89 protein; unnamed               protein product; hypothetical               protein       RC_AA428964_at   APPPP   serine protease-like protease;               serine protease homo-               log = NES1; normal epithelial               cell-specific 1       RC_T52813_s_at   APPPP   dJ28O10.2 (G0S2 (PUTATIVE               LYMPHOCYTE G0/G1               SWITCH PROTEIN 2; chrom 1       RC_AA075642_at   APPPP   gp-340 variant protein;               DMBT1/8kb.2 protein       RC_AA007218_at   APPPP   chrom 13 no homology       RC_N33920_at   APPPP   ubiquitin-like protein FAT10;               diubiquitin; dJ271M21.6 (Diu-               biquitin); chrom 6       RC_N71781_at   APPPP   KIAA1199 protein, chrom 15       RC_R67275_s_at   APPPP   alpha-1 (type XI) collagen pre-               cursor; collagen, type XI, alpha               1; collagen type XI alpha-1               isoform A; chrom 1       RC_W80763_at   APPPP   hypothetical protein; chrom 17       RP_AA443793_at   APPPP   chrom 7p22 AC006028 BAC               clone       RC_AA034499_s_at   APPPP   ZNF198 protein; zinc finger               protein; FIM protein; Cys-rich               protein; zinc finger protein 198;               chrom 13       RC_AA035482_at   APPPP   chrom 5; AK022505 clone;               CalcineurinB (weakly similar)       RC_AA024482_at   APPPP   hypothetical protein; unnamed               protein product; chrom 17       RC_H93021_at   APPPP   chrom 2; XM_004890 pep-               tidylprolyl isomerase A (cy-               clophilin A)       RC_AA427737_at   APPPP   no homology       RC_AA417078_at   APPPP   chrom 7q31; AF017104 clone       M29873_s_at   APPPP   cytochrome P450-IIB (hIIB3);               19q13.1-q13.2       RC_H27498_f_at   AAPPP       RC_T92363_s_at   AAPPP       RC_N89910_at   AAAPP       RC_W60516_at   AAAPP       RC_AA219699_at   AAAPP       RC_AA449450_at   AAAPP                  
 
       [0294] Or from  
                                        Homo sapiens  (clones “MDP4,” MDP7) microsomal   J05257       dipeptidase (MDP) “mRNA,” complete cds       “ Homo sapiens  reg gene” “homologue,” “compl t   L08010       cds”         H. sapiens  mRNA for prepro-alpha2(I) collagen   Z74616       “Human S-adenosylhomocysteine hydrolase (AHCY)”   M61832       “mRNA,” “complete cds”       Transcription Fact r Iiia   HG4312-           HT4582       Human gene for melanoma growth stimulatory activity   X54489       (MGSA)       Human stromelysin-3 mRNA   X57766       CDC25Hu2 = cdc25 + homolog “[human,” “mRNA,”   S78187       3118 nt]       Human mRNA for cripto protein   X14253       Human transformation-sensitive protein (IEF SSP 3521)   M86752       “mRNA,” complete cds       Human complement component 2 (C2) gene allele b   L09708         H. sapiens  mRNA for ITBA2 protein   X92896         H. sapiens  encoding CLA-1 mRNA   222555       “Human fibroblast growth factor receptor 4 (FGFR4)”   L03840       “mRNA,” “complete cds”       “”“Fibronectin,” “Alt. Splice 1”   HG3044-           HT3742       tyk2   X54667       Human mRNA for B-myb gene   X13293       “Human phosphofructokinase (PFKM)” “mRNA,”   U24183       “ complete cds”       Human pre-B cell enhancing factor (PBEF) “mRNA,” com-   U02020       plete cds       Human SH2-containing inositol 5-phosphatase (hSHIP)   U57650       “mRNA,” complete cds       Human interleukin 8 (IL8) “gene,” complete cds   M28130       “Human lamin B receptor (LBR)” “mRNA,”   L25931       “complete cds”         H. sapiens  mRNA for protein tyrosine phosphatase   Z48541       Human mRNA for unc-18 “homologue,” complete cds   D63851         H. sapiens  mRNA for Zn-alpha2-glycoprotein   X59766           Z25521       “Human asparagine synthetase” “mRNA,”   M27396       “complete cds”       Human hepatitis delta antigen interacting protein A (dipA)   U63825       “mRNA,” complete cds       Human splicesomal protein (SAP 61) “mRNA,” complete   U08815       cds       Human protein kinase C-binding protein RACK7 “mRNA,”   U48251       partial cds       Human MAC30 “mRNA,” 3′ end   L19183       Human thrombospondin 2 (THBS2) “mRNA,” complete cds   L12350       “Human nicotinamide N-methyltransferase (NNMT)”   U08021       “mRNA,” “complete cds”         H. sapiens  mRNA for type I interstitial collagenase   X54925       Human cytochrome b561 gene   U29463       Human H19 RNA “gene,” complete cds (spliced in sili-   M32053       co)       Human collagen type XVIII alpha 1 (COL18A1) “mRNA,”   L22548       partial cds       Human clone 23733 “mRNA,” complete cds.   U79274                  
 
       [0295] In another embodiment the probes consists of the sequences identified above.  
       [0296] The hybridization may be tested in vitro at conditions corresponding to in vivo conditions. Typically, hybridization conditions are of low to moderate stringency. These conditions favour specific interactions between completely complementary sequences, but allow some non-specific interaction between less than perfectly matched sequences to occur as well. After hybridization, the nucleic acids can be “washed” under moderate or high conditions of stringency to dissociate duplexes that are bound together by some non-specific interaction (the nucleic acids that form these duplexes are thus not completely complementary).  
       [0297] As is known in the art, the optimal conditions for washing are determined empirically, often by gradually increasing the stringency. The parameters that can be changed to affect stringency include, primarily, temperature and salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency. Washing can be initiated at a low temperature (for example, room temperature) using a solution containing a salt concentration that is equivalent to or lower than that of the hybridization solution. Subsequent washing can be carried out using progressively warmer solutions having the same salt concentration. As alternatives, the salt concentration can be lowered and the temperature maintained in the washing step, or the salt concentration can be lowered and the temperature increased. Additional parameters can also be altered. For example, use of a destabilizing agent, such as formamide, alters the stringency conditions.  
       [0298] In reactions where nucleic acids are hybridized, the conditions used to achieve a given level of stringency will vary. There is not one set of conditions, for example, that will allow duplexes to form between all nucleic acids that are 85% identical to one another; hybridization also depends on unique features of each nucleic acid. The length of the sequence, the composition of the sequence (for example, the content of purine-like nucleotides versus the content of pyrimidine-like nucleotides) and the type of nucleic acid (for example, DNA or RNA) affect hybridization. An additional consideration is whether one of the nucleic acids is immobilized (for example, on a filter).  
       [0299] An example of a progression from lower to higher stringency conditions is the following, where the salt content is given as the relative abundance of SSC (a salt solution containing sodium chloride and sodium citrate; 2×SSC is 10-fold more concentrated than 0.2×SSC). Nucleic acids are hybridized at 42° C. in 2×SSC/0.1% SDS (sodium dodecylsulfate; a detergent) and then washed in 0.2×SSC/0.1% SDS at room temperature (for conditions of low stringency); 0.2×SSC/0.1% SDS at 42° C. (for conditions of moderate stringency); and 0.1×SSC at 68° C. (for conditions of high stringency). Washing can be carried out using only one of the conditions given, or each of the conditions can be used (for example, washing for 10-15 minutes each in the order listed above). Any or all of the washes can be repeated. As mentioned above, optimal conditions will vary and can be determined empirically.  
       [0300] In another aspect a method of reducing tumoregeneicity relates to the use of antibodies against an expression product of a cell from the biological tissue. The antibodies may be produced by any suitable method, such as a method comprising the steps of  
       [0301] obtaining expression product(s) from at least one gene said gene being expressed as defined above for oncogenes,  
       [0302] immunising a mammal with said expression product(s) obtaining antibodies against the expression product.  
       [0303] Use  
       [0304] The methods described above may be used for producing an assay for diagnosing a biological condition in animal tissue, or for identification of the origin of a piece of tissue.  
       [0305] Furthermore, the invention relates to the use of a peptide as defined above for preparation of a pharmaceutical composition for the treatment of a biological condition in animal tissue.  
       [0306] Furthermore, the invention relates to the use of a gene as defined above for preparation of a pharmaceutical composition for the treatment of a biological condition in animal tissue.  
       [0307] Also, the invention relates to the use of a probe as defined above for preparation of a pharmaceutical composition for the treatment of a biological condition in animal tissue.  
       [0308] Gene Delivery Therapy  
       [0309] The genetic material discussed above for may be any of the described genes or functional parts thereof. The constructs may be introduced as a single DNA molecule encoding all of the genes, or different DNA molecules having one or more genes. The constructs may be introduced simultaneously or consecutively, each with the same or different markers.  
       [0310] The gene may be linked to the complex as such or protected by any suitable system normally used for transfection such as viral vectors or artificial viral envelope, liposomes or micellas, wherein the system is linked to the complex.  
       [0311] Numerous techniques for introducing DNA into eukaryotic cells are known to the skilled artisan. Often this is done by means of vectors, and often in the form of nucleic acid encapsidated by a (frequently virus-like) proteinaceous coat. Gene delivery systems may be applied to a wide range of clinical as well as experimental applications.  
       [0312] Vectors containing useful elements such as selectable and/or amplifiable markers, promoter/enhancer elements for expression in mammalian, particularly human, cells, and which may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art. Many are commercially available.  
       [0313] Various techniques have been developed for modification of target tissue and cells in vivo. A number of virus vectors, discussed below, are known which allow transfection and random integration of the virus into the host. See, for example, Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81:7529-7533; Kaneda et al., (1989) Science 243:375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86:3594-3598; Hatzoglu et al., (1990) J. Biol. Chem. 265:17285-17293; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381. Routes and modes of administering the vector include injection, e.g intravascularly or intramuscularly, inhalation, or other parenteral administration.  
       [0314] Advantages of adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms.  
       [0315] Another vector which can express the DNA molecule of the present invention, and is useful in gene therapy, particularly in humans, is vaccinia virus, which can be rendered non-replicating (U.S. Pat. Nos. 5,225,336; 5,204,243; 5,155,020; 4,769,330).  
       [0316] Based on the concept of viral mimicry, artificial viral envelopes (AVE) are designed based on the structure and composition of a viral membrane, such as HIV-1 or RSV and used to deliver genes into cells in vitro and in vivo. See, for example, U.S. Pat. No. 5,252,348, Schreier H. et al., J. Mol. Recognit., 1995, 8:59-62; Schreier H et al., J. Biol. Chem., 1994, 269:9090-9098; Schreier, H., Pharm. Acta Helv. 1994, 68:145-159; Chander, R et al. Life Sci., 1992, 50:481-489, which references are hereby incorporated by reference in their entirety. The envelope is preferably produced in a two-step dialysis procedure where the “naked” envelope is formed initially, followed by unidirectional insertion of the viral surface glycoprotein of interest. This process and the physical characteristics of the resulting AVE are described in detail by Chander et al., (supra). Examples of AVE systems are (a) an AVE containing the HIV-1 surface glycoprotein gp160 (Chander et al., supra; Schreier et al., 1995, supra) or glycosyl phosphatidylinositol (GPI)-linked gp120 (Schreier et al., 1994, supra), respectively, and (b) an AVE containing the respiratory syncytial virus (RSV) attachment (G) and fusion (F) glycoproteins (Stecenko, A. A. et al., Pharm. Pharmacol. Lett. 1:127-129 (1992)). Thus, vesicles are constructed which mimic the natural membranes of enveloped viruses in their ability to bind to and deliver materials to cells bearing corresponding surface receptors.  
       [0317] AVEs are used to deliver genes both by intravenous injection and by instillation in the lungs. For example, AVEs are manufactured to mimic RSV, exhibiting the RSV F surface glycoprotein which provides selective entry into epithelial cells. F-AVE are loaded with a plasmid coding for the gene of interest, (or a reporter gene such as CAT not present in mammalian tissue).  
       [0318] The AVE system described herein in physically and chemically essentially identical to the natural virus yet is entirely “artificial”, as it is constructed from phospholipids, cholesterol, and recombinant viral surface glycoproteins. Hence, there is no carryover of viral genetic information and no danger of inadvertant viral infection. Construction of the AVEs in two independent steps allows for bulk production of the plain lipid envelopes which, in a separate second step, can then be marked with the desired viral glycoprotein, also allowing for the preparation of protein cocktail formulations if desired.  
       [0319] Another delivery vehicle for use in the present invention are based on the recent description of attenuated Shigella as a DNA delivery system (Sizemore, D. R. et al., Science 270:299-302 (1995), which reference is incorporated by reference in its entirety). This approach exploits the ability of Shigellae to enter epithelial cells and escape the phagocytic vacuole as a method for delivering the gene construct into the cytoplasm of the target cell. Invasion with as few as one to five bacteria can result in expression of the foreign plasmid DNA delivered by these bacteria.  
       [0320] A preferred type of mediator of nonviral transfection in vitro and in vivo is cationic (ammonium derivatized) lipids. These positively charged lipids form complexes with negatively charged DNA, resulting in DNA charged neutralization and compaction. The complexes endocytosed upon association with the cell membrane, and the DNA somehow escapes the endosome, gaining access to the cytoplasm. Cationic lipid:DNA complexes appear highly stable under normal conditions. Studies of the cationic lipid DOTAP suggest the complex dissociates when the inner layer of the cell membrane is destabilized and anionic lipids from the inner layer displace DNA from the cationic lipid. Several cationic lipids are available commercially. Two of these, DMRI and DC-cholesterol, have been used in human clinical trials. First generation cationic lipids are less efficient than viral vectors. For d livery to lung, any inflammatory responses accompanying the liposome administration ar reduced by changing the delivery mode to aerosol administration which distributes the dose more evenly.  
       [0321] Drug Screening  
       [0322] Genes identified as changing in various stages of colorectal cancer can be used as markers for drug screening. Thus by treating colorectal cancer cells with test compounds or extracts, and monitoring the expression of genes identified as changing in the progression of colorectal cancers, one can identify compounds or extracts which change expression of genes to a pattern which is of an earlier stage or even of normal colorectal mucosa.  
       [0323] The following are non-limiting examples illustrating the present invention.  
       [0324] Experimentals  
       [0325] We have used two different approaches to identify tumor suppressors, oncogenes and classifiers. The first approach was based on a spreadsheet approach in which we used the fold change and the pattern of expression being present or absent in the different preparations of RNA. The second approach was based on a mathematical approach in which we used correlation to a predefined profile as selection criteria based on Pearsons correlation coefficient. 
     
    
    
     EXAMPLES  
     Example 1  
     [0326] Quantification of Gene Expression Using Microarrays  
     [0327] Material  
     [0328] Colon tumor and normal oral resection edge biopsies were sampled from each patient after informed consent was obtained, and after removal of the necessary amount of tissue for routine pathological examination. Number of Tissue examined was: Normal resection edge 6, Dukes A, 5; B, 6; C, 6; D,4. The six normal tissue samples were all from Dukes A individuals.  
     [0329] RNA from Different tumors of the same stage were combined to form each pool. Five isuch pools were prepared as Normal pool, Dukes A pool, Dukes B pool, Dukes C pool, Dukes D pool. All tumors and normal tissue specimens were from the sigmoid or upper rectum.  
     [0330] Preparation of mRNA  
     [0331] Total mRNA was isolated using the RNAzol B RNA isolation method (WAK-Chemie Medical GMBH). Poly (A)+ RNA was isolated by an oligo-dT selection step (Oligotex mRNA kit from Qiagen).  
     [0332] Preparation of cRNA  
     [0333] One μg mRNA was used as starting material for the cDNA preparation. The first and second strand cDNA synthesis was performed using the SuperScript Choice System (Life Technologies) according to the manufacturer&#39;s instructions, except that an oligo-dT primer containing a T7 RNA polymerase promoter site was used. Labeled cRNA was prepared using the MEGAscript In Vitro Transcription kit (Ambion). Biotin labeled CTP and UTP (Enzo) was used in the reaction together with unlabeled NTP&#39;s. Following the IVT reaction, the unincorporated nucleotides were removed using RNeasy columns (Qiagen).  
     [0334] Array Hybridization and Scanning  
     [0335] Ten μg of cRNA was fragmented at 94° C. for 35 min. In a fragmentation buffer containing 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Prior to hybridization, the fragmented cRNA in a 6×SSPE-T hybridization buffer (1 M NaCL, 10 mM Tris pH 7.6, 0.005% Triton) was heated to 95° C. for 5 min. And subsequently to 40° C. for 5 min. Before loading onto an Affymetrix probe array cartridge. The probe array was then incubated for 16 h at 40° C. at constant rotation (60 rpm). The washing and staining procedure was performed in the Affymetrix Fluidics Station. The probe array was exposed to 10 washes in 6×SSPE-T at 25° C. followed by 4 washes in 0.5×SSPE-T at 50° C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, 10 μg/ml (Molecular Probes, Eugene, Oreg.) in 6×SSPE-T for 30 min. at 25° C. followed by 10 washes in 6×SSPE-T at 25° C. The prove arrays were scanned at 560 nm using a confocal laser scanning microscope with an argon ion laser as the excitation source (made for Affymetrix by Molecular Dynamics). Following this scan, the array was incubated with an anti-avidin antibody and an biotinylated anti-immunoglobulin, and the streptavidin-phycoerythrin step was repeated.  
     [0336] The readings from the quantitative scanning were analyzed by the Affymetrix Gene Expression Analysis Software.  
     [0337] Normalization of Data  
     [0338] To compare samples, normalization of the data was necessary. For that purpose we compared scaling to total GAPDH intensity (sum of 3′, middle, 5′ probe sets) of 7000 units with scaling to a total array intensity (global scaling) of 281850 units (averaging 150 units per probe set). Both gave similar results with scaling factors that differed less than ten percent in a set of experiments. Based on this we chose the global scaling for all experiments.  
     Example 2  
     [0339] Change of Transcript Level During the Progression of Colon Cancer  
     [0340] Biopsies from human colon tumors were analyzed as pools of tumors representing the different stages in the progression of the colon cancer disease. A total of 4 tumor pools were used, each pool made by combining four to six tumors (see materials and methods). To generate a normal reference material, we pooled biopsies from normal colon mucosa from six volunteers.  
     [0341] From the biopsies RNA was extracted, reverse transcribed to cDNA and the cDNA transcribed into labelled cRNA, that was incubated on the array cartridges followed by scanning and scaling to a global array intensity amounting to 150 units per probe set. The scaling made it possible to compare individual experiments to each other. To verify the reproducibility, double determinations were made in selected cases and showed a good correlation.  
     [0342] The software GeneArray Analysis Suite 3.1 from Affymetrix, Inc. Was used to analyse the array data. In this software, increased levels indicat that the transcript is either up-regulated at the stated level or turned on de novo reaching a given fold above the background level. Decreased levels in a similar way indicate reduction or loss of transcript. Alterations of a single transcript during the progression of the colon cancer disease can follow several different pathways. Some of the transcript changes reflect the transition from normal cells to tumor cells, Others an increase in malignancy from Dukes A to Dukes B.  
     Example 2  
     [0343] A. Finding Classifiers of and Predictors etc. of Colorectal Cancer Based on a Spreadsheat Approach.  
     [0344] We used a spreadsheat to sort genes based on different parameters obtained from the Affymetrix analysis software.  
     [0345] The mRNA expression analysis on the AFFYMETRIX ARRAYs resulted in 42.843 datasets identifying individual genes (table I) or EST&#39;s (table II),altogether. These were obtained from the 6.8 k Arrays (7.129 datasets) and the EST ARRAYs (35.714 datasets)  
     [0346] Description of the Sorting Procedure for the Spreadsheat Sorting,  
     [0347] Per dataset the following was listed,  
     [0348] Probe Set No., Present or absent in Normal tissue or the different Duke&#39;s types, gene name or homoogy or number, “AvgDiff” which is the level of expression, “Abs Call” which determines if the gene is present (P) or absent (A), “Diff call” which determines the alteration as increasing (I) or decreasing (D), “fold change” the fold change from normal tissue expression level, and the “sort score” which determines the likelihood that it is real changes (if above 0.5).  
     [0349] The following steps were performed,  
     [0350] 1. exclude data if “Probe Set” is an AFFX-marker (58/array or sub-array)  
     [0351] 2. exclude data if “Diff Call” in all 4 comparisons is “NC” (no change)  
     [0352] 3. exclude data if “Abs Call” in all 4 comparisons is “A” (absent)  
     [0353] 4. exclude data if three “Abs call” ar “NC” and one is “Ml or MD” 
     [0354] 5. select data with absolute value of |sort score| arbitrarily set to &gt;=0,5  
     [0355] (At this step the sorting resulted in the following number of genes sorted as being of importance, 908 Genes (12,7%) and 4155 ESTs (11,6%)  
     [0356] 6. sort according to pattern of Abs Calls (e.g. PAAAA=lost from N to tumour Duke ABCD)  
     [0357] 7. select data with Avg Diff of &gt;=300 (500 for some ESTs) and/or fold change &gt;=3 (&gt;=5 for some ESTs)  
     [0358] Number of genes sorted out as being of interest after this final sorting, 130 Genes (1,8%), ≈240 ESTs (0,7%)  
     [0359] The following tables show the genes (Table I) and EST&#39;+s (Table II) that were identified by this approach, analyzing the hu 6.8K FI gene array. First a list of the potential tumor suppressors, then a list of the potential oncogenes, finally a list of genes that can be used to classify the different Dukes Stages. Genes that are in bold are those that we find are of the utmost interest.  
     [0360] The table (Table III) that follow this section are based on the hu EST arrays Hu35k Sub A,B,C,D. These are also divided into EST&#39;s that are supposed to be expressed from tumor suppressors, and oncogenes, as well as from genes that can be used as classifiers of the different Dukes stages. The most intersting Est&#39;s are shown in bold.  
               TABLE I                          Fold Change in comparison to normal       SUPPRESSOR CLASSIFIER                                 Gen nam   Acc No   Avg Diff   Avg Diff                                             CRC classifier genes lost PAAAA or PPAAA       N   A   B           “Human chromogranin A” “mRNA,” “complete cds”   J03915   831   lost   lost       Human adipsin/complement factor D “mRNA,” com-   M84526   822   lost   lost       plete cds         Homo sapiens  MLC-1V/Sb isoform gene   M24248   799   lost   lost       Human aminopeptidase N/CD13 mRNA encoding   M22324   657   lost   lost       aminopeptidase “N,” complete cds         H. sapiens  MT-1I mRNA   X76717   650   lost   lost         H. sapiens  GCAP-II gene   Z70295   572   lost   lost       Human somatostatin I gene and flanks   J00306   516   lost   lost       Human YMP “mRNA,” complete cds   U52101   459   lost   lost         H. sapiens  mRNA for beta subunit of epithelial amiloride-   X87159   439   lost   lost       sensitive sodium channel       Human K12 protein precursor “mRNA,” complete cds   U77643   429   121   lost       Human sulfate transporter (DTD) “mRNA,” complete cds   U14528   397   lost   lost       Human transcription factor hGATA-6 “mRNA,” complete   U66075   337   lost   lost       cds.         H. sapiens  SCAD “gene,” exon 1 and joining features   Z80345   326   lost   lost       Human S-lac lectin L-14-II (LGALS2) gene   M87860   301   lost   lost       Human mRNA for protein tyrosine phosphatase   D15049   277   43   lost         H. sapiens  mRNA for tetranectin   X64559   235   lost   lost       Human 11 kd protein “mRNA,” complete cds   U28249   233   47   lost       Human anti-mullerian hormone type II receptor precursor   U29700   223   lost   lost       “gene,” complete cds       Human heparin binding protein (HBp17) “mRNA,” com-   M60047   218   lost   lost       plete cds       Human ADP-ribosylation factor (hARF6) “mRNA,” com-   M57763   209   lost   lost       plete cds       beta -ADD = adducin beta subunit 63 kda iso-   S81083   188   lost   lost       form/membrane skeleton protein, beta -ADD = adducin       beta subunit 63 kda isoform/membrane skeleton protein       {alternatively spliced, exon 10 to 13 region} [human,       Genomic, 1851 nt, segment 3 of 3].       Zinc Finger Protein Znf155   HG4243-   186   lost   lost           HT4513       Human glucagon “mRNA,” complete cds   J04040   182   25   lost         H. sapiens  mRNA for hair “keratin,” hHb5   X99140   158   lost   lost       Human tubulin-folding cofactor E “mRNA,” complete cds   U61232   150   lost   lost       Human integrin alpha-3 chain”mRNA,” complete cds   M59911   126   lost   lost       Human NACP gene   U46901   123   lost   lost         H. sapiens  mRNA for flavin-containing monooxygenase 5   Z47553   110   lost   lost       (FMO5)       Human mRNA for ATF-a transcription factor   X52943   104   lost   lost         H. sapiens  intestinal VIP receptor related protein mRNA   X77777   93   lost   lost                                         Gene name   Acc No   Avg Diff   fold change to N                                             Only A Classifier       N   A                                               Homo sapiens  SKBIHs “mRNA,” complete cds.   AF015913   188   Lost               /gb = AF015913 /ntype = RNA       Mucin (Gb: M22406)   HG1067-   501   Lost           HT1067       Human platelet activating factor “acetylhydrolase,” brain   U72342   114   Lost       “isoform,” 45 kDa subunit (LIS1) gene         Homo sapiens  ERK activator kinase (MEK2) mRNA   L11285   1470   −5.2       Human 20-kDa myosin light chain (MLC-2) “mRNA,”   J02854   2047   −4.5       complete cds         H. sapiens  lysosomal acid phosphatase gene (EC   X15525   285   −4.4       3.1.3.2) Exon 1 (and joined CDS).       Human mRNA for matrix Gla protein   X53331   1069   −4.2         H. sapiens  mRNA for diacylglycerol kinase   X62535   362   −3.5       Human h at shock protein (hsp 70) gen, compl te cds.   M11717   405   −3.2       Human TRPM-2 protein gen   M63379   1594   −3       Only B Classifier       N   B       Human g ne for mitochondrial acetoacetyl-CoA thiolase   D10511   198   lost       Human mRNA for transcription factor “AREB6,” complete   D15050   232   lost       cds       Human mRNA for KIAA0248 “gen ,” partial cds   D87435   374   lost         Homo sapiens  (clone CC6) NADH-ubiquinone oxidore-   L04490   683   lost       ductase subunit “mRNA,” 3′ nd cds       Human phosphoglucomutase 1 (PGM1) “mRNA,”   M83088   1096   lost       complete cds         Homo sapiens  guanylin “mRNA,” complete cds   M97496   4983   lost       “Human trans-Golgi p230” “mRNA,”“complete cds”   U41740   131   lost         H. sapiens  mRNA for vacuolar proton “ATPase,” subunit   X71490   414   lost       D         H. sapiens  mRNA for 3-hydroxy-3-methylglutaryl   X83618   2196   lost       coenzyme A synthase       Human mRNA for KIAA0018 “gene,” complete cds   D13643   377   −7.7       “Mucin” “1.” “” “Epithelial,” “Alt. Splice 9”   HG371-   3296   −4.1           HT26388         H. sapiens  mRNA for L-3-hydroxyacyl-CoA dehydrogen-   X96752   252   −3       ase       Only C Classifier       N   C         Homo sapiens  colon mucosa-associated (DRA)   L02785   2978   Lost       “mRNA,” complete cds       Human Ig J chain gene   M12759   2193   Lost       Human selenium-binding protein (hSBP) “mRNA,”   U29091   1849   Lost       complete cds. /gb = U29091 /ntype = RNA         H. sapiens  mRNA for sigma 3B protein   X99459   722   Lost       Human ERK1 mRNA for protein serine/threonine   X60188   576   Lost       kinase       Human mRNA for mitochondrial 3-oxoacyl-CoA “thio-   D16294   529   Lost       lase,” complete cds       “Biliary” “Glycoprotein,”“Alt. Splice” “5,”“A”   HG2850-   489   Lost           HT4814       Human AQP3 gene for aquaporine 3 (water “channel),”   AB001325   413   Lost       partail cds       Human CD14 mRNA for myelid cell-specific leucine-rich   X13334   413   Lost       glycoprotein       Human thioredoxin “mRNA,” nuclear gene encoding   U78678   411   Lost       mitochondrial “protein,” complete cds       Human mitochondrial ATPase coupling factor 6 subunit   M37104   373   Lost       (ATP5A) “mRNA,” complete cds       “Human MHC class II HLA-DP light chain” “mRNA,”“   M57466   327   Lost       complete cds”       Human mRNA for early growth response protein 1   X52541   281   Lost       (hEGR1)       Human mRNA for mitochondrial 3-ketoacyl-CoA thiolase   D16481   268   Lost       beta-subunit of trifunctional “protein,” complete cds         Homo sapiens  laminin-related protein (LamA3) “mRNA,”   L34155   252   Lost       complete cds         H. sapiens  mRNA for selenoprotein P   Z11793   232   Lost       Human hkf-1 “mRNA,” complete cds   D76444   211   Lost         Homo sapiens  nuclear domain 10 protein (ndp52)   U22897   150   Lost       “mRNA,” complete cds       Human X104 “mRNA,” complete cds   L27476   149   Lost         H. sapien s cDNA for RFG   X77548   130   Lost         H. sapiens  mRNA for Progression Associated Protein   Y07909   128   Lost       Human liver “2,4-dienoyl-CoA” reductase “mRNA,” com-   U49352   101   Lost       plete cds       Human A33 antigen precursor “mRNA,” complete   U79725   1650   −6.9       cds         H. sapiens  pS2 protein gene   X52003   4298   −6       Human RASF-A PLA2 “mRNA,” complete cds   M22430   4983   −5.8         Homo sapiens  pstl mRNA for pancreatic secretory inhi-   Y00705   344   −3.1       bitor (expressed in neoplastic tissue).       Human CO-029   M35252   3500   −3       Only D Classifier       N   D       Human c mplement c mp nent C3 “mRNA,” alpha   K02765   744   lost       and beta “subunits,” complet cds         H. sapi ns  mRNA for adenosin “triphosphatase,”   Z69881   439   lost       calcium       Human skeletal muscle LIM-protein SLIM1 “mRNA,”   U60115   281   lost       complete cds       Human plat let-derived growth factor receptor alpha   M21574   187   lost       (PDGFRA) “mRNA,” complete cds       Human mRNA for KIAA0247 “gene,” complete cds   D87434   172   lost       Human mRNA for KIAA0171 “gen ,” complete cds   D79993   151   lost       Human Down syndrome critical regi n protein (DSCR1)   U28833   150   lost       “mRNA,” complete cds       Human Ki nuclear autoantigen “mRNA,” complete cds   U11292   125   lost       AB Classifier       N   A   B         Homo sapiens  chromosome 16 BAC clone CIT987SK-   AF001548   3513   −3.6   −4.3       815A9 complete sequence.       Human mRNA for ATP synthase alpha “subunit,” com-   D14710   3580   −3.8   −5.6       plete cds       BC Classifier       N   B   C       Human mRNA for IgG Fc binding “protein,” complete   D84239   3755   −19.3   −7.1       cds         H. sapiens  mRNA for carcinoembryonic “antigen,”   X98311   2456   −12   −6.5       CGM2       “ Homo sapiens  (clone lamda-hPEC-3) phosphoenol-   L05144   2630   −7.6   −14.7       pyruvate carboxykinase (PCK1)” “mRNA,”“complete       cds”       Human 11-beta-hydroxysteroid dehydrogenase type 2   U26726   1865   −7.1   −4.7       “mRNA,” complete cds       “Human intestinal mucin (MUC2)” “mRNA,”“complete   L21998   7803   −5.5   −4.2       cds”       Human mRNA for KIAA0106 “gene,” complete cds   D14662   766   −4.7   −3.2       metallothionein   V00594   5417   −4   −6.3                         Fold Change in comparison to normal       Oncogene CLASSSIFIER                                 Gene name   Acc No   Avg Diff   Avg Diff                                             CRC classifier genes gained APPPP or AAPPP       A   B                 Homo sapiens  (clones “MDP4,” MDP7) microsomal   J05257   1606   1403   gained       dipeptidase (MDP) “mRNA,” complete cds       “ Homo sapiens  reg gene” “homologue,”“complete   L08010   1165   294   gained       cds”         H. sapiens  mRNA for prepro-alpha2(I) collagen   Z74616   1003   905   gained       “Human S-adenosylhomocysteine hydrolase (AHCY)”   M61832   882   817   gained       “mRNA,” “complete cds”       Transcription Factor Iiia   HG4312-   837   948   gained           HT4582       Human gene for melanoma growth stimulatory activi-   X54489   731   330   gained       ty (MGSA)       Human stromelysin-3 mRNA   X57766   643   1116   gained       CDC25Hu2 = cdc25 + homolog “[human,” “mRNA,” 3118   S78187   603   627   gained       nt]       Human mRNA for cripto protein   X14253   532   293   gained       Human transformation-sensitive protein (IEF SSP   M86752   529   866   gained       3521) “mRNA,” complete cds       Human complement component 2 (C2) gene allele b   L09708   515   625   gained         H. sapiens  mRNA for ITBA2 protein   X92896   444   459   gained         H. sapiens  encoding CLA-1 mRNA   Z22555   422   549   gained       “Human fibroblast growth factor receptor 4 (FGFR4)”   L03840   359   276   gained       “mRNA,” “complete cds”       “” “Fibronectin,” “Alt. Splice 1”   HG3044-   354   261   gained           HT3742       tyk2   X54667   336   352   gain d       Human mRNA for B-myb gene   X13293   333   322   gained       “Human phosphofructokinase (PFKM)” “mRNA,”“com-   U24183   296   426   gain d       plete cds”       Human pre-B cell enhancing factor (PBEF) “mRNA,”   U02020   276   242   gained       compl te cds       Human SH2-containing inositol 5-phosphatase (hSHIP)   U57650   254   315   gained       “mRNA,” complete cds       Human Interteukin 8 (IL8) “gen ,” compl te cds   M28130   251   609   gain d       “Human lamin B receptor (LBR)” “mRNA,” “complete   L25931   239   193   gained       cds”         H. sapiens  mRNA for protein tyrosine phosphatase   Z48541   228   151   gained       Human mRNA for unc-18 “homologue,” complete cds   D63851   217   198   gained         H. sapiens  mRNA for Zn-alpha2-glycoprotein   X59766   215   156   gained           Z25521   215   127   gained       “Human asparagine synthetase” “mRNA,” “complete cds”   M27396   212   195   gained       Human hepatitis delta antigen interacting protein A (dipA)   U63825   211   231   gained       “mRNA,” complete cds       Human splicesomal protein (SAP 61) “mRNA,” complete   U08815   157   201   gained       cds       Human protein kinase C-binding protein RACK7   U48251   129   71   gained       “mRNA,” partial cds       Human MAC30 “mRNA,” 3′ end   L19183   128   224   gained       Human thrombospondin 2 (THBS2) “mRNA,” complete   L12350   111   126   gained       cds       “Human nicotinamide N-methyltransferase (NNMT)”   U08021   107   261   gained       “mRNA,” “complete cds”         H. sapiens  mRNA for type I interstitial collagenase   X54925   105   123   gained       Human cytochrome b561 gene   U29463   85   85   gained       Human H19 RNA “gene,” complete cds (spliced in   M32053   72   4498   gained       silico)       Human collagen type XVIII alpha 1 (COL18A1) “mRNA,”   L22548   67   275   gained       partial cds       Human clone 23733 “mRNA,” complete cds.   U79274   absent   162   gained                                     Gene name   Acc No   Avg Diff   fold change to N                                                 Only A Classifier       A                       Human migration inhibitory factor-related protein 8   M21005   120   GAINED       (MRP8) “gene,” complete cds       Human acyloxyacyl hydrolase “mRNA,” complete cds   M62840   130   GAINED       Human PEP19 (PCP4) “mRNA,” complete cds   U52969   174   GAINED         H. sapiens  Humig mRNA   X72755   118   GAINED         H. sapiens  PISSLRE mRNA   X78342   125   GAINED         H. sapiens  mRNA for twist “protein,” partial. /gb = Y11180   Y11180   121   GAINED       /ntype = RNA       Human mRNA for TGF-beta superfamily “protein,” com-   AB000584   1372   3.5       plete cds       Human mRNA for “MSS1,” complete cds   D11094   292   3.1       Human complement factor B “mRNA,” complete cds   L15702   2082   3.3       “ Homo sapiens  GTP-binding protein (RAB2)” “mRNA,”   M28213   289   3.1       “complete cds”       Human translational initiation factor 2 beta subunit (eIF-2-   M29536   956   4.1       beta) “mRNA,” complete cds       Human E16 “mRNA,” complete cds   M80244   278   3.8       IEX-1 = radiation-inducible immediate-early gene “[hu-   S81914   1531   3.6       man,” “placenta,” mRNA “Partial,” 1223 nt]       Human CDC16Hs “mRNA,” complete cds   U18291   244   6.1       Human DD96 “mRNA,” complete cds   U21049   625   3.2       Human (memc) “mRNA,” 3′UTR. /gb = U30999   U30999   256   3.8       /ntype = RNA       “Human ubiquitin-conjugating enzyme (UBE2I)”   U45328   448   10.6       “mRNA,” “complete cds”       “Human fetal brain glycogen phosphorylase B” “mRNA,”   U47025   2349   3.7       “complete cds”       “Human BTG2 (BTG2)” “mRNA,” “complete cds”   U72649   527   5.2       Human jun-B mRNA for JUN-B protein   X51345   1350   4.6       Only B Classifier       B       Human adipocyte lipid-binding “protein,” complete cds   J02874   268   GAINED       Human A1 protein “mRNA,” compl te cds   U29680   102   GAINED       Human LGN protein “mRNA,” complete cds   U54999   110   GAINED       Human sk letal muscle LIM-protein SLIM2 “mRNA,”   U60116   109   GAINED       partial cds       Human mRNA for alpha1-acid glycoprotein (orosomu-   X02544   156   GAINED       coid)       Human mRNA for fibronectin receptor alpha subunit   X06256   46   GAINED         H. sapiens  P1-Cdc21 mRNA   X74794   278   GAINED         H. sapiens  mRNA for fibulin-2   X82494   284   GAINED         H. sapiens  5T4 gene for 5T4 Oncofetal antigen   Z29083   152   GAINED         Homo sapiens  mRNA for osteoblast specific factor 2   D13666   324   7.6       (OSF-2os)       Mac25   HG987-HT987   2772   3.3       “Human lysozyme” “mRNA,” “complete cds with an Alu   J03801   920   3.7       repeat in the 3′ flank”       Human metalloproteinase (HME) “mRNA,” complete cds   L23808   794   7.4       Human alpha-1 collagen type IV gene, exon 52.   M26576   610   4.9       Human lumican “mRNA,” complete cds   U21128   1105   4.1       Human mRNA for fibronectin (FN precursor)   X02761   4181   5.5       Human mRNA fragment for elongation factor TU (N-   X03689   3515   3.1       terminus). /gb = X03689 /ntype = RNA       Human mRNA for type IV collagen alpha-2 chain   X05610   1531   3       Human mRNA for collagen VI alpha-1 C-terminal globu-   X15880   2062   3.5       lar domain       “ H. sapiens ,” gene for Membrane cofactor protein   X59405   272   3.4         H. sapiens  SOD-2 gene for manganese superoxide dis-   X65965   234   3.1       mutase. /gb = X65965 /ntype = DNA /annot = exon         H. sapiens  NMB mRNA   X76534   338   3.3         H. sapiens  vimentin gene   Z19554   3472   3.2       Only C Classifier       C       Ribosomal Protein L39 Homolog   HG2874-   102   GAINED           HT3018         Homo sapiens  (clone d2-115) kappa opioid receptor   L37362   168   GAINED       (OPRK1) “mRNA,” complete cds       Human kell blood group protein mRNA   M64934   143   GAINED           U73167   374   GAINED       Human cancellous bone osteoblast mRNA for serin   D87258   504   3.4       protease with IGF-binding “motif,” complete cds       Human interferon-inducible protein 27-Sep “mRNA,”   J04164   7717   3.8       complete cds       “Human sickle cell beta-globin” “mRNA,” “complete cds”   M25079   3090   4.6           M29277   1588   3.7       “Human spermidine synthase” “mRNA,” “complete cds”   M34338   866   4.1       Human copine 1 “mRNA,” complete cds   U83246   2079   3.7       Only D Classifier       D         Homo sapiens  FRG1 “mRNA,” complete cds   L76159   73   GAINED       Human cyclin protein “gene,” complete cds   M15796   149   GAINED       Human U2 small nuclear RNA-associated B″ antigen   M15841   194   GAINED       “mRNA,” complete cds       Human mRNA export protein Rae1 (RAE1) “mRNA,”   U84720   193   GAINED       complete cds.       Human protease-activated receptor 3 (PAR3) “mRNA,”   U92971   142   GAINED       complete cds.         H. sapiens  mRNA for mediator of receptor-induced toxi-   X84709   200   GAINED       city         H. sapiens  RFXAP mRNA   Y12812   230   GAINED       Human mRNA for “Qip1,” complete cds   AB002533   8881   2.7       Human mRNA for transferrin receptor   X01060   557   3       “metastasis-associated gene” “[human,” “highly metasta-   S79219   216   4       tic lung cell subline” “Anip[937],” “mRNA” “Partial,”“978       nt]”       AB Classifier       N   A   B       Human chaperonin 10 “mRNA,” complete cds   U07550   50   4.1   3.3         H. sapiens  RING4 cDNA   X57522   73   4.9   5.4         H. sapiens  genes TAP1, TAP2. LMP2, LMP7 and DOB.   X66401   134   3.2   3.1         H. sapiens  mRNA for alpha 4 prot in   Y08915   96   3.7   3.6         Homo sapiens  interl ukin-1 receptor-associat d kinase   L76191   285   3.1   3.1       (IRAK) “mRNA,” complet cds       “Human von Will brand factor” “mRNA,” “3′ end”   M10321   84   3.7   4.1       Human chromosome s gregation gen homolog CAS   U33286   86   4.8   3.6       “mRNA,” compl te cds       Human Bruton&#39;s tyrosine kinase-associated protein-135   U77948   68   3.4   4.9       “mRNA,” complete cds.       “Human KH type splicing regulatory protein KSRP”   U94832   52   3.2   3.2       “mRNA,” “complete cds.”         H. sapiens  ADE2H1 mRNA showing homologies to SAI-   X53793   40   3   3.1       CAR synthetase and AIR carboxylase of the purine       pathway (EC “6.3.2.6,” EC 4.1.1.21)       BC Classifier       N   B   C       “” “Globin,” “Beta”   HG1428-   504   3.1   4.3           HT1428       “Human alpha-1 collagen type 1” “gene,” “3′ end”   M55998   2706   3.1   3.7         H. sapiens  mRNA for SOX-4 protein   X70683   130   4.5   4.5       “Human mRNA for collagen binding protein” “2,” “com-   D83174   131   8.1   6.1       plete cds”       Human SPARC/osteonectin “mRNA,” complete cds   J03040   358   6.1   3.9       Human PRAD1 mRNA for cyclin   X59798   263   3.3   3.4       ABC Classifier       N   A   B   C       Human transforming growth factor-beta induced gene   M77349   426   4.7   6.7   4.4       product (BIGH3) “mRNA,” complete cds       “Human breast epithelial antigen BA46” “mRNA,” “com-   U58516   169   3.3   3.2   4.2       plete cds”           X57351   460   4.8   3.5   3.7         H. sapiens  NGAL gene   X99133   327   8.3   3.1   4.8       Human mRNA for MDNCF (monocyte-derived neu-   Y00787   87   5   9.2   13.4        trophil chemotactic factor)         H. sapiens  EF-1 delta gene encoding human elongation   Z21507   198   4.4   6.8   4.5       factor-1-delta         H. sapiens  mRNA for prepro-alpha1(I) collagen   Z74615   285   5   8.2   6.1       Nuclear Factor Nf-II6   HG3494-   246   4.3   4.4   4.2           HT3688           U29175   62   4.3   3.6   4.4       ABCD Classifier       N   A   B   C   D       “HNL = neutrophil lipocalin” “[human,” “ovarian can-   S75256   361   8.8   4.3   7.7   9       cer cell line” “OC6,” “mRNA” “Partial,” “534 nt].       /gb = S75256 /ntype = RNA”                  
 
     [0361]                                                       TABLE II                                  unknown genes, data                       Diff   Fold   Sort           Tumor   found after blast       Avg   Abs   Avg   Abs   Call   Change   Score       EST candidates   stages   search       Diff   Call   Diff   Call   Avs   Avs   Avs       Probe Set   NABCD   homologous to   EST name   N   N   A   A   N   N   N                         LOST from N to tumour (tumour suppressor)                                                         RC_H04768_at   PAAAA   chrom 15 no homology   yj51f03.s1   1078   P   127   A   D   −8.4   −7.99                     Homo                       sapiens                     cDNA                   clone                   152285 3′.       RC_Z39652_at   PAAAA   Y14593 APM-1 gene     H. sapiens     959   P   −157   A   D   ˜−19.7   −15.28               adipocyte-specific   partial               secretory protein:   cDNA               chrom 1q21.3-q23   sequence;                   clone                   c1fg03.       RC_H30270_at   PAAAA   chrom 18 PAAAA in   yp42e02.s1   941   P   366   A   D   −4.1   −3.11               colon &amp; bladder no     Homo                 homology     sapiens                     cDNA                   clone                   190106 3′                   similar to                   contains                   MSR1                   repetitive                   element;.       RC_T47089_s_at   PAAAA   tenascin-X; tenascin-   yb52b08.s1   815   P   −49   A   D   ˜−31.4   −18.16               X precursor; unidenti-     Homo                 fied protein     sapiens                     cDNA                   clone                   74775 3′                   similar to                   gb: M25813                   FIBRI-                   NOGEN-                   LIKE                   PROTEIN                   (HUMAN).       RC_W31906_at   PAAAA   secretagogin;   zc76c03.s1   736   P   243   A   D   −4.8   −3.12               dJ501N12.8 (putative   Pancre-               protein) chrom 6   atic Islet                     Homo                       sapiens                     cDNA                   clone                   328228 3′.       RC_AA279803_at   PAAAA   chrom 2 no homology   zs92a11.s1   643   P   164   A   D   −3.9   −2.35                   NCI_CGAP —                     GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   704924 3′.       RC_R01646_at   PAAAA   chrom 13q32.1-33.3;   ye79f11.s1   607   P   −75   A   D   ˜−17.7   −13.83               AL159152; homolo-     Homo                 gy to mouse Pcbp1 -     sapiens                 poly(rC)-binding   cDNA               protein 1   clone                   123981 3′.       RC_AA099820_at   PAAAA   BAC clone AC016778   zk87c05.s1   587   P   58   A   D   ˜−9.5   −6.53                   Soares                   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   489800 3′.       AA319615_at   PAAAA   secretory carrier   EST21862   568   P   −100   A   D   ˜−34.7   −15.76               membrane protein;   Adrenal               secretory carrier   gland               membrane protein 2;   tumor               chrom 15     Homo                       sapiens                     cDNA 5′                   end.       H07011_at   PAAAA   tetraspan NET-6   yl81e01.r1   324   P   123   A   D   −2.6   −0.78               mRNA; transmem-     Homo                 brane 4 superfamlly;     sapiens                 chrom 7   cDNA                   clone                   44466 5′.       RC_T68873_f_at   PPAAA       yc30f03.s1   3837   P   611   P   D   −6   −10.26                     Homo                       sapiens                     cDNA                   clone                   82205 3′                   similar to                   gb: J00272 —                     rna1                   Human                   metallothi-                   onein-II                   pseudo-                   gene (HU-                   MAN); con-                   tains L1                   repetitive                   element;.       RC_T40995_f_at   PPAAA       ya15e08.s3   1973   P   841   P   D   −2.7   −2.24                     Homo                       sapiens                     cDNA                   clone                   61574 3′.       RC_H81070_f_at   PPAAA       yu60h05.s1   1683   P   469   P   D   −4.3   −4.92                     Homo                       sapiens                     cDNA                   clone                   230553 3′                   similar to                   gb: X64177                     H. sapiens                     mRNA for                   metallothi-                   onein                   (HUMAN);.       RC_N30796_at   PPPAA       yw65d03.s1   1338   P   584   P   D   −2.7   −1.48                     Homo                       sapiens                     cDNA                   clone                   257093 3′.       RC_W37778_f_at   PPAAA       zc13b12.s1   945   P   371   P   D   −3.5   −2.53                   Soares                   parathy-                   roid tumor                   NbHPA                     Homo                       sapiens                     cDNA                   clone                   322175 3′                   similar to                   contains                   LTR2.t3                   LTR2                   repetitive                   element;.       RC_R70212_s_at   PPAAA       yj80d09.s1   718   P   227   P   D   −3.1   −1.57                     Homo                       sapiens                     cDNA                   clone                   155057 3′                   similar to                   gb: U05259 —                     rna1 MB-                   1 MEM-                   BRANE                   GLY-                   COPRO-                   TEIN                   PRECUR-                   SOR                   (HUMAN);.       RC_AA426330_at   PPAAA       zw11h09.s1   676   P   164   P   D   −4.1   −2.75                   Soares                   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   769025 3′.       RC_N33927_s_at   PPAAA       yv25e09.s1   599   P   312   P   D   −19   −0.46                     Homo                       sapiens                     cDNA                   clone                   243784 3′.       RC_T90190_s_at   PPAAA       yd38f12.s1   574   P   102   P   D   −4.8   −2.79                     Homo                       sapiens                     cDNA                   clone                   110543 3′.       RC_AA447145_at   PPPAA       zw93f08.s1   374   P   123   P   D   −3   −1.14                   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   784551 3′.       RC_H75860_at   PPAAA       yu60g09.s1   355   P   70   P   D   −4   −1.64                     Homo                       sapiens                     cDNA                   clone                   230560 3′.       RC_T71132_s_at   PPAAA       yd34a02.s1   325   P   131   P   D   −2.6   −0.81                     Homo                       sapiens                     cDNA                   clone                   110090 3′                   similar to                   gb: L13288                   VASOAC-                   TIVE                   INTESTI-                   NAL POL-                   YPEPTIDE                   RECEPTOR                   1 PRE-                   CURSOR                   (HUMAN);.                 GAINED from N to tumour       (oncogene)                                                         RC_AA609013_s —     APPPP   microsomal dipepti-   af05f12.s1   421   A   2176   P   I   5.1   6.42       at       dase (also on 6.8 k);   Soares               chrom 16   testis NHT                     Homo                       sapiens                     cDNA                   clone                   1030799 3′                   similar to                   gb: J05257                   MICRO-                   SOMAL                   DIPEPTI-                   DASE                   PRECUR-                   SOR                   (HUMAN);.       RC_AA232508_at   APPPP   CGI-89 protein; un-   zr28d08.s1   516   A   2511   P   I   4.5   5.56               named protein product;   Strata-               hypothetical protein   gene NT2                   neuronal                   precursor                   937230                     Homo                       sapiens                     cDNA                   clone                   664719 3′.       RC_AA428964_at   APPPP   serine protease-like   zw19b02.s1   395   A   559   P   I   1.4   0.13               protease; serine pro-   Soares               tease homolog = NES1;   ovary               normal epithelial cell-   tumor               specific 1   NbHOT                     Homo                       sapiens                     cDNA                   clone                   769707 3′                   similar to                   gb: X57025 —                     rna1                   INSULIN-                   LIKE                   GROWTH                   FACTOR                   IA PRE-                   CURSOR                   (HUMAN);.       RC_T52813_s_at   APPPP   dJ28O10.2 (G0S2   ya79h10.s1   191   A   471   P   I   3.1   1.2               (PUTATIVE LYM-     Homo                 PHOCYTE G0/G1     sapiens                 SWITCH PROTEIN 2;   cDNA               chrom 1   clone                   67939 3′                   similar to                   gb: M69199                   PUTA-                   TIVE LYM-                   PHOCYTE                   G0/G1                   SWITCH                   PROTEIN                   (HUMAN).       RC_AA075642_at   APPPP   gp-340 variant protein;   zm88a11.s1   264   A   3364   P   I   13   20.97               DMBT1/8kb.2 protein   Strata-                   gene                   ovarian                   cancer                   (#937219)                     Homo                       sapiens                     cDNA                   clone                   544988 3′.       RC_AA007218_at   APPPP   chrom 13 no homology   13cDNA54-   156   A   902   P   I   5.8   4.88                   3.seq                   Soares                   infant                   brain 1NIB                     Homo                       sapiens                     cDNA                   clone                   HY18-131                   3′.       RC_N33920_at   APPPP   ubiquitin-like protein   yv25b11.s1   15   A   1173   P   I   34.5   22.41               FAT10; diubiquitin;     Homo                 dJ271M21.6 (Diubiqui-     sapiens                 tin); chrom 6   cDNA                   clone                   243741 3′.       RC_N71781_at   APPPP   KIAA1199 protein,   yz94e06.s1   9   A   575   P   I   ˜16.7   10.24               chrom 15     Homo                       sapiens                     cDNA                   clone                   290722 3′.       RC_R67275_s_at   APPPP   alpha-1 (type XI)   yh01f11.s2   17   A   403   P   I   ˜10.9   6.01               collagen precursor;     Homo                 collagen, type XI,     sapiens                 alpha 1; collagen type   cDNA               XI alpha-1 isoform A;   clone               chrom 1   41676 3′                   similar to                   gb: J04177                   COLLA-                   GEN                   ALPHA                   1(XI)                   CHAIN                   PRECUR-                   SOR                   (HUMAN);.       RC_W80763_at   APPPP   hypothetical protein;   zd83g04.s1   193   A   571   P   I   3.6   1.97               chrom 17   Soares                   fetal heart                   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   347286 3′.       RC_AA443793_at   APPPP   chrom 7p22   zw86e11.s1   232   A   781   P   I   3.5   2.19               AC006028 BAC clone   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   783884 3′.       RC_AA034499_s —     APPPP   ZNF198 protein; zinc   zk23c04.s1   128   A   589   P   I   4.7   2.97       at       finger protein; FIM   Soares               protein; Cys-rich pro-   pregnant               tein; zinc finger protein   uterus               198; chrom 13   NbHPU                     Homo                       sapiens                     cDNA                   clone                   471366 3′.       RC_AA035482_at   APPPP   chrom 5; AK022505   zk27b07.s1   260   A   562   P   I   1.7   0.3               clone; CalcineurinB   Soares               (weakly similar)   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   471733 3′.       RC_AA024482_at   APPPP   hypothetical protein;   ze76a01.s1   −28   A   828   P   I   ˜44.0   19.93               unnamed protein   Soares               product; chrom 17   fetal heart                   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   364872 3′.       RC_H93021_at   APPPP   chrom 2; XM_004890   yv06a03.s1   274   A   345   P   I   3   1.29               peptidylprolyl isomer-     Homo                 ase A (cyclophilin A)     sapiens                     cDNA                   clone                   241900 3′                   similar to                   gb: X52851 —                     rna1 PEP-                   TIDYL-                   PROLYL                   CIS-                   TRANS                   ISOMER-                   ASE A                   (HUMAN);.       RC_AA427737_at   APPPP   no homology   zw30g12.s1   145   A   339   P   I   2.3   0.6                   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   770854 3′.       RC_AA417078_at   APPPP   chrom 7q31;   zu13c02.s1   59   A   405   P   I   3.2   1.21               AF017104 clone   Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   731714 3′.       M29873_s_at   APPPP   cytochrome P450-IIB   Human   19   A   640   P   I   ˜32.3   15.15               (hIIB3); 19q13.1-   cytochrome               q13.2   P450-                   IIB (hIIB3)                   mRNA,                   complete                   cds.       RC_H27498_f_at   AAPPP       yl57a08.s1   2557   A   1129   A   NC   −2.3   −1.52                     Homo                       sapiens                     cDNA                   clone                   162326 3′                   similar to                   gb: S55735                   IG AL-                   PHA-1                   CHAIN C                   REGION                   (HUMAN);.       RC_T92363_s_at   AAPPP       ye19h06.s1   68   A   255   A   NC   3.7   1.37                     Homo                       sapiens                     cDNA                   clone                   118235 3′.       RC_N89910_at   AAAPP       zb22f11.s1   205   A   226   A   NC   1.1   0.01                   Soares                   fetal lung                   NbHL 19W                     Homo                       sapiens                     cDNA                   clone                   302829 3′                   similar to                   SW: PGT —                     RAT                   Q00910                   PROSTAG                   LANDIN                   TRANS-                   PORTER;.       RC_W60516_at   AAAPP       zc99f02.s1   391   A   285   A   NC   −1.1   −0.02                   Pancreatic                   Islet  Homo                       sapiens                     cDNA                   clone                   339291 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_AA219699_at   AAAPP       zr03d01.s1   15   A   110   A   NC   ˜2.6   0.37                   Strata-                   gene NT2                   neuronal                   precursor                   937230                     Homo                       sapiens                     cDNA                   clone                   650401 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_AA449450_at   AAAPP       zx05e04.s1   237   A   315   A   NC   1.3   0.07                   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   785598 3′.                 A classifiers       Gained in Duke A and Inc; NC in other Dukes;       Avg Diff in A &gt; 300                                                         RC_AA599199_at   APAAA   ALU seq.   ag34a11.s1   4   A   833   P   I   ˜24.1   15.25                   Jia bone                   marrow                   stroma                     Homo                       sapiens                     cDNA                   clone                   1091420 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment; con-                   tains ele-                   ment                   TAR1                   repetitive                   element;.       RC_R12694_at   APAAA   unnamed protein   yf40f05.s1   115   A   361   P   I   3   1.02               product BAA91641,     Homo                 chrom 10     sapiens                     cDNA                   clone                   129345 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_H91325_s_at   APAAA   aldolase B; aldolase B   yu96g02.s1   92   A   318   P   I   3.4   1.33               (aa 1-364); chrom 9     Homo                       sapiens                     cDNA                   clone                   241106 3′                   similar to                   gb: X02747                   FRUC-                   TOSE-BIS-                   PHOS-                   PHATE                   ALDO-                   LASE B                   (HUMAN);.       RC_N51709_at   APAAA   chrom X   yy72e04.s1   466   A   440   P   I   −1.1   −0.01                     Homo                       sapiens                     cDNA                   clone                   279102 3′.       RC_N72610_at   APAAA   —   za46h03.s1   502   A   531   P   I   1.1   0.01                     Homo                       sapiens                     cDNA                   clone                   295637 3′.                 Lost in Duke A and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_N32411_f_at   PAPPP   Myc-associated zinc-   yw83b08.s1   4153   P   1319   A   D   −3.5   −5.06               finger protein of hu-     Homo                 man islet; chrom 16     sapiens                     cDNA                   clone                   258807 3′                   similar to                   gb: M94046                   Human                   zinc finger                   protein                   (HUMAN);.       RC_AA243858_at   PAPPP   KIAA0882 protein   zr68c02.s1   389   P   77   A   D   −5   −2.67                   Soares                   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   668546 3′.       RC_AA486283_at   PAPPP   ras-like protein; ras-   ab35h03.s1   4856   P   720   A   D   −6.2   −12.02               related C3 botulinum   Strata-               toxin substrate;   gene HeLa               dJ20J23   cell                   s3937216                     Homo                       sapiens                     cDNA                   clone                   842837 3′                   similar to                   gb: M29870                   RAS-                   RELATED                   C3 BOTU-                   LINUM                   TOXIN                   SUB-                   STRATE 2                   (HUMAN);.       RC_AA490930_at   PAPPP   chrom 18; KIAA1468   aa46e04.s1   552   P   242   A   D   −2.3   −0.72               protein   NCI CGAP —                     GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   823998 3′.                 PPPPP; Inc in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_N69263_at   PPPPP   chrom 10; AK026414   za38e10.s1   314   P   1239   P   I   3.4   2.35               clone (only 108 nt     Homo                 hom)     sapiens                     cDNA                   clone                   294858 3′.       RC_T15817_f_at   PPPPP   iNOS, inducible nitric   IB1935   484   P   1182   P   I   3.4   2.45               oxide synthase   Infant                   brain,                   Bento                   Soares                     Homo                       sapiens                     cDNA                   3′ end                   similar to                     H. sapiens                     inducible                   nitric oxide                   synthase                   mRNA.                 PPPPP; Dec in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_H54088_s_at   PPPPP   ribosomal protein L41   yq88g11.s1   5317   P   1302   P   D   −4.1   −7.26                     Homo                       sapiens                     cDNA                   clone                   202916 3′.       RC_H59052_f_at   PPPPP   fungal sterol-C5-   yr40g11.s1   5090   P   1086   P   D   −4.2   −7.05               desaturase homolog;     Homo                 ORF; thymosin beta-4     sapiens                     cDNA                   clone                   207812 3′                   similar to                   gb: M1773                   3 THY-                   MOSIN                   BETA-4                   (HUMAN);.       RC_R49198_s_at   PPPPP   —   yg58h09.s1   4836   P   1115   P   D   −4.3   −7.61                     Homo                       sapiens                     cDNA                   clone                   37141 3′                   similar to                   gb:                   X6915040S                   RIBOSO-                   MAL                   PROTEIN                   S18 (HU-                   MAN);.       RC_T73572_f_at   PPPPP   ferritin L-chain; L   yc36c10.s1   5727   P   1853   P   D   −3.1   −4.59               apoferritin     Homo                       sapiens                     cDNA                   clone                   82770 3′                   similar to                   gb: M10119                   FERRI-                   TIN LIGHT                   CHAIN                   (HUMAN);.       RC_AA477483_at   PPPPP   no matching est   zu44h02.s1   4494   P   1034   P   D   −3.8   −5.51                   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   740883 3′                   similar to                   SW: NU3M —                     HUMAN                   P03897                   NADH-UBI-                   QUINONE                   OXIDO-                   REDUC-                   TASE                   CHAIN 3;.                 B classifiers       Gained in Duke B and Inc; NC in other Dukes       Avg Diff in B &gt; 300                                                         RC_T67463_s_at   AAPAA   cathepsin O2; X; K   yd12c04.s1   132   A   240   A   NC   1.8   0.24                     Homo                       sapiens                     cDNA                   clone                   66918 3′                   similar to                   gb: X12451                   CATHEP-                   SIN L                   PRECUR-                   SOR                   (HUMAN);.       RC_W94688_at   AAPAA   perilipin   ze04f12.s1   139   A   137   A   NC   −1   0                   Soares                   fetal heart                   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   358031 3′.       RC_AA126743_at   AAPAA   Z97200 PAC chrom   zk95d02.s1   402   A   384   A   NC   4.3   2.1               1q24; PMX1 homeo-   Soares               box gene   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   490563 3′.       RC_AA236547_at   AAPAA   no homology   zs39a03.s1   223   A   −151   A   NC   −2.3   −0.48                   Soares                   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   687532 3′.       RC_AA255567_at   AAPAA   angiopoietin-related   zr85e11.s1   118   A   160   A   NC   1.3   0.05               protein-2; angiopoietin-   Soares               like 2   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   682508 3′.       RC_AA421256_at   AAPAA   —   zu06a01.s1   338   A   171   A   NC   −2   −0.38                   Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   731016 3′.                 Lost in Duke B and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_D59847_at   PPAPP   proSAAS; granin-like   Human   864   P   668   P   NC   −1.3   −0.1               neuroendocrine pep-   fetal brain               tide precursor   cDNA 3′-                   end GEN-                   070G07.       RC_F05038_at   PPAPP   polyamine modulated     H. sapiens     1683   P   1460   P   NC   −1.2   −0.05               factor-1; polyamine   partial               modulated factor 1   CDNA                   sequence;                   clone c-                   1gb09.       RC_N41059_at   PPAPP   chrom 3   yy53h01.s1   350   P   334   P   NC   −1   0                     Homo                       sapiens                     cDNA                   clone                   277297 3′.       RC_T23460_at   PPAPP   chrom 3; IFNAR2   seq3026   613   P   560   P   NC   −1.1   −0.01               21q22.11     Homo                       sapiens                     cDNA                   clone                   Hy18-                   Ch13-                   Charon40-                   cDNA-76                   3′.       RC_W42789_at   PPAPP   chrom 8 AF268037   zc25a06.s1   310   P   234   P   NC   −1.3   −0.07               C8ORF4 protein   Soares               (C8ORF4) chrom 8   senescent               ORF   fibroblasts                   NbHSF                     Homo                       sapiens                     cDNA                   clone                   323314 3′.       RC_AA460017_l —     PPAPP   BAC clone chrom 16   zx49h08.s1   1370   P   703   P   NC   −1.9   −0.73       at           Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   795615 3′.       RC_AA482127_at   PPAPP   KIAA1142 protein   zv43g10.s1   356   P   288   P   NC   −1.2   −0.04                   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   756450 3′.       RC_AA504806_at   PPAPP   chrom 2 AF052107   aa64a10.s1   421   P   384   P   NC   −1.4   −0.12               clone 23620 mRNA   NCI CGAP —                 sequence   GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   825690 3′.                 Inc in Duke B; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_AA386386_s —     PPPPP   —   EST81368   1672   P   2253   P   NC   1.4   0.3       at           Prostate                   gland I                     Homo                       sapiens                     cDNA 3′                   end similar                   to similar                   to prolyl 4-                   hydroxy-                   lase, beta                   sub-                   unit/protein                   disulfide                   isomer-                   ase/thyroid                   hormone-                   binding                   protein.       RC_AA452549_at   PPPPP   PR01659; hypotheti-   zx35f04.s1   400   P   368   P   NC   −1.1   −0.01               cal protein chrom 11   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   788479 3′.                 Dec in Duke B; NC in other dukes       Avg Diff N &gt;= 200; Fold change N to B &gt;= 3x                                                         RC_T90037_at   PPPPP   unnamed protein   ye13d01.s1   948   P   568   P   NC   −1.7   −.036               product, chrom 4     Homo                       sapiens                     cDNA                   clone                   117601 3′.       RC_AA432130_at   PPPPP   KIAA0867 protein,   zw69f10.s1   1007   P   475   P   NC   −2.1   −0.8               chrom 12   Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   781483 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment; con-                   tains ele-                   ment                   PTR7                   repetitive                   element;.                 C classifiers       Gained in Duke C and Inc; NC in other Dukes       Avg Diff in C &gt; 300                                                         RC_D45556_at   AAAPA   chrom 15; AL390085   Human   391   A   222   A   NC   −1.8   −0.28               clone   adult lung                   3′ directed                   Mbol                   CDNA,                   HUMGS02743,                   clone                   Ig1160.       RC_W86214_at   AAAPA       zh54a11.s1   88   A   132   A   NC   1.6   0.12                   Soares                   fetal liver                   spleen                   1NFLS S1                     Homo                       sapiens                     cDNA                   clone                   415868 3′.       RC_AA039439_s —     AAAPA   novel gene KIAA0134   zk39a11.s1   134   A   608   A   NC   3.8   1.94       at       protein 19q13.3   Soares                   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   485180 3′.       RC_AA128935_at   AAAPA       zo08e09.s1   162   A   270   A   NC   1.7   0.19                   Strata-                   gene neuro                   epithelium                   NT2RAMI                   937234                     Homo                       sapiens                     cDNA                   clone                   567112 3′.       RC_AA134158_s —     AAAPA   class I homeodomain;   zo18b11.s1   255   A   200   A   NC   3.7   1.17       at       homeobox protein,   Strata-               chrom 7   gene colon                   (#937204)                     Homo                       sapiens                     cDNA                   clone                   587229 3′                   similar to                   gb: S41211                   HOMEO-                   BOX                   PROTEIN                   HOX-A10                   (HUMAN);.       RC_AA232646_at   AAAPA   chrom 17, AF266756   zr45f08.s1   123   A   238   A   NC   1.9   0.3               sphingosine kinase   Soares               (SPHK1   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   666375 3′.       RC_AA401184_at   AAAPA   no homology   zu52b06.s1   348   A   62   A   NC   ˜−1.2   −0.01                   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   741587 3′.       RC_AA436840_at   AAAPA       zv57g11.s1   399   A   −13   A   NC   ˜−6.1   −2                   Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   757796 3′.       RC_AA488655_at   AAAPA       ab40c03.s1   −4   A   176   A   NC   ˜6.8   2.59                   Strata-                   gene HeLa                   cell s3                   937216                     Homo                       sapiens                     cDNA                   clone                   843268 3′.                 L st in Duke C and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_N30231_at   PPPAP   Lsm4 protein; U6   yw64c08.s1   1496   P   885   P   NC   −2.2   −1.26               snRNA-associated     Homo                 Sm-like protein LSm4;     sapiens                 glycine-rich protein   cDNA                   clone                   257006 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_W73790_f_at   PPPAP   immunoglobulin-   zd50g12.s1   3320   P   1638   P   NC   −2   −1.27               related protein 14.1;   Soares               lambda L-chain C   fetal heart               region; omega protein,   NbHH19W               chrom 22     Homo                       sapiens                     cDNA                   clone                   344134 3′                   similar to                   gb: M27749                   IMMU-                   NOGLOB-                   ULIN-                   RELATED                   14.1                   PROTEIN                   PRECUR-                   SOR                   (HUMAN);.       RC_AA412184_at   PPPAP   chrom 1p36; d89060   zt93f09.s1   714   P   570   P   NC   −1.4   −0.15               dolichyl-   Soares               diphosphooligosaccha-   testis NHT               ride-protein glycosyl-     Homo                 transferase     sapiens                     cDNA                   clone                   729929 3′                   similar to                   WP: EEED                   8.9                   CE01893;.       RC_AA521303_at   PPPAP   methionine adenosyl-   aa79f11.s1   777   P   760   P   NC   −1   0               transferase regulatory   NCI_CGAP —                 beta subunit; dTDP-4-   GCB1               keto-6-deoxy-D-     Homo                 glucose 4-reductase,     sapiens                 chrom 5   cDNA                   clone                   IMAGE:                   827181 3′.                 Inc in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to C &gt;= 3x                                                         RC_AA181902_at   PPPPP   AC007201 on chrom   zp63d06.s1   478   P   679   P   NC   1.6   0.29               19 (only 80 nt hom)   Strata-                   gene                   endothelial                   cell                   937223                     Homo                       sapiens                     cDNA                   clone                   624875 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.                 Dec in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to D &gt;= 3x                                                         RC_AA461174_at   PPPPP   8p21.3-p22 AB020860   zx70c04.s1   327   P   198   P   NC   −1.5   −0.12               anti-oncogene   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   796806 3′.       AA393432_s_at   PPPPP   chrom 2, Unknown;   zt71a04.r1   319   P   222   P   NC   −1.4   −0.11               unnamed protein   Soares               product AAD20029   testis NHT                     Homo                       sapiens                     cDNA                   clone                   727758 5′.                 D classifier       Gained in Duke D and Inc; NC in other Dukes       Avg Diff in D &gt; 300                                                         RC_N91920_at   AAAAP   chrom 16p12-p11.2;   zb48e07.s1   −76   A   58   A   NC   ˜4.7   0.62               XN_007994 retinobla-   Soares               stoma binding protein   fetal lung                   NbHL19W                     Homo                       sapiens                     cDNA                   clone                   306852 3′.       RC_AA621601_at   AAAAP   chrom 17 XM_009868   af47g08.s1   −46   A   232   A   NC   ˜−3.5   −0.18               RAB36 ARS oncogene   Soares               family   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   1034846 3′                   similar to                   TR:                   G240986                   G240986                   LMW G-                   PROTEIN.                   ;.                 Avg Diff in N &gt; 500       Lost in Duke D and Dec; NC in other Dukes                                                         RC_R72886_s_at   PPPPA   KIAA0422; adenylyl   yl10f04.s1   1768   P   880   P   NC   −2   −0.91               cyclase type VI, chrom     Homo                 12     sapien s                   cDNA                   clone                   157855 3′.       RC_AA026030_at   PPPPA   chrom 1   ze84d01.s1   1478   P   1163   P   NC   −1.3   −0.11                   Soares                   fetal heart                   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   365665 3′                   similar to                   PIR: A48764                   A48764                   calpain;.       RC_Z39006_at   PPPPA   hypothetical protein,     H. sapiens     1251   P   691   P   NC   −1.4   −0.14               chrom 17   partial                   cDNA                   sequence;                   clone c-                   Owe07.       RC_AA435908_at   PPPPA   chrom 19; ac011491   zt87a11.s1   1173   P   494   P   NC   −1.6   −0.28               clone and 20 nt hom.   Soares               RAB2, RAS oncogene   testis NHT               family     Homo                       sapiens                     cDNA                   clone                   729308 3′.       RC_AA057829_s —     PPPPA   growth-arrest-specific   zl95c02.s1   1136   P   534   P   NC   −1.5   −0.26       at       protein; growth arrest-   Stratagene               specific 6; AXL stimu-   corneal               latory factor, chrom 13   stroma                   (#937222)                     Homo                       sapiens                     cDNA                   clone                   512354 3′                   similar to                   TR:                   G401767                   G401767                   GROWTH-                   ARREST-                   SPECIFIC                   PROTEIN.;.       RC_R72087_at   PPPPA   chrom 5 EST; hom to   yj87e06.s1   923   P   768   P   NC   −1.2   −0.06               chrom 20 AL356652     Homo                 clone     sapiens                     cDNA                   clone                   155746 3′.       RC_H04242_at   PPPPA   ras related protein   yj46c08.s1   869   P   661   P   NC   −1.1   −0.01               Rab5b; RAB5B, mem-     Homo                 ber RAS oncogene     sapiens                 family   cDNA                   clone                   151790 3′.       RC_R97304_f_at   PPPPA   HLA-drb5; cell surface   yq52c02.s1   858   P   1822   P   NC   1.5   0.25               glycoprotein; MHC     Homo                 HLA-DR-beta chain     sapiens                 precursor chrom 6   cDNA                   clone                   199394 3′                   similar to                   gb: M33600                   HLA                   CLASS II                   HISTO-                   COMPATI-                   BILITY                   ANTIGEN,                   DR-1                   BETA                   CHAIN                   (HUMAN);.       RC_N48609_at   PPPPA   chrom 11; AC004584   yy74f08.s1   770   P   612   P   NC   −1.1   −0.02               chrom 17     Homo                       sapiens                     cDNA                   clone                   279303 3′.       RC_W86850_f_at   PPPPA   chrom 22 ? X96924   zh59d02.s1   644   P   273   P   NC   −1.4   −0.09               mitochondrial citrate   Soares               tranbsport region   fetal liver                   spleen                   1NFLS S1                     Homo                       sapiens                     cDNA                   clone                   416355 3′.       RC_AA130603_at   PPPPA   ak024908 clone   zo10a01.s1   640   P   568   P   NC   −1.1   −0.02                   Strata-                   gene neuro                   epithelium                   NT2RAMI                   937234                     Homo                       sapiens                     cDNA                   clone                   567240 3′.       RC_AA479610_at   PPPPA   singleton ak025344   zu31a12.s1   640   P   571   P   NC   −1.1   −0.02               clone   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   739582 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_AA490593_l_at   PPPPA   chrom 17 ? Synapto-   aa47e09.s1   540   P   536   P   NC   −1.3   −0.07               brevin2 (VAMP2)   NCI_CGAP —                 AF135372   GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   824104 3′.       RC_AA054321_s —     PPPPA   6p21 HLA class i   zl68c01.s1   509   P   472   P   NC   −1.1   −0.01       at       region; AC004202   Stratagene               clone   colon                   (#937204)                     Homo                       sapiens                     cDNA                   clone                   509760 3′.                 Inc in Duke D; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         D79052_s_at   PPPPP   Sec61 gamma   Human   772   P   680   P   NC   −1.3   −0.08                   placenta                   cDNA 5′-                   end GEN-                   530B11.       RC_T40439_s_at   PPPPP   U2 small nuclear   ya01c08.s1   673   P   911   P   NC   1.2   0.06               ribonucleoprotein B″;     Homo                 dJ705D16.1     sapiens                     cDNA                   clone                   60206 3′                   similar to                   SP: RU2B —                     HUMAN                   P08579                   U2 SMALL                   NUCLEAR                   RIBONU-                   CLEOPRO-                   TEIN,.       RC_AA251829_at   PPPPP   CGI-29 protein   zs09c12.s1   202   P   670   P   NC   2.8   1.19                   NCI_CGAP —                     GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   684694 3′.                 Dec in Duke D; NC in other Dukes       Avg Diff N &gt;= 500; Fold change N to D &gt;= −5x                                                         RC_D60328_at   PPPPP   chrom 6, unknown;   Human   1345   P   731   P   NC   −1.7   −0.4               ring finger protein 5   fetal brain                   cDNA 3′-                   end GEN-                   099F03.       RC_H96850_at   PPPPP   oligosaccharyltransfer-   yw03b12.s1   4876   P   3348   P   NC   −1.5   −0.45               ase d89060 1p36.1   Soares               (also C-class)   melano-                   cyte                   2NbHM                     Homo                       sapiens                     cDNA                   clone                   251135 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment;.       RC_127444_at   PPPPP   chrom 1 no homology   zn92e09.s1   626   P   353   P   NC   −1.8   −0.36                   Strata-                   gene lung                   carcinoma                   937218                     Homo                       sapiens                     cDNA                   clone                   565672 3′                   similar to                   TR:                   G563829                   G563829                   FATTY                   ACID                   TRANS-                   PORT                   PROTEIN.;.       RC_AA242824_at   PPPPP   chrom 11; ac005233   zr65e11.s1   710   P   178   P   NC   −2.2   −0.52               PAC clone chrom 22   Soares                   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   668300 3′.       AA405775_s_at   PPPPP   similar to CAA16821   zu57c10.r1   1047   P   692   P   NC   −1.5   −0.25               (PID: g3255952)   Soares                   ovary                   tumor                   NbHOT                     Homo                       sapiens                     cDNA                   clone                   742098 5′.                 AB classifiers       Gained in Duke AB and Inc; NC in other Dukes       Avg Diff in AB &gt;= 300                                                         RC_AA136269_at   APPAA   HSPC314, chrom 12   zk93b07.s1   184   A   319   P   I   2.9   1.25                   Soares                   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   490357 3′.                 Avg Diff in N &gt; 300       Lost in Duke AB and Dec; NC in other Dukes                                                         RC_T40895_at   PAAPP   protein tyrosine phos-   ya13f02.s1   1154   P   244   A   D   −3.1   −1.64               phatase PTPCAAX1;     Homo                 protein tyrosine phos-     sapiens                 phatase hPRL-1N; iva-   cDNA               1, chrom 6   clone                   61371 3′.       RC_AA424400_at   PAAPP   chrom 14   zv82e05.s   344   P   123   A   D   −2.8   −0.91                   1 Soares                   total fetus                   Nb2HF8                   9w  Homo                       sapiens                     cDNA                   clone                   760160 3′.                 PPPPP; Inc in Duke AB       Avg Diff AB &gt;= 300; Fold change N to AB &gt;= 5x                                                         RC_N22015_at   PPPPP   unnamed protein   yw31h10.s1   638   P   3335   P   I   5.2   8.09               product, chrom 17;     Homo                 BC class.     sapiens                     cDNA                   clone                   253891 3′.       RC_N50971_at   PPPPP   —   yy94b07.s1   129   P   1031   P   I   10   10.43                     Homo                       sapiens                     CDNA                   clone                   281173 3′.       RC_AA194833_at   PPPPP   senescence-   zr31b04.s1   185   P   999   P   I   5.7   5.26               associated epithelial   Soares               membrane protein;   NhHMPu               Claudin-1, chrom 3   S1  Homo                       sapiens                     cDNA                   clone                   664975 3′.       RC_AA393803_at   PPPPP   chrom 6   zv64c05.s1   69   P   310   P   I   5.7   2.8                   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   758408 3′.                 BC classifiers       Gained in Duke BC and Inc; NC in other Dukes       Avg Diff in BC &gt; 300                                                         RC_AA148923_at   AAPPA   DEPP; decidual pro-   zl27g11.s1   233   A   212   A   NC   1.2   0.04               tein induced by proges-   Soares               terone; hypothetical   pregnant               protein, chrom 10   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   503204 3′.                 Lost in Duke BC and Dec       Avg Diff in N &gt; 300                                                         RC_N91919_s_at   PPAAP   mineralocorticoid   zb48e05.s1   618   P   206   P   D   −2.4   −0.82               receptor; mineralocor-   Soares               ticoid receptor (al-   fetal lung               dosterone receptor);   NbHL19W               chrom 4     Homo                       sapiens                     cDNA                   clone                   306848 3′                   similar to                   gb: M16801                   MINE-                   RALO-                   CORTI-                   COID                   RECEP-                   TOR                   (HUMAN);.       RC_T99196_s_at   PPAAP   chrom 5 AF010235   ye62f11.s1   529   P   274   P   D   −2.2   −0.67                 Homo sapiens  mRNA     Homo                 from chromosome     sapiens                 5q31-33 region   cDNA                   clone                   122349 3′.       RC_W72366_at   PPAAP   chrom 1 clone RP11-   Zd62f01.s1   323   P   183   P   NC   −1.8   −0.26               404F10 on chromo-   Soares               some 1q23.1-24.1,   fetal heart               CD48 hom   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   345241 3′                   similar to                   gb: M81181                   SODI-                   UM/POTAS-                   SIUM-                   TRANS-                   PORTING                   ATPASE                   BETA-2                   (HU-                   MAN); con-                   tains Alu                   repetitive                   element;.       RC_AA255903_at   PPAAP   CD39L4; CD39-like 4;   zs28d05.s1   840   P   260   P   NC   −3.4   −2.13               chrom 14   NCI_CGAP —                     GCB1                     Homo                       sapiens                     cDNA                   clone                   IMAGE:                   686505 3′.                 PPPPP; Inc in Duke BC       Avg Diff N &gt;= 300; Fold change N to BC &gt;= 5x                                                         J03464_s_at   PPPPP   collagen alpha-2 type   Human   352   P   1494   P   I   4.2   4.09               I; mRNA COL2A1   collagen                   alpha-2                   type I                   mRNA,                   complete                   cds, clone                   pHCOL2A1       RC_N22015_at   PPPPP   unnamed protein   yw31h10.s1   638   P   3335   P   I   5.2   8.09               product, chrom 17     Homo                       sapiens                     CDNA                   clone                   253891 3′.       RC_AA055896_at   PPPPP   chrom 9   zf20e03.s1   590   P   1835   P   I   2.9   2.15                   Soares                   fetal heart                   NbHH19W                     Homo                       sapiens                     cDNA                   clone                   377500 3′.       RC_AA121315_at   PPPPP   KIAA1077 protein;   zk91g08.s1   429   P   1764   P   I   4.9   5.57               hypothetical protein,   Soares               chrom 8   pregnant                   uterus                   NbHPU                     Homo                       sapiens                     cDNA                   clone                   490238 3′.       RC_AA122386_at   PPPPP   procollagen alpha   zn36f10.s1   466   P   1403   P   I   3   2.15               2(V); pro-alpha   Stratagene               (V)collagen (AA 1099),   endothelial               chrom 2   cell                   937223                     Homo                       sapiens                     cDNA                   clone                   549547 3′.                 PPPPP; DEC in Duke BC       Avg Diff N &gt;= 500; Fold change N to BC &gt;= 5x                                                         C02386_s_at   PPPPP       HUMGS00   3429   P   1667   P   D   −2.1   −1.35                   10652,                   Human                   Gene                   Signature,                   3′-directed                   cDNA                   sequence.       M12272_s_at   PPPPP   alcohol dehydrogen-     Homo     3368   P   904   P   D   −3.4   −4.07               ase class I gamma     sapiens                 subunit (ADH3)   alcohol                   dehydro-                   genase                   class I                   gamma                   subunit                   (ADH3)                   mRNA,                   complete                   cds       M12759_at   PPPPP   Ig J chain gene   Human Ig   2628   P   686   P   D   −3.2   −3.14                   J chain                   gene       M83670_s_at   PPPPP   carbonic anhydrase   Human   2138   P   432   P   D   −5.1   −6               IV   carbonic                   anhydrase                   IV mRNA,                   complete                   cds       N91087_at   PPPPP   dJ991C6.1 (novel   za18b11.r1   923   P   217   P   D   −4.4   −3.43               protein similar to  C.     Soares                 elegans  F55A12.9   fetal liver               (Tr: P91086)); un-   spleen               named protein   1NFLS               product; chrom 6     Homo                       sapiens                     cDNA                   clone                   292893 5′.       RC_H77597_f_at   PPPPP   unident. EST chrom ///   ys08a06.s1   5411   P   1118   P   D   −4.4   −8.23               AFFY = similar metal-     Homo                 lothionein     sapiens                     cDNA                   clone                   214162 3′                   similar to                   gb: X64177                     H. sapiens                     mRNA for                   metallothi-                   onein                   (HUMAN);.       RC_N23665_s_at   PPPPP   L11708 ESTRADIOL   yw40b12.s1   1150   P   246   P   D   −4.1   −3.18               17 BETA-     Homo                 DEHYDROGENASE     sapiens                 2, chrom 16   cDNA                   clone                   254687 3′                   similar to                   gb: L11708                   ESTRA-                   DIOL 17                   BETA-                   DEHYDRO-                   GENASE                   2 (HU-                   MAN);.       RC_N79237_at   PPPPP   unnamed protein   za63a11.s1   7342   P   2621   P   D   −2.8   −4.25               product, hom to     Homo                 S49589 cortical gran-     sapiens                 ule lectin - African   cDNA               clawed frog;.   clone                   297212 3′                   similar to                   PIR: S49589                   S49589                   cortical                   granule                   lectin -                   African                   clawed                   frog;.       RC_N80129_f_at   PPPPP   MT-11 protein; metal-   za65a05.s1   3623   P   1009   P   D   −3.6   −4.83               lothionein IX; metal-     Homo                 lothionein 1L, chrom     sapiens                 16 &amp; chrom 1   cDNA                   clone                   297392 3′                   similar to                   gb: X76717                     H. sapiens                     MT-1I                   mRNA.                   (HUMAN);.       RC_T24011_at   PPPPP   —   seq2167   900   P   317   P   D   −2.8   −1.53                     Homo                       sapiens                     cDNA                   clone                   3HFLSK20-                   53 3′.       RC_T90492_at   PPPPP   IGJ, P01591 IMMU-   ye15c08.s1   4498   P   1829   P   D   −2.5   −2.47               NOGLOBULIN J;.     Homo                 chrom 4     sapiens                     cDNA                   clone                   117806 3′                   similar to                   SP: IGJ —                     HUMAN                   P01591                   IMMU-                   NOGLOB-                   ULIN J;.       RC_AA058357_s —     PPPPP   singleton; no hom.,   zl67e01.s1   5524   P   1757   P   D   −3.1   −4.66       at       NONSPECIFIC   Stratagene               CROSSREACTING   colon               ANTIGEN.;.   (#937204)                     Homo                       sapiens                     cDNA                   clone                   509688 3′                   similar to                   TR:                   G189087                   G189087                   NONSPE-                   CIFIC                   CROSS-                   REAC-                   TING                   ANTIGEN.;.       RC_AA133469_at   PPPPP   cytokeratin 20, chrom   zo13e11.s1   3504   P   1231   P   D   −2.4   −1.87               17   Strata-                   gene colon                   (#937204)                     Homo                       sapiens                     cDNA                   clone                   586796 3′.       RC_AA151674_at   PPPPP   carbonic anhydrase   zo29e02.s1   1820   P   357   P   D   −5.1   −5.9               XII precursor, chrom   Strata-               15   gene colon                   (#937204)                     Homo                       sapiens                     cDNA                   clone                   588314 3′.       RC_AA405715_at   PPPPP   unknown   zu66d01.s1   4188   P   1080   P   D   −3.9   −5.93                   Soares                   testis NHT                     Homo                       sapiens                     cDNA                   clone                   742945 3′.       RC_AA428410_at   PPPPP   KIAA0828 protein,   zw57g04.s1   964   P   277   P   D   −3   −1.72               chrom 7   Soares                   total fetus                   Nb2HF89w                     Homo                       sapiens                     cDNA                   clone                   774198 3′                   similar to                   contains                   Alu repeti-                   tive ele-                   ment; con-                   tains ele-                   ment                   PTR7                   repetitive                   element;.       RC_AA429253_at   PPPPP   KIAA0803 protein;   zv50c08.s1   555   P   279   P   D   −2   −0.49               Centrosome- and   Soares               Golgi-localized PKN-   ovary               associated protein   tumor               (CG-NAP); AKAP450,   NbHOT               A-kinase anchoring     Homo                 protein AKAP350,     sapiens                 chrom 7   cDNA                   clone                   757070 3′.       RC_AA487468_at   PPPPP   chrom 7   ab23b07.s1   820   P   263   P   D   −2.6   −1.13                   Strata-                   gene lung                   (#937210)                     Homo                       sapiens                     cDNA                   clone                   841621 3′.       RC_AA621680_at   PPPPP   zinc finger transcrip-   af48e09.s1   1201   P   295   P   D   −4.1   −3.43               tion factor GKLF; EZF;   Soares               hEZF; endothelial   total fetus               Kruppel-like zinc finger   Nb2HF89w               protein, transforming     Homo                 oncogene     sapiens                     cDNA                   clone                   1034920                   3′.       Y09616_at   PPPPP   putative carboxyles-     H. sapiens     1776   P   605   P   D   −3   −2.48               terase   mRNA for                   putative                   carboxyl-                   esterase       AA171913_at   PPPPP   carbonic anhydrase   zo95d05.r1   1117   P   148   P   D   −7.2   −6.85               XII (CA12) chrom 15   Strata-                   gene                   ovarian                   cancer                   (#937219)                     Homo                       sapiens                     cDNA                   clone                   594633 5′.       AA253330_s_at   PPPPP   hypothetical protein;   zr72g02.r1   2590   P   740   P   D   −3.5   −3.91               unnamed protein   Soares               product, chrom 15   NhHMPu                   S1  Homo                       sapiens                     cDNA                   clone                   668978 5′.                                                                         unknown genes, data           Diff   Fold   Sort           Diff           Tumor   found after blast   Avg   Abs   Call   Change   Score   Avg   Abs   Call       EST candidates   stages   search   Diff   Call   Bvs   Bvs   Bvs   Diff   Call   Cvs       Probe Set   NABCD   homologous to   B   B   N   N   N   C   C   N                         LOST from N to tumour (tumour suppressor)                                                         RC_H04768_at   PAAAA   chrom 15 no homology   104   A   D   −10.2   −9.57   133   A   D       RC_Z39652_at   PAAAA   Y14593 APM-1 gene   −212   A   D   ˜−22.9   −18.17   −269   A   D               adipocyte-specific               secretory protein:               chrom 1q21.3-q23       RC_H30270_at   PAAAA   chrom 18 PAAAA in   287   A   D   −7.1   −6.36   240   A   D               colon &amp; bladder no               homology       RC_T47089_s_at   PAAAA   tenascin-X; tenascin-   79   A   D   −10.4   −8.51   15   A   D               X precursor; unidenti-               fied protein       RC_W31906_at   PAAAA   secretagogin;   148   A   D   −2.6   −1.18   263   A   D               dJ501N12.8 (putative               protein) chrom 6       RC_AA279803_at   PAAAA   chrom 2 no homology   217   A   D   −3   −1.4   91   A   D       RC_R01646_at   PAAAA   chrom 13q32.1-33.3;   92   A   D   −10.1   −8.86   67   A   D               AL159152; homolo-               gy to mouse Pcbp1 -               poly(rC)-binding               protein 1       RC_AA099820_at   PAAAA   BAC clone AC016778   17   A   D   ˜−10.0   −6.91   38   A   D       AA319615_at   PAAAA   secretory carrier   −198   A   D   ˜−39.6   −17.52   −103   A   D               membrane protein;               secretory carrier               membrane protein 2;               chrom 15       H07011_at   PAAAA   tetraspan NET-6   5   A   D   ˜−14.9   −6.63   122   A   D               mRNA; transmem-               brane 4 superfamlly;               chrom 7       RC_T68873_f_at   PPAAA       269   A   D   −13.5   −22.23   14   A   D       RC_T40995_f_at   PPAAA       −8   A   D   ˜−73.5   −38.45   533   A   D       RC_H81070_f_at   PPAAA       −228   A   D   ˜−87.0   −43.73   −100   A   D       RC_N30796_at   PPPAA       217   P   D   −5   −4.48   345   A   D       RC_W37778_f_at   PPAAA       174   A   D   −6.5   −6.21   237   A   D       RC_R70212_s_at   PPAAA       84   A   D   −8.5   −6.7   109   A   D       RC_AA426330_at   PPAAA       36   A   D   ˜−19.0   −11.89   −227   A   D       RC_N33927_s_at   PPAAA       151   A   D   −4   −2.33   222   A   D       RC_T90190_s_at   PPAAA       33   A   D   −14.8   −8.64   56   A   D       RC_AA447145_at   PPPAA       64   P   D   −5.9   −3.21   60   A   D       RC_H75860_at   PPAAA       13   A   D   ˜−11.1   −6.17   5   A   D       RC_T71132_s_at   PPAAA       54   A   D   −6.4   −3.4   73   A   D                 GAINED from N to tumour (oncogene)                                                         RC_AA609013_s —     APPPP   microsomal dipepti-   3117   P   I   6.7   10.33   3163   P   I       at       dase (also on 6.8 k);               chrom 16       RC_AA232508_at   APPPP   CGI-89 protein; un-   2138   P   I   4.1   4.71   1942   P   I               named protein product;               hypothetical protein       RC_AA428964_at   APPPP   serine protease-like   1894   P   I   5.2   6.55   969   P   I               protease; serine pro-               tease homolog = NES1;               normal epithelial cell-               specific 1       RC_T52813_s_at   APPPP   dJ28O10.2 (G0S2   1827   P   I   8.4   9.84   1597   P   I               (PUTATIVE LYM-               PHOCYTE G0/G1               SWITCH PROTEIN 2;               chrom 1       RC_AA075642_at   APPPP   gp-340 variant protein;   893   P   I   9   7.83   5271   P   I               DMBT1/8kb.2 protein       RC_AA007218_at   APPPP   chrom 13 no homology   878   P   I   5.6   4.65   884   P   I       RC_N33920_at   APPPP   ubiquitin-like protein   811   P   I   ˜27.4   17.37   425   P   I               FAT10; diubiquitin;               dJ271M21.6 (Diubiqui-               tin); chrom 6       RC_N71781_at   APPPP   KIAA1199 protein,   775   P   I   ˜22.0   13.85   305   P   I               chrom 15       RC_R67275_s_at   APPPP   alpha-1 (type XI)   699   P   I   ˜17.6   10.87   468   P   I               collagen precursor;               collagen, type XI,               alpha 1; collagen type               XI alpha-1 isoform A;               chrom 1       RC_W80763_at   APPPP   hypothetical protein;   658   P   I   4.2   2.67   789   P   I               chrom 17       RC_AA443793_at   APPPP   chrom 7p22   594   P   I   3.6   2.27   847   P   I               AC006028 BAC clone       RC_AA034499_s —     APPPP   ZNF198 protein; zinc   594   P   I   4.7   2.98   536   P   I       at       finger protein; FIM               protein; Cys-rich pro-               tein; zinc finger protein               198; chrom 13       RC_AA035482_at   APPPP   chrom 5; AK022505   506   P   I   1.9   0.39   572   P   I               clone; CalcineurinB               (weakly similar)       RC_AA024482_at   APPPP   hypothetical protein;   445   P   I   ˜26.6   12.37   360   P   I               unnamed protein               product; chrom 17       RC_H93021_at   APPPP   chrom 2; XM_004890   436   P   I   3.7   2.12   384   P   I               peptidylprolyl isomer-               ase A (cyclophilin A)       RC_AA427737_at   APPPP   no homology   419   P   I   2.9   1.07   515   P   I       RC_AA417078_at   APPPP   chrom 7q31;   411   P   I   3.3   1.34   462   P   I               AF017104 clone       M29873_s_at   APPPP   cytochrome P450-IIB   372   P   I   5.2   2.7   760   P   I               (hIIB3); 19q13.1-               q13.2       RC_H27498_f_at   AAPPP       6246   P   I   2.4   2.87   9295   P   I       RC_T92363_s_at   AAPPP       730   P   I   10.7   8.3   238   P   I       RC_N89910_at   AAAPP       149   A   NC   1.1   0.01   301   P   I       RC_W60516_at   AAAPP       328   A   NC   1   0   437   P   I       RC_AA219699_at   AAAPP       109   A   MI   ˜2.6   0.35   114   P   I       RC_AA449450_at   AAAPP       526   A   NC   2.2   0.65   411   P   I                 A classifiers       Gained in Duke A and Inc; NC in other Dukes;       Avg Diff in A &gt; 300                                                         RC_AA599199_at   APAAA   ALU seq.   284   A   NC   ˜8.8   4.36   176   A   NC       RC_R12694_at   APAAA   unnamed protein   200   A   NC   1.9   0.27   208   A   NC               product BAA91641,               chrom 10       RC_H91325_s_at   APAAA   aldolase B; aldolase B   157   A   NC   1.7   0.16   71   A   NC               (aa 1-364); chrom 9       RC_N51709_at   APAAA   chrom X   378   A   NC   −1.2   −0.05   473   A   NC       RC_N72610_at   APAAA   —   338   A   NC   1.5   0.13   618   A   NC                 Lost in Duke A and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_N32411_f_at   PAPPP   Myc-associated zinc-   3491   P   NC   −1.2   −0.11   4943   P   NC               finger protein of hu-               man islet; chrom 16       RC_AA243858_at   PAPPP   KIAA0882 protein   548   P   NC   1.4   0.13   334   P   NC       RC_AA486283_at   PAPPP   ras-like protein; ras-   3548   P   NC   −1.4   −0.33   5135   P   NC               related C3 botulinum               toxin substrate;               dJ20J23       RC_AA490930_at   PAPPP   chrom 18; KIAA1468   214   P   NC   −2.6   −0.97   225   P   NC               protein                 PPPPP; Inc in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_N69263_at   PPPPP   chrom 10; AK026414   909   P   NC   2.5   1.12   681   P   NC               clone (only 108 nt               hom)       RC_T15817_f_at   PPPPP   iNOS, inducible nitric   606   P   NC   1.3   0.06   865   P   NC               oxide synthase                 PPPPP; Dec in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_H54088_s_at   PPPPP   ribosomal protein L41   4157   P   NC   −1.3   −0.22   6623   P   NC       RC_H59052_f_at   PPPPP   fungal sterol-C5-   4077   P   NC   −1.2   −0.18   5764   P   NC               desaturase homolog;               ORF; thymosin beta-4       RC_R49198_s_at   PPPPP   —   3144   P   NC   −1.5   −0.58   4785   P   NC       RC_T73572_f_at   PPPPP   ferritin L-chain; L   5148   P   NC   −1.1   −0.06   6119   P   NC               apoferritin       RC_AA477483_at   PPPPP   no matching est   3186   P   NC   −1.4   −0.37   4175   P   NC                 B classifiers       Gained in Duke B and Inc; NC in other Dukes       Avg Diff in B &gt; 300                                                         RC_T67463_s_at   AAPAA   cathepsin O2; X; K   713   P   I   5.4   3.97   226   A   NC       RC_W94688_at   AAPAA   perilipin   393   P   I   2.8   1   84   A   NC       RC_AA126743_at   AAPAA   Z97200 PAC chrom   734   P   I   1.8   0.43   98   A   NC               1q24; PMX1 homeo-               box gene       RC_AA236547_at   AAPAA   no homology   402   P   I   1.8   0.3   285   A   NC       RC_AA255567_at   AAPAA   angiopoietin-related   408   P   I   3.4   1.5   207   A   NC               protein-2; angiopoietin-               like 2       RC_AA421256_at   AAPAA   —   382   P   I   1.1   0.02   236   A   NC                 Lost in Duke B and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_D59847_at   PPAPP   proSAAS; granin-like   336   A   D   −2.6   −1.2   308   P   NC               neuroendocrine pep-               tide precursor       RC_F05038_at   PPAPP   polyamine modulated   972   A   D   −1.9   −0.84   1644   P   NC               factor-1; polyamine               modulated factor 1       RC_N41059_at   PPAPP   chrom 3   246   A   D   −1.4   −0.11   378   P   NC       RC_T23460_at   PPAPP   chrom 3; IFNAR2   402   A   D   −2   −0.53   570   P   NC               21q22.11       RC_W42789_at   PPAPP   chrom 8 AF268037   29   A   D   ˜−5.6   −2.73   129   P   NC               C8ORF4 protein               (C8ORF4) chrom 8               ORF       RC_AA460017_L —     PPAPP   BAC clone chrom 16   725   A   D   −1.9   −0.66   666   P   NC       at       RC_AA482127_at   PPAPP   KIAA1142 protein   138   A   D   −2.6   −0.78   211   P   NC       RC_AA504806_at   PPAPP   chrom 2 AF052107   135   A   D   −4.5   −2.02   351   P   NC               clone 23620 mRNA               sequence                 Inc in Duke B; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         RC_AA386386_s —     PPPPP   —   4776   P   I   3   3.89   1559   P   NC       at       RC_AA452549_at   PPPPP   PR01659; hypotheti-   1255   P   I   3.1   2.22   422   P   NC               cal protein chrom 11                 Dec in Duke B; NC in other dukes       Avg Diff N &gt;= 200; Fold change N to B &gt;= 3x                                                         RC_T90037_at   PPPPP   unnamed protein   204   P   D   −4.6   −3.72   395   P   NC               product, chrom 4       RC_AA432130_at   PPPPP   KIAA0867 protein,   226   P   D   −5.4   −5.23   534   P   NC               chrom 12                 C classifiers       Gained in Duke C and Inc; NC in other Dukes       Avg Diff in C &gt; 300                                                         RC_D45556_at   AAAPA   chrom 15; AL390085   407   A   NC   1   0   536   P   I               clone       RC_W86214_at   AAAPA       305   A   NC   2.2   0.39   409   P   I       RC_AA039439_s —     AAAPA   novel gene KIAA0134   320   A   NC   3.2   1.39   600   P   I       at       protein 19q13.3       RC_AA128935_at   AAAPA       245   A   NC   1.5   0.12   416   P   I       RC_AA134158_s —     AAAPA   class I homeodomain;   273   A   NC   5   2.21   988   P   I       at       homeobox protein,               chrom 7       RC_AA232646_at   AAAPA   chrom 17, AF266756   601   A   NC   4.9   3.21   608   P   I               sphingosine kinase               (SPHK1       RC_AA401184_at   AAAPA   no homology   224   A   NC   ˜−2.2   −0.1   502   P   I       RC_AA436840_at   AAAPA       367   A   NC   1.4   0.1   458   P   I       RC_AA488655_at   AAAPA       257   A   NC   ˜9.5   4.43   360   P   I                 L st in Duke C and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_N30231_at   PPPAP   Lsm4 protein; U6   1161   P   NC   −1.3   −0.13   −978   A   D               snRNA-associated               Sm-like protein LSm4;               glycine-rich protein       RC_W73790_f_at   PPPAP   immunoglobulin-   728   P   NC   −4.2   −5.76   759   A   D               related protein 14.1;               lambda L-chain C               region; omega protein,               chrom 22       RC_AA412184_at   PPPAP   chrom 1p36; d89060   488   P   NC   −1.5   −0.18   437   A   D               dolichyl-               diphosphooligosaccha-               ride-protein glycosyl-               transferase       RC_AA521303_at   PPPAP   methionine adenosyl-   479   P   NC   −1.6   −0.29   338   A   D               transferase regulatory               beta subunit; dTDP-4-               keto-6-deoxy-D-               glucose 4-reductase,               chrom 5                 Inc in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to C &gt;= 3x                                                         RC_AA181902_at   PPPPP   AC007201 on chrom   614   P   NC   1.5   0.21   1347   P   I               19 (only 80 nt hom)                 Dec in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to D &gt;= 3x                                                         RC_AA461174_at   PPPPP   8p21.3-p22 AB020860   169   P   NC   −1.7   −0.23   84   P   D               anti-oncogene       AA393432_s_at   PPPPP   chrom 2, Unknown;   159   P   NC   −2   −0.38   97   P   D               unnamed protein               product AAD20029                 D classifier       Gained in Duke D and Inc; NC in other Dukes       Avg Diff in D &gt; 300                                                         RC_N91920_at   AAAAP   chrom 16p12-p11.2;   123   A   NC   ˜6.6   1.88   −70   A   NC               XN_007994 retinobla-               stoma binding protein       RC_AA621601_at   AAAAP   chrom 17 XM_009868   185   A   NC   ˜1.3   0.01   18   A   NC               RAB36 ARS oncogene               family                 Lost in Duke D and Dec; NC in other Dukes       Avg Diff in N &gt; 500                                                         RC_R72886_s_at   PPPPA   KIAA0422; adenylyl   1052   P   NC   −1.7   −0.53   1528   P   NC               cyclase type VI, chrom               12       RC_AA026030_at   PPPPA   chrom 1   1074   P   NC   −1.4   −0.19   1263   P   NC       RC_Z39006_at   PPPPA   hypothetical protein,   824   P   NC   −1.5   −0.28   876   P   NC               chrom 17       RC_AA435908_at   PPPPA   chrom 19; ac011491   673   P   NC   −1.2   −0.04   394   P   NC               clone and 20 nt hom.               RAB2, RAS oncogene               family       RC_AA057829_s —     PPPPA   growth-arrest-specific   1572   P   NC   1.6   0.44   936   P   NC       at       protein; growth arrest-               specific 6; AXL stimu-               latory factor, chrom 13       RC_R72087_at   PPPPA   chrom 5 EST; hom to   726   P   NC   −1.3   −0.09   971   P   NC               chrom 20 AL356652               clone       RC_H04242_at   PPPPA   ras related protein   365   P   NC   −1.4   −0.1   475   P   NC               Rab5b; RAB5B, mem-               ber RAS oncogene               family       RC_R97304_f_at   PPPPA   HLA-drb5; cell surface   556   P   NC   −1.5   −0.25   680   P   NC               glycoprotein; MHC               HLA-DR-beta chain               precursor chrom 6       RC_N48609_at   PPPPA   chrom 11; AC004584   884   P   NC   1.1   0.04   834   P   NC               chrom 17       RC_W86850_f_at   PPPPA   chrom 22 ? X96924   −63   P   NC   ˜−23.9   −13.75   474   P   NC               mitochondrial citrate               tranbsport region       RC_AA130603_at   PPPPA   ak024908 clone   681   P   NC   1.1   0.01   598   P   NC       RC_AA479610_at   PPPPA   singleton ak025344   618   P   NC   −1   0   726   P   NC               clone       RC_AA490593_l_at   PPPPA   chrom 17 ? Synapto-   610   P   NC   −1.1   −0.02   518   P   NC               brevin2 (VAMP2)               AF135372       RC_AA054321_s —     PPPPA   6p21 HLA class i   346   P   NC   −1.5   −0.15   441   P   NC       at       region; AC004202               clone                 Inc in Duke D; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                         D79052_s_at   PPPPP   Sec61 gamma   856   P   NC   −1   0   868   P   NC       RC_T40439_s_at   PPPPP   U2 small nuclear   934   P   NC   1.2   0.07   1125   P   NC               ribonucleoprotein B″;               dJ705D16.1       RC_AA251829_at   PPPPP   CGI-29 protein   440   P   NC   2.2   0.57   366   P   NC                 Dec in Duke D; NC in other Dukes       Avg Diff N &gt;= 500; Fold change N to D &gt;= −5x                                                         RC_D60328_at   PPPPP   chrom 6, unknown;   738   P   NC   −1.8   −0.58   722   P   NC               ring finger protein 5       RC_H96850_at   PPPPP   oligosaccharyltransfer-   4062   P   NC   −1.2   −0.13   3685   P   NC               ase d89060 1p36.1               (also C-class)       RC_AA127444_at   PPPPP   chrom 1 no homology   364   P   NC   −1.7   −0.33   427   P   NC       RC_AA242824_at   PPPPP   chrom 11; ac005233   246   P   NC   −1.3   −0.07   144   P   NC               PAC clone chrom 22       AA405775_s_at   PPPPP   similar to CAA16821   804   P   NC   −1.3   −0.11   791   P   NC               (PID: g3255952)                 AB classifiers       Gained in Duke AB and Inc; NC in other Dukes       Avg Diff in AB &gt;= 300                                                         RC_AA136269_at   APPAA   HSPC314, chrom 12   266   P   I   2.9   1.25   85   A   NC                 Lost in Duke AB and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                         RC_T40895_at   PAAPP   protein tyrosine phos-   41   A   D   ˜−12.7   −9.62   720   P   D               phatase PTPCAAX1;               protein tyrosine phos-               phatase hPRL-1N; iva-               1, chrom 6       RC_AA424400_at   PAAPP   chrom 14   120   A   D   −2.9   −0.97   180   P   NC                 PPPPP; Inc in Duke AB       Avg Diff AB &gt;= 300; Fold change N to AB &gt;= 5x                                                         RC_N22015_at   PPPPP   unnamed protein   3608   P   I   5.3   8.6   2944   P   I               product, chrom 17;               BC class.       RC_N50971_at   PPPPP   —   581   P   I   6.8   4.78   761   P   I       RC_AA194833_at   PPPPP   senescence-   1316   P   I   7.6   8.35   328   P   I               associated epithelial               membrane protein;               Claudin-1, chrom 3       RC_AA393803_at   PPPPP   chrom 6   503   P   I   9.2   6.04   359   P   I                 BC classifiers       Gained in Duke BC and Inc; NC in other Dukes       Avg Diff in BC &gt; 300                                                         RC_AA148923_at   AAPPA   DEPP; decidual pro-   731   P   I   4   2.7   507   P   I               tein induced by proges-               terone; hypothetical               protein, chrom 10                 Lost in Duke BC and Dec       Avg Diff in N &gt; 300                                                         RC_N91919_s_at   PPAAP   mineralocorticoid   16   A   D   ˜−13.4   −7.34   39   A   D               receptor; mineralocor-               ticoid receptor (al-               dosterone receptor);               chrom 4       RC_T99196_s_at   PPAAP   chrom 5 AF010235   143   A   D   −3.7   −1.94   197   A   D                 Homo sapiens  mRNA               from chromosome               5q31-33 region       RC_W72366_at   PPAAP   chrom 1 clone RP11-   69   A   D   −4.7   −2.2   115   A   D               404F10 on chromo-               some 1q23.1-24.1,               CD48 hom       RC_AA255903_at   PPAAP   CD39L4; CD39-like 4;   −36   A   D   ˜−25.4   −15.91   153   A   D               chrom 14                 PPPPP; Inc in Duke BC       Avg Diff N &gt;= 300; Fold change N to BC &gt;= 5x                                                         J03464_s_at   PPPPP   collagen alpha-2 type   3072   P   I   8.7   14.19   2027   P   I               I mRNA COL2A1       RC_N22015_at   PPPPP   unnamed protein   3608   P   I   5.3   8.6   2944   P   I               product, chrom 17       RC_AA055896_at   PPPPP   chrom 9   3885   P   I   6.4   11.26   2467   P   I       RC_AA121315_at   PPPPP   KIAA1077 protein;   3493   P   I   9.8   16.81   1724   P   I               hypothetical protein,               chrom 8       RC_AA122386_at   PPPPP   procollagen alpha   2356   P   I   5.1   6.63   1725   P   I               2(V); pro-alpha               (V)collagen (AA 1099),               chrom 2                 PPPPP; DEC in Duke BC       Avg Diff N &gt;= 500; Fold change N to BC &gt;= 5x                                                         C02386_s_at   PPPPP       469   P   D   −7.1   −11.91   640   P   D       M12272_s_at   PPPPP   alcohol dehydrogen-   266   P   D   −11.6   −18.17   120   P   D               ase class I gamma               subunit (ADH3)       M12759_at   PPPPP   Ig J chain gene   239   P   D   −8.6   −11.42   107   P   D       M83670_s_at   PPPPP   carbonic anhydrase   249   P   D   −9.6   −12.13   342   P   D               IV       N91087_at   PPPPP   dJ991C6.1 (novel   173   P   D   −5.3   −4.45   167   P   D               protein similar to  C.                   elegans  F55A12.9               (Tr: P91086)); un-               named protein               product; chrom 6       RC_H77597_f_at   PPPPP   unident. EST chrom ///   762   P   D   −7.1   −15.26   619   P   D               AFFY = similar metal-               lothionein       RC_N23665_s_at   PPPPP   L11708 ESTRADIOL   84   P   D   −12   −10.65   68   P   D               17 BETA-               DEHYDROGENASE               2, chrom 16       RC_N79237_at   PPPPP   unnamed protein   237   P   D   −29.6   −48.42   458   P   D               product, hom to               S49589 cortical gran-               ule lectin - African               clawed frog;.       RC_N80129_f_at   PPPPP   MT-11 protein; metal-   323   P   D   −11.2   −19.21   516   P   D               lothionein IX; metal-               lothionein 1L, chrom               16 &amp; chrom 1       RC_T24011_at   PPPPP   —   177   P   D   −5.1   −4.15   157   P   D       RC_T90492_at   PPPPP   IGJ, P01591 IMMU-   468   P   D   −10.3   −20.59   321   P   D               NOGLOBULIN J;.               chrom 4       RC_AA058357_s —     PPPPP   singleton; no hom.,   232   P   D   −20.3   −32.79   460   P   D       at       NONSPECIFIC               CROSSREACTING               ANTIGEN.;.       RC_AA133469_at   PPPPP   cytokeratin 20, chrom   453   P   D   −6.5   −10.18   375   P   D               17       RC_AA151674_at   PPPPP   carbonic anhydrase   188   P   D   −8.8   −10.51   116   P   D               XII precursor, chrom               15       RC_AA405715_at   PPPPP   unknown   542   P   D   −7.7   −14.69   521   P   D       RC_AA428410_at   PPPPP   KIAA0828 protein,   141   P   D   −8.6   −6.99   53   P   D               chrom 7       RC_AA429253_at   PPPPP   KIAA0803 protein;   103   P   D   −5.4   −3.5   111   P   D               Centrosome- and               Golgi-localized PKN-               associated protein               (CG-NAP); AKAP450,               A-kinase anchoring               protein AKAP350,               chrom 7       RC_AA487468_at   PPPPP   chrom 7   106   P   D   −6.5   −4.92   95   P   D       RC_AA621680_at   PPPPP   zinc finger transcrip-   110   P   D   −10.9   −10.81   124   P   D               tion factor GKLF; EZF;               hEZF; endothelial               Kruppel-like zinc finger               protein, transforming               oncogene       Y09616_at   PPPPP   putative carboxyles-   299   P   D   −6.1   −7.47   260   P   D               terase       AA171913_at   PPPPP   carbonic anhydrase   78   P   D   −13.6   −11.99   85   P   D               XII (CA12) chrom 15       AA253330_s_at   PPPPP   hypothetical protein;   448   P   D   −6   −8.89   420   P   D               unnamed protein               product, chrom 15                                                                     unknown genes, data   Fold   Sort           Diff   Fld   Sort           Tumor   found after blast   Change   Score   Avg   Abs   Call   Change   Score       EST candidates   stages   search   Cvs   Cvs   Diff   Call   Dvs   Dvs   Dvs       Probe Set   NABCD   homologous to   N   N   D   D   N   N   N                         LOST from N to tumour (tumour suppressor)                                                     RC_H04768_at   PAAAA   chrom 15 no homology   −9.3   −9.54   46   A   D   ˜−7.8   −0.22       RC_Z39652_at   PAAAA   Y14593 APM-1 gene   ˜−21.5   −16.51   16   A   D   ˜−7.0   −0.53               adipocyte-specific               secretory protein:               chrom 1q21.3-q23       RC_H30270_at   PAAAA   chrom 18 PAAAA in   −8.1   −7.28   114   A   D   ˜−5.7   −8.11               colon &amp; bladder no               homology       RC_T47089_s_at   PAAAA   tenascin-X; tenascin-   ˜−21.5   −14.77   −42   A   D   ˜−21.7   −4.93               X precursor; unidenti-               fied protein       RC_W31906_at   PAAAA   secretagogin;   −2.8   −1.34   147   A   D   ˜−17.0   −0.4               dJ501N12.8 (putative               protein) chrom 6       RC_AA279803_at   PAAAA   chrom 2 no homology   −4.3   −2.73   161   A   D   −11.1   −12.8       RC_R01646_at   PAAAA   chrom 13q32.1-33.3;   −13.8   −11.32   −37   A   D   ˜−6.5   −5.64               AL159152; homolo-               gy to mouse Pcbp1 -               poly(rC)-binding               protein 1       RC_AA099820_at   PAAAA   BAC clone AC016778   ˜−9.8   −6.76   −5   A   D   ˜−4.7   −0.52       AA319615_at   PAAAA   secretory carrier   ˜−34.8   −15.82   −225   A   D   ˜−35.4   −14.53               membrane protein;               secretory carrier               membrane protein 2;               chrom 15       H07011_at   PAAAA   tetraspan NET-6   −2.6   −0.79   193   A   D   −1.7   −9.87               mRNA; transmem-               brane 4 superfamlly;               chrom 7       RC_T68873_f_at   PPAAA       ˜−61.8   −46.51   440   A   D   −8.3   −14.71       RC_T40995_f_at   PPAAA       −4.3   −5.18   856   A   D   −4.2   −4.98       RC_H81070_f_at   PPAAA       ˜−60.5   −38.36   −183   A   D   ˜−64.2   −39.64       RC_N30796_at   PPPAA       −3.7   −3.04   711   A   D   −2.6   −1.48       RC_W37778_f_at   PPAAA       −4.6   −3.92   280   A   D   −4.3   −3.58       RC_R70212_s_at   PPAAA       −7.4   −6.17   115   A   D   −6.2   −4.77       RC_AA426330_at   PPAAA       ˜−23.3   −14.18   −23   A   D   ˜−19.4   −20.17       RC_N33927_s_at   PPAAA       −4.1   −2.44   109   A   D   ˜−3.8   −2.11       RC_T90190_s_at   PPAAA       −8.8   −5.67   170   A   D   −3.4   −1.73       RC_AA447145_at   PPPAA       −6.2   −3.44   130   A   D   −2.9   −1.04       RC_H75860_at   PPAAA       ˜−10.8   −5.85   29   A   D   ˜−8.6   −7.11       RC_T71132_s_at   PPAAA       ˜−8.5   −4.61   9   A   D   ˜−8.6   −4.57                 GAINED from N to tumour       (oncogene)                                                     RC_AA609013_s —     APPPP   microsomal dipepti-   7.5   12.38   2447   P   I   5.8   8.12       at       dase (also on 6.8 k);               chrom 16       RC_AA232508_at   APPPP   CGI-89 protein; un-   3.1   2.5   3179   P   I   5.7   8.6               named protein product;               hypothetical protein       RC_AA428964_at   APPPP   serine protease-like   3.1   2.19   1037   P   I   4.3   3.56               protease; serine pro-               tease homolog = NES1;               normal epithelial cell-               specific 1       RC_T52813_s_at   APPPP   dJ28O10.2 (G0S2   7.3   8.03   604   P   I   1.4   0.14               (PUTATIVE LYM-               PHOCYTE G0/G1               SWITCH PROTEIN 2;               chrom 1       RC_AA075642_at   APPPP   gp-340 variant protein;   20.1   34.59   7085   P   I   9.7   23.8               DMBT1/8kb.2 protein       RC_AA007218_at   APPPP   chrom 13 no homology   5.7   4.71   711   P   I   ˜5.4   4.47       RC_N33920_at   APPPP   ubiquitin-like protein   ˜12.5   7.18   524   P   I   ˜5.8   3.73               FAT10; diubiquitin;               dJ271M21.6 (Diubiqui-               tin); chrom 6       RC_N71781_at   APPPP   KIAA1199 protein,   ˜9.4   4.81   579   P   I   ˜6.6   4.59               chrom 15       RC_R67275_s_at   APPPP   alpha-1 (type XI)   ˜11.8   6.7   411   P   I   ˜4.3   2.06               collagen precursor;               collagen, type XI,               alpha 1; collagen type               XI alpha-1 isoform A;               chrom 1       RC_W80763_at   APPPP   hypothetical protein;   5   3.82   747   P   I   3.9   2.5               chrom 17       RC_AA443793_at   APPPP   chrom 7p22   3.7   2.51   473   P   I   1.4   0.12               AC006028 BAC clone       RC_AA034499_s —     APPPP   ZNF198 protein; zinc   4.3   2.56   464   P   I   ˜3.2   1.47       at       finger protein; FIM               protein; Cys-rich pro-               tein; zinc finger protein               198; chrom 13       RC_AA035482_at   APPPP   chrom 5; AK022505   2.2   0.67   592   P   I   3   1.37               clone; CalcineurinB               (weakly similar)       RC_AA024482_at   APPPP   hypothetical protein;   ˜21.9   10.07   443   P   I   ˜22.7   11.3               unnamed protein               product; chrom 17       RC_H93021_at   APPPP   chrom 2; XM_004890   3.3   1.58   590   P   I   2.7   1.3               peptidylprolyl isomer-               ase A (cyclophilin A)       RC_AA427737_at   APPPP   no homology   3.5   1.77   461   P   I   3.2   1.37       RC_AA417078_at   APPPP   chrom 7q31;   3.7   1.71   337   P   I   2.7   0.83               AF017104 clone       M29873_s_at   APPPP   cytochrome P450-IIB   ˜38.3   17.75   400   P   I   5.5   3.08               (hIIB3); 19q13.1-               q13.2       RC_H27498_f_at   AAPPP       3.6   7.91   7876   P   I   3.1   5.35       RC_T92363_s_at   AAPPP       3.5   1.18   311   P   I   ˜2.4   0.53       RC_N89910_at   AAAPP       1.5   0.12   250   P   NC   −2.3   −0.74       RC_W60516_at   AAAPP       1.1   0.02   404   P   NC   −2.7   −1.51       RC_AA219699_at   AAAPP       ˜2.7   0.4   293   P   I   ˜2.6   0.57       RC_AA449450_at   AAAPP       1.7   0.27   395   P   NC   −1.7   −0.3                 A classifiers       Gained in Duke A and Inc; NC in other Dukes;       Avg Diff in A &gt; 300                                                     RC_AA599199_at   APAAA   ALU seq.   ˜5.8   2.11   568   A   NC   −2.1   −0.83       RC_R12694_at   APAAA   unnamed protein   1.8   0.22   213   A   NC   ˜1.6   0.11               product BAA91641,               chrom 10       RC_H91325_s_at   APAAA   aldolase B; aldolase B   −1.3   −0.03   96   A   NC   1   0               (aa 1-364); chrom 9       RC_N51709_at   APAAA   chrom X   1   0   435   A   NC   −3   −2.05       RC_N72610_at   APAAA   —   1.2   0.06   373   A   NC   −3.7   −3.23                 Lost in Duke A and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                     RC_N32411_f_at   PAPPP   Myc-associated zinc-   1.2   0.12   3403   P   NC   −1.3   −0.27               finger protein of hu-               man islet; chrom 16       RC_AA243858_at   PAPPP   KIAA0882 protein   −1.2   −0.03   322   P   NC   −3.4   −2.36       RC_AA486283_at   PAPPP   ras-like protein; ras-   1.1   0.02   5504   P   NC   1.1   0.07               related C3 botulinum               toxin substrate;               dJ20J23       RC_AA490930_at   PAPPP   chrom 18; KIAA1468   −2.5   −0.86   175   P   NC   −3.2   −1.48               protein                 PPPPP; Inc in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                     RC_N69263_at   PPPPP   chrom 10; AK026414   1.9   0.43   660   P   NC   1.8   0.35               clone (only 108 nt               hom)       RC_T15817_f_at   PPPPP   iNOS, inducible nitric   1.8   0.44   753   P   NC   −1.3   −0.11               oxide synthase                 PPPPP; Dec in Duke A; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                     RC_H54088_s_at   PPPPP   ribosomal protein L41   1.2   0.21   5372   P   NC   1   0       RC_H59052_f_at   PPPPP   fungal sterol-C5-   1.1   0.08   5216   P   NC   1   0.01               desaturase homolog;               ORF; thymosin beta-4       RC_R49198_s_at   PPPPP   —   −1   0   5478   P   NC   1.1   0.07       RC_T73572_f_at   PPPPP   ferritin L-chain; L   1.1   0.03   5457   P   NC   −1   −0.02               apoferritin       RC_AA477483_at   PPPPP   no matching est   −1.1   −0.03   4052   P   NC   −1.1   −0.05                 B classifiers       Gained in Duke B and Inc; NC in other Dukes       Avg Diff in B &gt; 300                                                     RC_T67463_s_at   AAPAA   cathepsin O2; X; K   1.7   0.19   275   A   NC   ˜1.3   0.05       RC_W94688_at   AAPAA   perilipin   −1   0   92   A   NC   −1.5   −0.09       RC_AA126743_at   AAPAA   Z97200 PAC chrom   1.1   0.02   310   A   NC   −1.3   −0.07               1q24; PMX1 homeo-               box gene       RC_AA236547_at   AAPAA   no homology   1.3   0.05   191   A   NC   −2.1   −0.6       RC_AA255567_at   AAPAA   angiopoietin-related   1.7   0.2   235   A   NC   −1.4   −0.1               protein-2; angiopoietin-               like 2       RC_AA421256_at   AAPAA   —   −1.1   −0.01   269   A   NC   −3.8   −2.9                 Lost in Duke B and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                     RC_D59847_at   PPAPP   proSAAS; granin-like   −1.9   −0.56   423   P   NC   −1.5   −0.22               neuroendocrine pep-               tide precursor       RC_F05038_at   PPAPP   polyamine modulated   −1   0   1068   P   NC   −4.4   −7.65               factor-1; polyamine               modulated factor 1       RC_N41059_at   PPAPP   chrom 3   1.1   0.01   287   P   NC   −3.4   −2.28       RC_T23460_at   PPAPP   chrom 3; IFNAR2   −1.3   −0.08   318   P   NC   −1.9   −0.47               21q22.11       RC_W42789_at   PPAPP   chrom 8 AF268037   −2.4   −0.61   489   P   NC   1.6   0.21               C8ORF4 protein               (C8ORF4) chrom 8               ORF       RC_AA460017_l —     PPAPP   BAC clone chrom 16   −2.1   −0.85   952   P   NC   −1.4   −0.23       at       RC_AA482127_at   PPAPP   KIAA1142 protein   −1.7   −0.23   332   P   NC   −1.1   −0.01       RC_AA504806_at   PPAPP   chrom 2 AF052107   −1.2   −0.04   414   P   NC   −3.5   −2.64               clone 23620 mRNA               sequence                 Inc in Duke B; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                     RC_AA386386_s —     PPPPP   —   −1.1   −0.02   1731   P   NC   1.1   0.04       at       RC_AA452549_at   PPPPP   PR01659; hypotheti-   1.1   0.01   257   P   NC   −1.6   −0.17               cal protein chrom 11                 Dec in Duke B; NC in other dukes       Avg Diff N &gt;= 200; Fold change N to B &gt;= 3x                                                     RC_T90037_at   PPPPP   unnamed protein   −2.4   −1.07   466   P   NC   −2   −0.69               product, chrom 4       RC_AA432130_at   PPPPP   KIAA0867 protein,   −1.9   −0.56   437   P   NC   −6.4   −9.82               chrom 12                 C classifiers       Gained in Duke C and Inc; NC in other Dukes       Avg Diff in C &gt; 300                                                     RC_D45556_at   AAAPA   chrom 15; AL390085   1.4   0.11   362   A   NC   −3   −1.89               clone       RC_W86214_at   AAAPA       3.5   1.34   247   A   NC   −1.4   −0.1       RC_AA039439_s —     AAAPA   novel gene KIAA0134   4.5   2.81   259   A   NC   ˜1.7   0.19       at       protein 19q13.3       RC_AA128935_at   AAAPA       2.6   0.84   412   A   NC   ˜1.8   0.26       RC_AA134158_s —     AAAPA   class I homeodomain;   3.9   2.87   416   A   NC   −1.7   −0.34       at       homeobox protein,               chrom 7       RC_AA232646_at   AAAPA   chrom 17, AF266756   5   3.27   −18   A   NC   ˜1.1   0.01               sphingosine kinase               (SPHK1       RC_AA401184_at   AAAPA   no homology   1.4   0.14   78   A   NC   ˜0.4   0.63       RC_AA436840_at   AAAPA       1.7   0.3   57   A   NC   −1.5   −0.08       RC_AA488655_at   AAAPA       ˜9.6   5.32   124   A   NC   ˜4.1   1.01                 L st in Duke C and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                     RC_N30231_at   PPPAP   Lsm4 protein; U6   ˜−59.3   −36   808   P   NC   −1.9   −0.67               snRNA-associated               Sm-like protein LSm4;               glycine-rich protein       RC_W73790_f_at   PPPAP   immunoglobulin-   −4   −5.39   989   P   NC   −8.6   −23.38               related protein 14.1;               lambda L-chain C               region; omega protein,               chrom 22       RC_AA412184_at   PPPAP   chrom 1p36; d89060   −2   −0.54   545   P   NC   −1.4   −0.13               dolichyl-               diphosphooligosaccha-               ride-protein glycosyl-               transferase       RC_AA521303_at   PPPAP   methionine adenosyl-   −2.3   −0.87   741   P   NC   −1   −0.01               transferase regulatory               beta subunit; dTDP-4-               keto-6-deoxy-D-               glucose 4-reductase,               chrom 5                 Inc in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to C &gt;= 3x                                                     RC_AA181902_at   PPPPP   AC007201 on chrom   3.1   2.3   565   P   NC   1.2   0.04               19 (only 80 nt hom)                 Dec in Duke C; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to D &gt;= 3x                                                     RC_AA461174_at   PPPPP   8p21.3-p22 AB020860   −3.5   −1.3   170   P   NC   −1.7   −0.22               anti-oncogene       AA393432_s_at   PPPPP   chrom 2, Unknown;   −3.3   −1.22   377   P   NC   −1.3   −0.05               unnamed protein               product AAD20029                 D classifier       Gained in Duke D and Inc; NC in other Dukes       Avg Diff in D &gt; 300                                                     RC_N91920_at   AAAAP   chrom 16p12-p11.2;   ˜1.2   0.01   371   P   I   ˜6.9   3.52               XN_007994 retinobla-               stoma binding protein       RC_AA621601_at   AAAAP   chrom 17 XM_009868   ˜4.2   0.23   478   P   I   ˜12.5   5.32               RAB36 ARS oncogene               family                 Lost in Duke D and Dec; NC in other Dukes       Avg Diff in N &gt; 500                                                     RC_R72886_s_at   PPPPA   KIAA0422; adenylyl   −1.5   −0.31   410   A   D   −4.4   −4.68               cyclase type VI, chrom               12       RC_AA026030_at   PPPPA   chrom 1   −1.2   −0.06   514   A   D   −2.9   −2.02       RC_Z39006_at   PPPPA   hypothetical protein,   −1.4   −0.21   450   A   D   −2.8   −1.72               chrom 17       RC_AA435908_at   PPPPA   chrom 19; ac011491   −2   −0.6   314   A   D   −7   −9.6               clone and 20 nt hom.               RAB2, RAS oncogene               family       RC_AA057829_s —     PPPPA   growth-arrest-specific   −1   0   276   A   D   −2.7   −1.49       at       protein; growth arrest-               specific 6; AXL stimu-               latory factor, chrom 13       RC_R72087_at   PPPPA   chrom 5 EST; hom to   1.1   0.01   362   A   D   −2.5   −1.22               chrom 20 AL356652               clone       RC_H04242_at   PPPPA   ras related protein   −1.5   −0.17   298   A   D   −2.3   −0.86               Rab5b; RAB5B, mem-               ber RAS oncogene               family       RC_R97304_f_at   PPPPA   HLA-drb5; cell surface   −1.3   −0.08   425   A   D   ˜−24.3   −16.4               glycoprotein; MHC               HLA-DR-beta chain               precursor chrom 6       RC_N48609_at   PPPPA   chrom 11; AC004584   1.1   0.01   265   A   D   −2.4   −0.99               chrom 17       RC_W86850_f_at   PPPPA   chrom 22 ? X96924   −1.9   −0.59   −257   A   D   ˜−22.8   −14.3               mitochondrial citrate               tranbsport region       RC_AA130603_at   PPPPA   ak024908 clone   −1.1   −0.01   241   A   D   −2.7   −1.11       RC_AA479610_at   PPPPA   singleton ak025344   1.1   0.03   337   A   D   −5.3   −6.14               clone       RC_AA490593_l_at   PPPPA   chrom 17 ? Synapto-   −1.4   −0.12   308   A   D   −6.5   −7.34               brevin2 (VAMP2)               AF135372       RC_AA054321_s —     PPPPA   6p21 HLA class i   −1.2   −0.03   63   A   D   ˜−3.6   −1.67       at       region; AC004202               clone                 Inc in Duke D; NC in other Dukes       Avg Diff N &gt;= 200; Fold change N to A &gt;= 3x                                                     D79052_s_at   PPPPP   Sec61 gamma   1.1   0.03   3411   P   I   3.9   5.13       RC_T40439_s_at   PPPPP   U2 small nuclear   1.7   0.39   2813   P   I   4.2   5.48               ribonucleoprotein B″;               dJ705D16.1       RC_AA251829_at   PPPPP   CGI-29 protein   1.8   0.3   1445   P   I   6.7   7.21                 Dec in Duke D; NC in other Dukes       Avg Diff N &gt;= 500; Fold change N to D &gt;= −5x                                                     RC_D60328_at   PPPPP   chrom 6, unknown;   −1.9   −0.63   663   P   D   −5.7   −9.71               ring finger protein 5       RC_H96850_at   PPPPP   oligosaccharyltransfer-   −1.3   −0.27   2211   P   D   −6.2   −20.52               ase d89060 1p36.1               (also C-class)       RC_AA127444_at   PPPPP   chrom 1 no homology   −1.5   −0.17   264   P   D   −6.6   −8.01       RC_AA242824_at   PPPPP   chrom 11; ac005233   −2.1   −0.46   188   P   D   −8.4   −8.95               PAC clone chrom 22       AA405775_s_at   PPPPP   similar to CAA16821   −1.3   −0.12   135   P   D   −6.1   −4.92               (PID: g3255952)                 AB classifiers       Gained in Duke AB and Inc; NC in other Dukes       Avg Diff in AB &gt;= 300                                                     RC_AA136269_at   APPAA   HSPC314, chrom 12   1.8   0.28   154   A   NC   1.7   0.2                 Lost in Duke AB and Dec; NC in other Dukes       Avg Diff in N &gt; 300                                                     RC_T40895_at   PAAPP   protein tyrosine phos-   −1.6   −0.34   1079   P   NC   −1.1   −0.01               phatase PTPCAAX1;               protein tyrosine phos-               phatase hPRL-1N; iva-               1, chrom 6       RC_AA424400_at   PAAPP   chrom 14   −1.9   −0.35   166   P   D   −5.8   −5.07                 PPPPP; Inc in Duke AB       Avg Diff AB &gt;= 300; Fold change N to AB &gt;= 5x                                                     RC_N22015_at   PPPPP   unnamed protein   4.4   5.98   2893   P   I   4.5   6.17               product, chrom 17;               BC class.       RC_N50971_at   PPPPP   —   5.9   4.62   599   P   I   3.1   1.71       RC_AA194833_at   PPPPP   senescence-   3.7   1.78   1281   P   I   7.3   7.88               associated epithelial               membrane protein;               Claudin-1, chrom 3       RC_AA393803_at   PPPPP   chrom 6   6.6   3.59   400   P   I   7.3   4.28                 BC classifiers       Gained in Duke BC and Inc; NC in other Dukes       Avg Diff in BC &gt; 300                                                     RC_AA148923_at   AAPPA   DEPP; decidual pro-   2.8   1.11   235   A   NC   1.2   0.02               tein induced by proges-               terone; hypothetical               protein, chrom 10                 Lost in Duke BC and Dec       Avg Diff in N &gt; 300                                                     RC_N91919_s_at   PPAAP   mineralocorticoid   ˜−12.0   −7.51   136   P   D   −3.7   −1.89               receptor; mineralocor-               ticoid receptor (al-               dosterone receptor);               chrom 4       RC_T99196_s_at   PPAAP   chrom 5 AF010235   −2.7   −1.04   221   P   D   −2.7   −1.13                 Homo sapiens  mRNA               from chromosome               5q31-33 region       RC_W72366_at   PPAAP   chrom 1 clone RP11-   −2.8   −0.89   140   P   D   −6.4   −5.55               404F10 on chromo-               some 1q23.1-24.1,               CD48 hom       RC_AA255903_at   PPAAP   CD39L4; CD39-like 4;   −5.7   −4.69   204   P   MD   −11.5   −15.73               chrom 14                 PPPPP; Inc in Duke BC       Avg Diff N &gt;= 300; Fold change N to BC &gt;= 5x                                                     J03464_s_at   PPPPP   collagen alpha-2 type   5.8   7.31   2206   P   I   6.3   8.46               I mRNA COL2A1       RC_N22015_at   PPPPP   unnamed protein   4.4   5.98   2893   P   I   4.5   6.17               product, chrom 17       RC_AA055896_at   PPPPP   chrom 9   3.9   4.33   1553   P   MI   2.5   1.48       RC_AA121315_at   PPPPP   KIAA1077 protein;   4.8   5.33   1220   P   NC   3.4   2.57               hypothetical protein,               chrom 8       RC_AA122386_at   PPPPP   procollagen alpha   3.7   3.51   1716   P   I   3.7   3.47               2(V); pro-alpha               (V)collagen (AA 1099),               chrom 2                 PPPPP; DEC in Duke BC       Avg Diff N &gt;= 500; Fold change N to BC &gt;= 5x                                                     C02386_s_at   PPPPP       −5.4   −8.64   969   P   D   −3.5   −4.58       M12272_s_at   PPPPP   alcohol dehydrogen-   −25.7   −30   290   P   D   −10.6   −16.96               ase class I gamma               subunit (ADH3)       M12759_at   PPPPP   Ig J chain gene   −19.2   −20.94   190   P   D   −11.7   −15.55       M83670_s_at   PPPPP   carbonic anhydrase   −6.6   −8.32   249   P   D   −9.4   −11.84               IV       N91087_at   PPPPP   dJ991C6.1 (novel   −5.5   −4.68   350   P   D   −2.6   −1.32               protein similar to  C.                   elegans  F55A12.9               (Tr: P91086)); un-               named protein               product; chrom 6       RC_H77597_f_at   PPPPP   unident. EST chrom ///   −8.7   −18.86   979   P   D   −15.4   −49.46               AFFY = similar metal-               lothionein       RC_N23665_s_at   PPPPP   L11708 ESTRADIOL   −14   −11.67   143   P   D   −22.4   −28.45               17 BETA-               DEHYDROGENASE               2, chrom 16       RC_N79237_at   PPPPP   unnamed protein   −15.7   −34.48   499   P   D   −40.1   −92.94               product, hom to               S49589 cortical gran-               ule lectin - African               clawed frog;.       RC_N80129_f_at   PPPPP   MT-11 protein; metal-   −24.5   −31.69   737   P   D   −14.8   −40.79               lothionein IX; metal-               lothionein 1L, chrom               16 &amp; chrom 1       RC_T24011_at   PPPPP   —   −5.7   −4.85   371   P   D   −2.8   −1.54       RC_T90492_at   PPPPP   IGJ, P01591 IMMU-   −15   −27.44   623   P   D   −21.6   −57.11               NOGLOBULIN J;.               chrom 4       RC_AA058357_s —     PPPPP   singleton; no hom.,   −11.3   −23.01   1392   P   D   −3.7   −6.14       at       NONSPECIFIC               CROSSREACTING               ANTIGEN.;.       RC_AA133469_at   PPPPP   cytokeratin 20, chrom   −7.9   −12.52   1359   P   D   −2.6   −2.44               17       RC_AA151674_at   PPPPP   carbonic anhydrase   −13.7   −14.72   327   P   D   −5.6   −6.63               XII precursor, chrom               15       RC_AA405715_at   PPPPP   unknown   −8   −15.29   1120   P   D   −10.4   −32.45       RC_AA428410_at   PPPPP   KIAA0828 protein,   −14.8   −10.91   79   P   D   −9.9   −8               chrom 7       RC_AA429253_at   PPPPP   KIAA0803 protein;   −5   −3.15   213   P   D   −7.3   −8.35               Centrosome- and               Golgi-localized PKN-               associated protein               (CG-NAP); AKAP450,               A-kinase anchoring               protein AKAP350,               chrom 7       RC_AA487468_at   PPPPP   chrom 7   −7.2   −5.56   319   P   D   −2.6   −1.17       RC_AA621680_at   PPPPP   zinc finger transcrip-   −9.7   −9.77   371   P   D   −3.2   −2.3               tion factor GKLF; EZF;               hEZF; endothelial               Kruppel-like zinc finger               protein, transforming               oncogene       Y09616_at   PPPPP   putative carboxyles-   −7   −8.77   333   P   D   −5.5   −6.52               terase       AA171913_at   PPPPP   carbonic anhydrase   −51.6   −23.46   148   P   D   −7.2   −6.84               XII (CA12) chrom 15       AA253330_s_at   PPPPP   hypothetical protein;   −6.4   −9.59   697   P   D   −3.7   −4.33               unnamed protein               product, chrom 15                    
     [0362] B. Finding Potential Classifier Genes for c l rectal canc r (Dukes A, B, C &amp; D) by Sorting acc rding to Pearson Correlation co fficient  
     [0363] Primary Selection Criteria for Classifier Genes:  
     [0364] 1. All genes with a score of A (AbsCall) or NC (DiffCall) for all groups (N, A, B, C &amp; D) were removed.  
     [0365] 2. Genes with a fold change below 5 and a Sort Score below 0.5 were removed.  
     [0366] 3. If DiffCall were NC for a gene in a particular experiment the FC were set to 1.  
     [0367] Secondary Selection Criteria for Classifier Genes:  
     [0368] Based on Pearson correlation coefficient (FIG. 1) genes similar to a predefined profile were selected. 
 
 
     [0369] Classifier Genes for Dukes A, B, C and D:  
               TABLE III                          A classifiers (Profile 1, 0, 0, 0),       Pearson correlations approach                     Hu6800           D87444_at   Human mRNA for KIAA0255 “gene,”           complete cds       U18291_at   Human CDC16Hs “mRNA,” complete cds       L76568_xpt3_f_at   S26 from  Homo sapiens  excision and           cross link repair protein (ERCC4)           “gene,” complete genomic sequence.           /gb = L76568 /ntype = DNA /annot = exon       U45328_s_at   “Human ubiquitin-conjugating enzyme           (UBE2I)” “mRNA,” “complete cds”       Z14982_ma1_at     H. sapiens  gene for major histocom-           patibility complex encoded proteasome           subunit LMP7.       AD000092_cds7_s_at   RAD23A gene (human RAD23A homolog)           extracted from  Homo sapiens  DMA from           chromosome 19p13.2 cosmids “R31240,”           R30272 and R28549 containing the           “EKLF,” “GCDH,” “CRTC,”           and RAD23A “genes,” genomic sequence       D86973_at   Human mRNA for KIAA0219 “gene,”           partial cds       X81636_at     H. sapiens  clathrin light chain a gene       M59916_at   Human acid sphingomyelinase (ASM)           “mRNA,” complete cds       X85781_s_at   “ H. sapiens  NOS2” “gene,”           “exon 27 /gb = X85781 /ntype = DNA           /annot = exon”       M57731_s_at   “Human gro-beta” “mRNA,”           “complete cds”       U49188_at   Human placenta (Diff33) “mRNA,”           complete cds       X53800_s_at   Human mRNA for macrophage           inflammatory protein-2beta (MIP2beta)       U56816_at   Human kinase Myt1 (Myt1) “mRNA,”           complete cds.       HG1067-HT1067_r_at   Mucin (Gb: M22406)       EST:       RC_F03077_f   Chromosom 17, clone hRPC.15       RC_AA599199   Alu seq       RC_AA207015   clone RP4-733M16 on chromosom           1p36.11-36.23       RC_AA234916   Chromosome 19 clone CTC-461H2       RC_N92239_a   Wnt inhibitory factor-1 (WIF-1),           chromosome 12       RC_N93958_s   Phospholipase A2, group X (PLA2G10),       U95301_at   Phospholipase A2, group X (PLA2G10),       RC_AA426330   Chromosome 17, clone hRPC.1110_E_20       RC_AA024658   clone SCb-254N2 (UWGC: rg254N02)           from 6p21       RC_H88540_a   heat shock protein 90, 1q21.2-q22                 B classifiers (Profile 0, 1, 0, 0)                     Hu6800:           U57316_at   Human GCN5 (hGCNS) “gene,”           complete cds       X66839_at     H. sapiens  MaTu MN mRNA for p54/58N           protein       J04599_at   Human hPGI mRNA encoding bone small           proteoglycan I “(biglycan),”           complete cds       X57579_s_at     H. sapiens  activin beta-A subunit           (exon 2)       J02874_at   Human adipocyte lipid-binding           “protein,” complete cds       M11749_at   Human Thy-1 glycoprotein “gene,”           complete cds       U06863_at   Human follistatin-related protein           precursor “mRNA,” complete cds       U51010_s_at   “Human nicotinamide N-methyltrans-           ferase” “gene,” “exon 1 and 5′           flanking region. /gb = U51010 /ntype = DNA           /annot = exon”       U08021_at   “Human nicotinamide N-methyltrans-           ferase (NNMT)” “mRNA,”           “complete cds”       HG3044-   “”“Fibronectin,” “Alt. Splice 1”       HT3742_s_at       X02761_s_at   Human mRNA for fibronectin (FN precursor)       X02544_at   Human mRNA for alpha1-acid glycoprotein           (orosomucoid)       M62505_at   Human C5a anaphylatoxin receptor           “mRNA,” complete cds       J05070_at   Human type IV collagenase “mRNA,”           complete cds       U16306_at   Human chondroitin sulfate proteoglycan           versican V0 splice-variant precursor           peptide “mRNA,” complete cds       M14218_at   Human argininosuccinate lyase “mRNA,”           complete cds       L77567_s_at   “ Homo sapiens  mitochondrial citrate           transport protein (CTP)” “mRNA,”           “3′ end”       M63391_ma1_at   Human desmin gene, complete cds.       D13643_at   Human mRNA for KIAA0018 “gene,”           complete cds       D79985_at   Human mRNA for KIAA0163 “gene,”           complete cds       EST:       M63262_at   5-lipoxygenase activating protein           (FLAP), 13q12       R67290_at   Interleukine 14       N36619_at       L19161_at   Translation initiation factor 2,           subunit 3″, Xp22.2-22.1       RC_AA496035   Chromosome 1? (TIGR)       L29217_s_at   CDC-like kinase 3 (CLK3), 15q24       RC_W73194_a   Dermatoponin, 1q12-q23       RC_N69507_a   Hypothetical protein PRO1847 (Alu           according to TIGR)       RC_H15814_s   adipose most abundant gene transcript 1       M84526_at   D component of complement (adipsin)                 C classifiers (Profile 0, 0, 1, 0)                     Hu6800:           M20681_at   Human glucose transporter-like protein-           III “(GLUTS),” complete cds       D50914_at   Human mRNA for KIAA0124 “gene,”           partial cds       L37362_at     Homo sapiens  (clone d2-115) kappa           opioid receptor (OPRK1) “mRNA,”           complete cds       X66114_ma1_at     H. sapiens  g n for 2-oxoglutarat           carrier protein.       M32053_at   Human H19 RNA “gene,” complete           cds (spliced in silico)       Y00787_s_at   Human mRNA for MDNCF (monocyte-derived           neutrophil chemotactic factor)       U64444_at   Human ubiquitin fusion-degradation           prot in (UFD1L) “mRNA,” complete           cds       X95325_s_at     H. sapiens  mRNA for DNA binding protein           A variant       X02419_ma1_s_at     H. sapiens  uPA gene       X57522_at     H. sapiens  RING4 cDNA       AB001325_at   Human AQP3 gene for aquaporine 3 (water           “channel),” partail cds       AB002315_at   Human mRNA for KIAA0317 “gene,”           complete cds. /gb = AB002315           /ntype = RNA       L12760_s_at   “Human phosphoenolpyruvate           carboxykinase (PCK1)” “gene,”           “complete cds with repeats”       M80899_at   Human novel protein AHNAK “mRNA,”           partial sequence       EST:       RC_AA122350   Chromosome 8       AA374109_at   spondin 2, extracellular matrix           protein, chromosome 4       RC_AA621755   Transcription factor Dp-2, 3q23       RC_AA442069   sodium channel 2, 12q12       RC_T40767_a   Chromosome 19       RC_AA488655   Mus?       RC_AA398908       RC_AA447764   Hypothetical protein, chromosome 4       RC_N69136_a                 D classifiers (Profile 0, 0, 0, 1)                     X17644_s_at   Human GST1-Hs mRNA for GTP-binding           protein       Y12812_at     H. sapiens  RFXAP mRNA       X60486_at     H. sapiens  H4/g gene for H4 histone       X52221_at     H. sapiens  ERCC2 “gene,” exons           1 &amp; 2 (partial)       L06175_at     Homo sapiens  P5-1 “mRNA,” complete cds       Z48481_at     H. Sapiens  mRNA for membrane-type           matrix metalloproteinase 1       X54232_at   Human mRNA for heparan sulfate           proteaglycan (glypican)       L08010_at   “ Homo sapiens  reg gene” “homologue,”           “complete cds”       L27706_at   Human chaperonin protein (Tcp20)           gene complete cds       L15533_ma1_at     Homo sapiens  pancreatits-associated           protein (PAP) gene, complete cds.       X51408_at   Human mRNA for n-chimaerin       K02765_at   Human complement component C3 “mRNA,”           alpha and beta “subunits,” complete cds       U38904_at   Human zinc finger protein C2H2-25           “mRNA,” complete cds       EST:       RC_AA121433   Axin, chromosome 16       RC_N91920_a   RB protein binding protein, chromosome 16       RC_AA621601   GTP-binding protein Rab36, chromosome 17       RC_AA454020   NADPH quinone oxidoreductase homolog;           p53 induced, chromosome 2       RC_Z39652_a   APM-1 gene, chromosome 18                  
 
     [0370] Conclusion.  
     [0371] As can be seen from these tables we have identified a number of genes and EST&#39;s, based on two different aporoaches, that we believe are either of importance for initiating and developing colorectal cancer, or can be used to classify the disease. These genes and EST&#39;s are subdivided into potential tumor suppressors that have a reduced level during progressi n of the diseas—or that ev n completely los their expression; potential oncogenes that increas th ir level during disease progression, or ven are gain d de novo, not being xpressed at arly stages or in normal mucosa; and finally classifiers of the disease that can be us d to identify the different Dukes stages, .g. being only expressed at a certain stage.