Abstract:
The invention discloses a method for classification of cancer in an individual having contracted cancer. The method of classification involves the determination of microsatellite status and a prognostic marker by examining gene expression patterns. The invention also relates to various methods of treatment of cancer. Additionally, the present invention concerns a pharmaceutical composition for treatment of cancer and uses of the present invention. The invention also relates to an assay for classification of cancer.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to a method for classification of cancer in an individual, wherein the microsatellite status and a prognostic marker are determined by examining gene expression patterns. The invention also relates to various methods of treatment of cancer. Additionally, the present invention concerns a pharmaceutical composition for treatment of cancer and uses of the present invention. The invention also relates to an assay for classification of cancer. 
       BACKGROUND OF INVENTION 
       [0002]    Studies of differential gene expression in diseased and normal tissues have been greatly facilitated by the building of large databases of the human genome sequences. Gene expression alterations are important factors in the progression from normal tissue to diseased tissue. In order to obtain a profile of transcriptional status in a certain cell type or tissue, array-based screening of thousands of genes simultaneously is an invaluable tool. Array-based screening even allows for the identification of key genes that alone, or in combination with other genes, regulate the behaviour of a cell or tissue. Candidate genes for future therapeutic intervention may thus also be identified. 
         [0003]    Colorectal cancer generally occurs in 1 out of every 20 individuals at some point during their lifetime. In the United States alone about 150,000 new cases are diagnosed each year which amount to 15% of the total number of new cancer diagnoses. Unfortunately, colorectal cancer causes about 56,000 deaths a year in the United States. 
         [0004]    The malignant transformation from normal tissue to cancer is believed to be a multistep process. Two molecular pathways are known to be involved in the development of colorectal cancer (Lengauer C, Kinzler K W, Vogelstein B., 1998) namely the microsatellite stable (MSS) pathway and the microsatellite instable (MSI) pathway. MSS is associated with high frequency of allelic losses, abnormalities of cytogenetic nature and abnormal tumor content of DNA. MSI however is associated with defects in the DNA mismatch repair system which leads to increased rate of point mutations and minor chromosomal insertions or deletions. 
         [0005]    MSI tumors can be of hereditary or sporadic nature. Ninety percent of MSI tumours are of sporadic origin. Sporadic tumours are presumably MSI due to epigenetic hypermethylation of the MLH1 gene promoter. The hereditary tumours account for 10% of the MSI tumors. Mutations of for example the MLH1 or MSH 2 genes are often the cause of hereditary tumor development. 
         [0006]    The ability of being able to determine the sporadic or hereditary nature of a MSI tumor is highly valuable. In case a tumor is characterized as being MSI, and certain clinical criteria are fulfilled such as age below 50 or three first degree relatives with colon cancer, a screening programme of family members for early diagnosis and treatment of potential colon or endometrial cancer development is initiated. The human and economic costs in relation to screening programmes are severe. Consequently, a need for identifying colon cancers with a hereditary character exists. Further, these patients have a poor prognosis, as they have an increased risk of metachronous colon tumors and a highly increased risk of getting cancer in the endometrium (females), upper urinary tract and a number of other organs. Thus, one may regard the determination of a colon tumor as being sporadic or hereditary as determination of a prognostic factor. 
         [0007]    Tumors appearing to be similar—morphologically, histochemically or microscopically—can be profoundly different. They can have 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. Determination of microsatellite status using an array-based methodology is faster than conventional DNA based methods, as it does not require microdissection, and forms a set of genes that can be combined with other sets of genes on a colon cancer array that can be used to determine microsatellite status as well as e.g. predict disease course by identifying hereditary cases or other prognostic important factors, and finally predict therapy response. 
       SUMMARY OF INVENTION 
       [0008]    In one aspect the present invention relates to a method of classifying cancer in an individual having contracted cancer comprising 
         [0000]    in a sample from the individual having contracted cancer determining the microsatellite status of the tumor and
 
in a sample from the individual having contracted cancer, said sample comprising a plurality of gene expression products the presence and/or amount which forms a pattern, determining from said pattern a prognostic marker, wherein the microsatellite status and the prognostic marker is determined simultaneously or sequentially
 
classifying said cancer from the microsatellite status and the prognostic marker.
 
         [0009]    The cancer may be any cancer known to be microsatellite instable in at least a fraction of the cases, such as colon cancer, uterine cancer, ovary cancer, stomach cancer, cancer in the small intestine, cancer in the biliary system, urinary tract cancer, brain cancer or skin cancer. These cancers are part of the spectrum of cancers that belong to the hereditary non-polyposis colon cancer syndrome, but the invention is not limited to this syndrome. 
         [0010]    Gene expression patterns may be formed by only a few genes, but it is also a preferred embodiment that a multiplicity of genes form the expression pattern whereby information for classification of cancer can be obtained. 
         [0011]    Furthermore, the invention relates to a method for classification of cancer in an individual having contracted cancer, wherein the microsatellite status is determined by a method comprising the steps of 
         [0000]    in a sample from the individual having contracted cancer, said sample comprising a plurality of gene expression products the presence and/or amount of which forms a pattern that is indicative of the microsatellite status of said cancer,
 
determining the presence and/or amount of said gene expression products forming said pattern,
 
obtaining an indication of the microsatellite status of said cancer in the individual based on the step above.
 
         [0012]    Yet another aspect of the invention relates to a method for classification cancer in an individual having contracted cancer, wherein the hereditary or sporadic nature is determined by a method comprising the steps of 
         [0000]    in a sample from the individual having contracted cancer, said sample comprising a plurality of gene expression products the presence and/or amount of which forms a pattern that is indicative of the hereditary or sporadic nature of said cancer,
 
determining the presence and/or amount of said gene expression products forming said pattern,
 
obtaining an indication of the hereditary or sporadic nature of said cancer in the individual based on the step above.
 
         [0013]    The present invention further concerns a method for treatment of an individual comprising the steps of 
         [0000]    selecting an individual having contracted a colon cancer, wherein the microsatellite status is stable, determined according to any of the methods as defined herein
 
treating the individual with anti cancer drugs.
 
         [0014]    Another aspect of the present invention relates to a method for treatment of an individual comprising the steps of 
         [0000]    selecting an individual having contracted a colon cancer, wherein the microsatellite status is instable, determined according to any of the methods as defined herein
 
treating the individual with anti cancer drugs.
 
         [0015]    Yet another aspect of the present invention relates to a method for reducing malignancy of a cell, said method comprising 
         [0000]    contacting a tumor cell in question with at least one peptide expressed by at least one gene selected from genes being expressed at least two-fold higher in tumor cells than the amount expressed in said tumor cell in question. 
         [0016]    Additionally, the present invention concerns a method for reducing malignancy of a tumor cell in question comprising, 
         [0000]    obtaining at least one gene selected from genes being expressed at least two fold lower in tumor cells than the amount expressed in normal cells
 
introducing said at least one gene into the tumor cell in question in a manner allowing expression of said gene(s).
 
         [0017]    The invention also relates to a method for reducing malignancy of a cell in question, said method comprising 
         [0000]    obtaining at least one nucleotide probe capable of hybridising with at least one gene of a tumor cell in question, said at least one gene being selected from genes being expressed in an amount at least two-fold higher in tumor cells than the amount expressed in normal cells, and
 
introducing said at least one nucleotide probe into the tumor cell in question in a manner allowing the probe to hybridise to the at least one gene, thereby inhibiting expression of said at least one gene.
 
         [0018]    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 
         [0000]    obtaining expression product(s) from at least one gene said gene being expressed as defined herein
 
immunising a mammal with said expression product(s) obtaining antibodies against the expression product.
 
         [0019]    The present invention also concerns a method for treatment of an individual comprising the steps of 
         [0000]    selecting an individual having contracted a colon cancer, wherein the microsatellite status is stable, determined according to any of the methods as defined herein
 
introducing at least one gene into the tumor cell in a manner allowing expression of said gene(s).
 
         [0020]    The present invention further relates to a pharmaceutical composition for the treatment of a classified cancer comprising at least one antibody as defined herein. 
         [0021]    In yet another aspect the invention concerns a pharmaceutical composition for the treatment of a classified cancer comprising at least one polypeptide as defined herein. 
         [0022]    Further, the invention relates to a pharmaceutical composition for the treatment of a classified cancer comprising at least one nucleic acid and/or probe as defined herein. 
         [0023]    In an additional aspect the present invention relates to an assay for classification of cancer in an individual having contracted cancer, comprising 
         [0000]    at least one marker capable of determining the microsatellite status in a sample and at least one marker in a sample determining the prognostic marker, wherein the microsatellite status and the prognostic marker is determined simultaneously or sequentially. 
     
    
     
       DETAILED DESCRIPTION OF THE DRAWINGS 
         [0024]    
         FIG. 1 
       
           [0025]    Unsupervised Hierarchical Clustering of Colorectal Tumors Based on the 1239 Genes with the Highest Variation Across all Tumors. 
           [0026]    The phylogenetic tree shows the spontaneous clustering of tumor samples and normal biopsies. Germline mutation indicates samples with hereditary mutations in either MLH1 or MSH2 genes. In columns referring to results of immunohistochemistry a plus indicates a positive antibody staining. Tumor location indicates right-sided or left-sided location in the colon of the tumor. 
           [0027]    
         FIG. 2 
       
           [0028]    Summary of the Performance of the Microsatellite Instability Classifier Based on Microarray Data. 
           [0029]    Panel A shows the number of classification errors as a function of the number of genes used. Panel B shows log 2  of the ratio of the distance between a tumor to the centers of the microsatellite instable group and the microsatellite stable tumors. A value of +2 indicates that the distance of a tumor to the microsatellite instable group is 4 times the distance to the microsatellite stable group. Open bars are MSI tumors and solid bars are MSS tumors. Panel C shows the result of the permutation analysis for estimation of the stability of the classifier. This was estimated by generating one hundred new classifiers based on randomly chosen datasets from the 101 tumors each consisting of 30 microsatellite stable and 25 microsatellite instable samples. In each case the classifier was tested with the remaining 46 samples. The performance for each set was evaluated and averaged over all 100 training and test sets. 
           [0030]    
         FIG. 3 
       
           [0031]    Classification of MSI Tumors as Hereditary or Sporadic Cases Based on Two Genes. 
           [0032]    Panel A shows the number of classification errors as a function of the number of genes used. In crossvalidation we found a minimum number of one error using two genes and adding more genes increased the number of errors to a maximum number of twelve. Both genes were used in at least 36 of the 37 crossvalidation loops. Panel B shows log 2  of the ratio of the distance between a tumor to the centers of the sporadic microsatellite instable group and the hereditary microsatellite instable group. Panel C shows microarray signal values for MLH1 and PIWIL1 genes for all tumors. Asterisk indicates the misclassified tumor 
           [0033]    
         FIG. 4 
       
           [0034]    Classification of Microsatellite-Instability Status Based on Real-Time PCR. 
           [0035]    Panel A shows a cluster analysis of 18 of the 101 tumors samples and 9 genes based on the microarray data and compared to real-time PCR data from same samples and genes. Dark colors indicate relative low expression and light/light grey color palette high expression. Panel B shows the result of 47 new independent samples based on PCR data from 7 of the 9 genes. Relative distances are explained in the legend to  FIG. 2 . The two misclassified tumors are indicated with an asterisk. For PCR primers and hybridization probes see supplement to methods. 
           [0036]    
         FIG. 5 
       
           [0037]    Kaplan-Meier estimates of crude survival among patient with Stage II and Stage III colorectal cancer according to microsatellite status of the tumor, determined by gene expression. Open triangles indicate censored samples. The patients left at risk are denoted in brackets. The P values were calculated with use of the log-rank test. 
           [0038]    
         FIG. 6 
       
           [0039]    Phylogenetic tree resulting from unsupervised hierarchical clustering. Cluster analysis of colon specimens with associated clinicopathological features. 
           [0040]    
         FIG. 7 
       
           [0041]    Multidimentional scaling plot showing distances between groups of tumors. 
           [0042]    
         FIG. 8 
       
           [0043]    Performance of prediction of survival before and after separation in MSI-H and MSS 
           [0044]    
         FIG. 9 
       
           [0045]    Performance of the classifier for identification of hereditary disease. 
           [0046]    
         FIG. 10 
       
           [0047]    Kaplan Meier estimates of overall survival among patients with Dukes&#39; B and Dukes&#39; C colon cancer according to microsatellite-instability status of the tumor, determined by gene expression. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Classification of Cancer 
       [0048]    The present inventors have, using large-scale array-based screenings, found a pool of genes, the expression products of which may be used to classify cancer in an individual. The presence of expression products and level of expression products provides an expression pattern which is correlated to a specific status and/or prognostic marker of the cancer. Characterization of the genes or functional analysis of the gene expression products as such is not required to classify the cancer based on the present method. Thus, the expression products of the plurality of genes can be used as markers for the classification of disease. 
         [0049]    One aspect of the present invention concerns a method for classifying cancer in an individual having contracted cancer by determining the microsatellite status and a prognostic marker in a sample. Determination of the microsatellite status and the prognostic marker may be performed simultaneously or sequentially. In one embodiment of the present invention the microsatellite status is determined. The prognostic marker is determined in a sample, wherein the presence and/or the amount of a number of gene expression products form a pattern wherefrom the prognostic marker is determined. Based on the information gathered from the microsatellite status and the prognostic marker the cancer can be classified. In a preferred embodiment the prognostic marker is the hereditary or sporadic nature of the cancer. The hereditary or sporadic nature of the cancer can be determined through a number of steps comprising determining the presence and/or amount of gene expression products forming a pattern in a sample. The sample comprises a number of gene expression products the presence and/or amount of which forms a pattern that is indicative of the hereditary or sporadic nature of the cancer. Hereby, an indication of the hereditary or sporadic nature of the cancer is obtained. 
         [0050]    In one embodiment of the invention the microsatellite status is determined using conventional analysis of microsatellite status as described elsewhere herein. 
         [0051]    In another embodiment of the present invention the microsatellite status is determined by gene expression patterns wherein the presence and/or the amount of the gene expression products form a pattern that is indicative of the microsatellite status. 
         [0052]    Classification of cancer provides knowledge of the survival chances of an individual having contracted cancer. In case of cancer which according to the present invention has been classified as a hereditary cancer, screening programmes of family members to the individual having the classified cancer can be initiated. Such screening programmes can comprise conventional screening programmes employing sequencing and other methods as described elsewhere. Thus, individuals at risk of developing cancer may be identified and action taken accordingly to detect developing cancer at an early stage of the disease greatly improving the chances of successful intervention and thus survival rates. 
         [0053]    Classification of cancer also provides insights on which sort of treatment should be offered to the individual having contracted cancer, thus providing an improved treatment response of the individual. Likewise, the individual may be spared treatment that is inefficient in treating the particular class of cancer and thus spare the individual severe side effects associated with treatment that may even not be suitable for the class of cancer. 
       Microsatellite Status 
       [0054]    The use of highly variable repetitive sequences found in microsatellite regions adjacent to genes or other areas of interest may be used as markers for linkage analysis, DNA fingerprinting, or other diagnostic application. 
         [0055]    Microsatellites are defined as loci (or regions within DNA sequences) where short sequences of DNA are repeated in tandem repeats. This means that the sequences are repeated one right after the other. The lengths of sequences used most often are di-, tri-, or tetra-nucleotides. At the same location within the genomic DNA the number of times the sequence (ex. AC) is repeated often varies between individuals, within populations, and/or between species. Due to the many repeats the microsatellites are prone to alter if there is a reduced repair of mismatches in the genome. In the present invention the traditional method of determining microsatellite status by employing microsatellite markers is replaced by determination of gene expression patterns. 
         [0056]    An important factor in multi-step carcinogenesis is genomic instability. The development of some cancer forms is known to follow two distinct molecular routes. One route is the microsatellite stable, MSS, (and chromosomal instable pathway) which is often associated with a high frequency of allelic losses, cytogenetic abnormalities and abnormal DNA tumor contents. The second route is the microsatellite instable pathway MSI that is characterized by defects in the DNA mismatch repair system which leads to a high rate of point mutations and small chromosomal insertions and deletions. The small chromosomal insertions and deletions can be detected as mono and dinucleotide repeats (Boland C R, Thibodeau S N, Hamilton S R, et al., Cancer Res 1998; 58(22):5248-57). 
         [0057]    One aspect of the present invention relates to the classification of cancer in an individual having contracted cancer by determining the microsatellite status and a prognostic marker. One embodiment of the invention relates to microsatellite status determined by conventional methods employing microsatellite analysis as described above. Another embodiment of the invention relates to establishing the microsatellite status by determining the presence and/or amount of gene expression products of a sample which comprises a plurality of gene expression products forming a pattern which is indicative of the microsatellite status. 
         [0058]    The expression products of genes according to the present invention are not necessarily identical to the genes that are analysed by microsatellite markers in conventional methods of determining microsatellite status. The pattern of the gene expression products according to the present invention however correlates with information on microsatellite status that can be obtained using traditional methods. 
         [0059]    The determination of the microsatellite status and the prognostic marker of the cancer may be performed sequentially. However, the determinations may also be performed simultaneously. 
       Prognostic Marker 
       [0060]    Together with knowledge of the microsatellite status in a sample of an individual having contracted cancer a prognostic marker is employed for classifying the cancer. The prognostic marker may be any marker that provides knowledge of the cancer type when combined with knowledge of microsatellite status. Consequently the prognostic marker may provide additional information on the cancer type when the microsatellite status is stable and similarly when the microsatellite status is instable. In a preferred embodiment of the present invention the prognostic marker is the hereditary or sporadic nature of a cancer given that the microsatellite status is instable. The prognostic marker may in another embodiment be a prognostic marker for any feature or trait that provides further possibilities of classifying cancer. The prognostic marker is determined in a sample comprising a number of gene expression products wherein the presence and/or amounts of gene expression products form a pattern that is indicative of the prognostic marker. 
       Hereditary and Sporadic Nature of Cancer 
       [0061]    Hereditary nonpolyposis colon cancer (HNPCC) is a hereditary cancer syndrome which carries a very high risk of colon cancer and an above-normal risk of other cancers (uterus, ovary, stomach, small intestine, biliary system, urinary tract, brain, and skin). The HNPCC syndrome is due to mutation in a gene in the DNA mismatch repair system, usually the MLH1 or MSH2 gene or less often the MSH6 or PMS2 genes. Families with HNPCC account for about 5% of all cases of colon cancer and typically have the following features (called the Amsterdam clinical criteria): 
         [0062]    Three or more first relative family members with colorectal cancer; affected family members in two or more generations; and at least one person with colon cancer diagnosed before the age of 50. 
         [0063]    The highest risk with HNPCC is for colon cancer. A person with HNPCC has about an 80% lifetime risk of colon cancer. Two-thirds of these tumors occur in the proximal colon. Women with HNPCC have a 20-60% lifetime risk of endometrial cancer. In HNPCC, the gastric cancer is usually intestinal-type adenocarcinoma. The ovarian cancer in HNPCC may be diagnosed before age 40. Other HNPCC-related cancers have characteristic features: the urinary tract cancers are transitional carcinoma of the ureter and renal pelvis; the small bowel cancer is most common in the duodenum and jejunum; and the most common type of brain tumor is glioblastoma. The diagnosis of HNPCC may be made on the basis of the Amsterdam clinical criteria (listed above) or on the basis of molecular genetic testing for mutations in a mismatch repair gene (MLH1, MSH2, MSH6 or PMS2). Mutations in MLH1 and MSH2 account for 90% of HNPCC. Mutations in MSH6 and PMS2 account for the rest. 
         [0064]    HNPCC is inherited in an autosomal dominant manner. Each child of an individual with HNPCC has a 50% chance of inheriting the mutation. Most people diagnosed with HNPCC have inherited the condition from a parent. However, not all individuals with an HNPCC gene mutation have a parent who had cancer. Prenatal diagnosis for pregnancies at increased risk for HNPCC is possible. 
         [0065]    In tumors that are microsatellite instable it is often found that the DNA mismatch repair proteins that are encoded by the MLH1 or MSH2 genes are inactivated. In case of microsatellite instable hereditary non-polyposis colorectal cancers germline mutation in MLH1 and MSH2 and somatic loss of function of the normal allele has been found to be associated with the disease. 
         [0066]    For most sporadic MSI tumors epigenetic hypermethylation of the MLH1 promoter can be found to be associated with the cancer (Cunningham J M, Christensen E R, Tester D J, et al., Cancer Res 1998; 58(15):3455-60., Kane M F, Loda M, Gaida G M, et al., Cancer Res 1997; 57(5):808-11., Herman J G, Umar A, Polyak K, et al., Proc Natl Acad Sci USA 1998; 95(12):6870-5., Kuismanen S A, Holmberg M T, Salovaara R, de la Chapelle A, Peltomaki P., Am J Pathol 2000; 156(5):1773-9). 
       Forms of Cancer 
       [0067]    Cancer leads to a change in the expression of one or more genes. The methods according to the invention may be used for classifying cancer according to the microsatellite status and/or the hereditary or sporadic nature of the cancer. Thus, the cancer may be any malignant condition in which genomic instability is involved in the development of cancer, such as cancers related to hereditary non-polyposis colorectal cancer, such as endometrial cancer, gastric cancer, small bowel cancer, ovarian cancer, kidney cancer, pelvic renal cancer or tumors of the nervous system, such as glioblastoma. 
         [0068]    One particular form of cancer according to the present invention is that of the colon/rectum. 
         [0069]    The cancer may be of any tumor type, such as an adenocarcinoma, a carcinoma, a teratoma, a sarcoma, and/or a lymphoma. 
         [0070]    In relation to the gastrointestinal tract, the biological condition may also be colitis ulcerosa, Mb. Crohn, diverticulitis, adenomas. 
       Colorectal Tumors 
       [0071]    The data presented herein relates to colorectal tumors and therefore the description has focused on the gene expression level as one manner of identifying genes involved in the prediction of survival in cancer tissue. The malignant progression of cancer of colon or rectum may be described using Dukes stages where normal mucosa may progress to Dukes A superficial tumors to Dukes B, slightly invasive tumors, to Dukes C that have spread to lymph nodes and finally to Dukes D that have metastasized to other organs. 
         [0072]    The grade of a tumor can also be expressed on a scale of I-IV. The grade reflects the cytological appearance of the cells. Grade I cells are almost normal, whereas grade II cells deviate slightly from normal. Grade III appear clearly abnormal, whereas grade IV cells are highly abnormal. 
         [0073]    The phrase colon cancer is in this application meant to be equivalent to the phrase colorectal cancer. Colon cancers may be located in the right side of the colon, the left side of the colon, the transverse part of the colon and/or in the rectum. 
       Samples 
       [0074]    The samples according to the present invention may be any cancer tissue. The sample may be in a form suitable to allow analysis by the skilled artisan, such as a biopsy of the tissue, or a superficial sample scraped from the tissue. In one embodiment of the invention it is preferred that the sample is from a resected colon cancer tumor. In another embodiment the sample may be prepared by forming a suspension of cells made from the tissue. The sample may, however, also be an extract obtained from the tissue or obtained from a cell suspension made from the tissue. The sample may be fresh or frozen, or treated with chemicals. 
       Expression Pattern 
       [0075]    Expression of one gene or more genes in a sample forms a pattern that is characteristic of the state of the cell. In a sample from an individual having contracted cancer a plurality of gene expression products are present. By expression pattern is meant the presence of a combination of a number of expression products and/or the amount of expression products specific for a given biological condition, such as cancer. The pattern is produced by determining the expression products of selected genes that together reveals a pattern that is indicative of the biological condition. Thus, a selection of the genes that carry information about a specific condition is developed. Selection of the genes is achieved by analyzing large numbers of genes and their expression products to find the genes that will enable the desired differentiation between various conditions, such as microsatellite status (MSS or MSI) and/or prognostic marker, such as for example the sporadic or hereditary nature of a given cancer sample. The criteria for selection of the best genes for the pattern to be indicative of given biological conditions include confidence levels i.e. how accurate are the selected genes forming an expression pattern in giving correct information of the biological condition. Thus, in one aspect of the present invention a specific pattern of gene expression profiles can be used to determine the microsatellite status in the sample. In a second aspect of the present invention the microsatellite status is determined and a specific pattern of the presence of a plurality of gene expression products and/or amount wherefrom a prognostic marker is determined. 
       Determination of the Microsatellite Status Employing Gene Expression Patterns 
       [0076]    One aspect of the invention specifically relates to a method for determining the microsatellite status in a sample of an individual having contracted cancer based on determination of the expression pattern of at least two genes, such as at least three genes, such as at least four genes, such as at least 5 genes, such as at least 6 genes, such as at least 7 genes, such as at least 8 genes, such as at least 9 genes, such as at least 10 genes, such as at least 15 genes, such as at least 20 genes, such as at least 30 genes, such as at least 40 genes, such as at least 50 genes, such as at least 60 genes, such as at least 70 genes, such as at least 80 genes, such as at least 90 genes, such as at least 126 genes selected from the group of genes listed in Table 1 below 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                 SEQ ID 
               
               
                 Gene name 
                 Ref seq 
                 Gene symbol 
                 NO.: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 chemokine (C-C motif) ligand 5 
                 NM_002985 
                 CCL5 
                 1 
               
               
                 tryptophanyl-tRNA synthetase 
                 NM_004184 
                 WARS 
                 2 
               
               
                 proteasome (prosome, macropain) activator 
                 NM_006263 
                 PSME1 
                 3 
               
               
                 subunit 1 (PA28 alpha) 
               
               
                 bone marrow stromal cell antigen 2 
                 NM_004335 
                 BST2 
                 4 
               
               
                 ubiquitin-conjugating enzyme E2L 6 
                 NM_004223 
                 UBE2L6 
                 5 
               
               
                 A kinase (PRKA) anchor protein 1 
                 NM_003488 
                 AKAP1 
                 6 
               
               
                 proteasome (prosome, macropain) activator 
                 NM_002818 
                 PSME2 
                 7 
               
               
                 subunit 2 (PA28 beta) 
               
               
                 carcinoembryonic antigen-related cell adhesion 
                 NM_004363 
                 CEACAM5 
                 8 
               
               
                 molecule 5 
               
               
                 FERM, RhoGEF (ARHGEF) and pleckstrin domain 
                 NM_005766 
                 FARP1 
                 9 
               
               
                 protein 1 (chondrocyte-derived) 
               
               
                 myosin X 
                 NM_012334 
                 MYO10 
                 10 
               
               
                 heterogeneous nuclear ribonucleoprotein L 
                 NM_001533 
                 HNRPL 
                 11 
               
               
                 autocrine motility factor receptor 
                 NM_001144 
                 AMFR 
                 12 
               
               
                 dimethylarginine dimethylaminohydrolase 2 
                 NM_013974 
                 DDAH2 
                 13 
               
               
                 tumor necrosis factor, alpha-induced protein 2 
                 NM_006291 
                 TNFAIP2 
                 14 
               
               
                 mutL homolog 1, colon cancer, nonpolyposis 
                 NM_000249 
                 MLH1 
                 15 
               
               
                 type 2 ( E. coli ) 
               
               
                 thymidylate synthetase 
                 NM_001071 
                 TYMS 
                 16 
               
               
                 intercellular adhesion molecule 1 (CD54), human 
                 NM_000201 
                 ICAM1 
                 17 
               
               
                 rhinovirus receptor 
               
               
                 general transcription factor IIA, 2, 12 kDa 
                 NM_004492 
                 GTF2A2 
                 18 
               
               
                 Rho-associated, coiled-coil containing protein 
                 NM_004850 
                 ROCK2 
                 19 
               
               
                 kinase 2 
               
               
                 ATP binding protein associated with cell differentiation 
                 NM_005783 
                 TXNDC9 
                 20 
               
               
                 NCK adaptor protein 2 
                 NM_003581 
                 NCK2 
                 21 
               
               
                 phytanoyl-CoA hydroxylase (Refsum disease) 
                 NM_006214 
                 PHYH 
                 22 
               
               
                 metastais-associated gene family, member 2 
                 NM_004739 
                 MTA2 
                 23 
               
               
                 amiloride binding protein 1 (amine oxidase (copper- 
                 NM_001091 
                 ABP1 
                 24 
               
               
                 containing)) 
               
               
                 biliverdin reductase A 
                 NM_000712 
                 BLVRA 
                 25 
               
               
                 phospholipase C, beta 4 
                 NM_000933 
                 PLCB4 
                 26 
               
               
                 chemokine (C—X—C motif) ligand 9 
                 NM_002416 
                 CXCL9 
                 27 
               
               
                 purine-rich element binding protein A 
                 NM_005859 
                 PURA 
                 28 
               
               
                 quinolinate phosphoribosyltransferase (nicotinate- 
                 NM_014298 
                 QPRT 
                 29 
               
               
                 nucleotide pyrophosphorylase (carboxylating)) 
               
               
                 retinoic acid receptor responder (tazarotene 
                 NM_004585 
                 RARRES3 
                 30 
               
               
                 induced) 3 
               
               
                 chemokine (C-C motif) ligand 4 
                 NM_002984 
                 CCL4 
                 31 
               
               
                 forkhead box O3A 
                 NM_001455 
                 FOXO3A 
                 32 
               
               
                 interferon, alpha-inducible protein (clone IFI-6- 
                 NM_002038 
                 G1P3 
                 34 
               
               
                 16) 
                 NM_022873 
                   
                 123 
               
               
                 chemokine (C—X—C motif) ligand 10 
                 NM_001565 
                 CXCL10 
                 35 
               
               
                 metallothionein 1G 
                 NM_005950 
                 MT1G 
                 36 
               
               
                   
                 NM_005950 
               
               
                 tumor necrosis factor receptor superfamily, 
                 NM_000043 
                 TNFRSF6 
                 37 
               
               
                 member 6 
                 NM_152877 
                   
                 133 
               
               
                   
                 NM_152876 
                   
                 132 
               
               
                   
                 NM_152875 
                   
                 134 
               
               
                   
                 NM_152872 
                   
                 130 
               
               
                   
                 NM_152873 
                   
                 33 
               
               
                   
                 NM_152871 
                   
                 129 
               
               
                   
                 NM_152874 
                   
                 131 
               
               
                 endothelial cell growth factor 1 (platelet-derived) 
                 NM_001953 
                 ECGF1 
                 38 
               
               
                 SCO cytochrome oxidase deficient homolog 2 
                 NM_005138 
                 SCO2 
                 39 
               
               
                 (yeast) 
               
               
                 chemokine (C—X—C motif) ligand 13 (B-cell 
                 NM_006419 
                 CXCL13 
                 40 
               
               
                 chemoattractant) 
               
               
                 Granulysin 
                 NM_006433 
                 GNLY 
                 41 
               
               
                 CD2 antigen (p50), sheep red blood cell receptor 
                 NM_001767 
                 CD2 
                 42 
               
               
                 splicing factor, arginine/serine-rich 6 
                 NM_006275 
                 SFRS6 
                 43 
               
               
                 teratocarcinoma-derived growth factor 1 
                 NM_003212 
                 TDGF1 
                 44 
               
               
                 metallothionein 1H 
                 NM_005951 
                 MT1H 
                 45 
               
               
                 cytochrome P450, family 2, subfamily B, poly- 
                 NM_000767 
                 CYP2B6 
                 46 
               
               
                 peptide 6 
               
               
                 tumor necrosis factor (ligand) superfamily, member 9 
                 NM_003811 
                 TNFSF9 
                 47 
               
               
                 RNA binding motif protein 12 
                 NM_006047 
                 RBM12 
                 48 
               
               
                   
                 NM_006047 
               
               
                 heat shock 105 kDa/110 kDa protein 1 
                 NM_006644 
                 HSPH1 
                 49 
               
               
                 staufen, RNA binding protein ( Drosophila ) 
                 NM_004602 
                 STAU 
                 50 
               
               
                   
                 NM_017452 
                   
                 125 
               
               
                   
                 NM_017453 
                   
                 126 
               
               
                 lymphocyte antigen 6 complex, locus G6D 
                 NM_021246 
                 LY6G6D 
                 51 
               
               
                 calcium binding protein P22 
                 NM_007236 
                 CHP 
                 52 
               
               
                 CDC14 cell division cycle 14 homolog B ( S. cerevisiae ) 
                 NM_003671 
                 CDC14B 
                 53 
               
               
                   
                 NM_033331 
                   
                 115 
               
               
                 epiplakin 1 
                 XM_372063 
                 EPPK1 
                 54 
               
               
                 metallothionein 1X 
                 NM_005952 
                 MT1X 
                 55 
               
               
                 transforming growth factor, beta receptor II 
                 NM_003242 
                 TGFBR2 
                 56 
               
               
                 (70/80 kDa) 
               
               
                 protein kinase C binding protein 1 
                 NM_012408 
                 PRKCBP1 
                 57 
               
               
                   
                 NM_183047 
                   
                 124 
               
               
                 transmembrane 4 superfamily member 6 
                 NM_003270 
                 TM4SF6 
                 58 
               
               
                 pleckstrin homology domain containing, family B 
                 NM_021200 
                 PLEKHB1 
                 59 
               
               
                 (evectins) member 1 
               
               
                 apolipoprotein L, 1 
                 NM_003661 
                 APOL1 
                 60 
               
               
                   
                 NM_145343 
                   
                 120 
               
               
                 indoleamine-pyrrole 2,3 dioxygenase 
                 NM_002164 
                 INDO 
                 61 
               
               
                 forkhead box A2 
                 NM_021784 
                 FOXA2 
                 62 
               
               
                 granzyme H (cathepsin G-like 2, protein h- 
                 NM_033423 
                 GZMH 
                 63 
               
               
                 CCPX) 
               
               
                 baculoviral IAP repeat-containing 3 
                 NM_001165 
                 BIRC3 
                 64 
               
               
                 Homo sapiens metallothionein 1H-like protein 
                   
                 AF333388 
                 135 
               
               
                   
                   
                 (Hs 382039) 
               
               
                 KIAA0182 protein 
                 NM_014615 
                 KIAA0182 
                 117 
               
               
                 G protein-coupled receptor 56 
                 NM_005682 
                 GPR56 
                 65 
               
               
                   
                 NM_201524 
                   
                 116 
               
               
                 metallothionein 2A 
                 NM_005953 
                 MT2A 
                 66 
               
               
                 F-box only protein 21 
                 NM_015002 
                 FBXO21 
                 67 
               
               
                 erythrocyte membrane protein band 4.1-like 1 
                 NM_012156, 
                 EPB41L1 
                 68 
               
               
                   
                 NM_012156 
               
               
                 hypothetical protein MGC21416 
                 NM_173834 
                 MGC21416 
                 69 
               
               
                 protein O-fucosyltransferase 1 
                 NM_015352, 
                 POFUT1 
                 70 
               
               
                   
                 NM_015352 
               
               
                 metallothionein 1E (functional) 
                 NM_175617 
                 MT1E 
                 71 
               
               
                 troponin T1, skeletal, slow 
                 NM_003283 
                 TNNT1 
                 72 
               
               
                 chimerin (chimaerin) 2 
                 NM_004067 
                 CHN2 
                 73 
               
               
                 heterogeneous nuclear ribonucleoprotein H1 (H) 
                 NM_005520 
                 HNRPH1 
                 74 
               
               
                 ATP synthase, H+ transporting, mitochondrial F1 
                 NM_004046 
                 ATP5A1 
                 75 
               
               
                 complex, alpha subunit, isoform 1, cardiac muscle 
               
               
                 eukaryotic translation initiation factor 5A 
                 NM_001970 
                 EIF5A 
                 76 
               
               
                 perforin 1 (pore forming protein) 
                 NM_005041 
                 PRF1 
                 77 
               
               
                 OGT(O-Glc-NAc transferase)-interacting protein 
                 NM_014965 
                 OIP106 
                 78 
               
               
                 106 KDa 
               
               
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 27 
                 NM_017895 
                 DDX27 
                 79 
               
               
                 vacuolar protein sorting 35 (yeast) 
                 NM_018206 
                 VPS35 
                 80 
               
               
                 tripartite motif-containing 44 
                 NM_017583 
                 TRIM44 
                 81 
               
               
                 transmembrane, prostate androgen induced 
                 NM_020182 
                 TMEPAI 
                 82 
               
               
                 RNA 
                 NM_199169 
                   
                 127 
               
               
                   
                 NM_199170 
                   
                 128 
               
               
                 dynein, cytoplasmic, light polypeptide 2A 
                 NM_014183 
                 DNCL2A 
                 83 
               
               
                   
                 NM_177953 
                   
                 122 
               
               
                 leucine aminopeptidase 3 
                 NM_015907 
                 LAP3 
                 84 
               
               
                 chromosome 20 open reading frame 35 
                 NM_018478 
                 C20orf35 
                 85 
               
               
                   
                 NM_033542 
                   
                 118 
               
               
                 solute carrier family 38, member 1 
                 NM_030674 
                 SLC38A1 
                 86 
               
               
                 CGI-85 protein 
                 NM_016028 
                 CGI-85 
                 87 
               
               
                 death associated transcription factor 1 
                 NM_022105, 
                 DATF1 
                 88 
               
               
                   
                 NM_080796 
                   
                 121 
               
               
                 hepatocellular carcinoma-associated antigen 
                 NM_018487 
                 HCA112 
                 89 
               
               
                 112 
               
               
                 sestrin 1 
                 NM_014454 
                 SESN1 
                 90 
               
               
                 hypothetical protein FLJ20315 
                 NM_017763 
                 FLJ20315 
                 91 
               
               
                 hypothetical protein FLJ20647 
                 NM_017918 
                 FLJ20647 
                 92 
               
               
                 membrane protein expressed in epithelial-like 
                 NM_024792 
                 CT120 
                 93 
               
               
                 lung adenocarcinoma 
               
               
                 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 
                 NM_014314 
                 RIG-I 
                 94 
               
               
                 keratin 23 (histone deacetylase inducible) 
                 NM_015515, 
                 KRT23 
                 95 
               
               
                 UDP-N-acetyl-alpha-D- 
                 NM_007210 
                 GALNT6 
                 96 
               
               
                 galactosamine:polypeptide N- 
               
               
                 acetylgalactosaminyltransferase 6 (GalNAc-T6) 
               
               
                 aryl hydrocarbon receptor nuclear translocator- 
                 NM_020183 
                 ARNTL2 
                 97 
               
               
                 like 2 
               
               
                 apobec-1 complementation factor 
                 NM_014576, 
                 ACF 
                 98 
               
               
                   
                 NM_138932 
                   
                 119 
               
               
                 hypothetical protein FLJ20232 
                 NM_019008 
                 FLJ20232 
                 99 
               
               
                 apolipoprotein L, 2 
                 NM_030882, 
                 APOL2 
                 100 
               
               
                   
                 NM_145343 
                   
                 120 
               
               
                 mitochondrial solute carrier protein 
                 NM_016612 
                 MSCP 
                 101 
               
               
                 hypothetical protein FLJ20618 
                 NM_017903 
                 FLJ20618 
                 102 
               
               
                 SET translocation (myeloid leukaemia- 
                 NM_003011.1 
                 SET 
                 103 
               
               
                 associated) 
               
               
                 ATPase, class II, type 9a 
                 Xm_030577.9 
                 ATP9a 
                 104 
               
               
                   
               
             
          
         
       
     
         [0077]    One embodiment of the invention concerning the determination of microsatellite status is based on the expression pattern of at least 2 genes, such as at least 3 genes, such as at least 4 genes, such as at least 5 genes, such as at least 6 genes, such as at least 7 genes, such as at least 8 genes, such as at least 9 genes, such as at least 10 genes, such as at least 15 genes, such as at least 20 genes, such as at least 25 genes selected from the group of genes listed in Table 2. 
         [0000]                                                  TABLE 2                           SEQ ID       Gene name   Ref seq   Gene symbol   NO.:                                chemokine (C-C motif) ligand 5   NM_002985   CCL5   1       tryptophanyl-tRNA synthetase   NM_004184   WARS   2       proteasome (prosome, macropain) activator   NM_006263   PSME1   3       subunit 1 (PA28 alpha)       bone marrow stromal cell antigen 2   NM_004335   BST2   4       ubiquitin-conjugating enzyme E2L 6   NM_004223   UBE2L6   5       A kinase (PRKA) anchor protein 1   NM_003488   AKAP1   6       proteasome (prosome, macropain) activator   NM_002818   PSME2   7       subunit 2 (PA28 beta)       carcinoembryonic antigen-related cell adhesion   NM_004363   CEACAM5   8       molecule 5       FERM, RhoGEF (ARHGEF) and pleckstrin domain   NM_005766   FARP1   9       protein 1 (chondrocyte-derived)       myosin X   NM_012334   MYO10   10       heterogeneous nuclear ribonucleoprotein L   NM_001533   HNRPL   11       autocrine motility factor receptor   NM_001144   AMFR   12       dimethylarginine dimethylaminohydrolase 2   NM_013974   DDAH2   13       tumor necrosis factor, alpha-induced protein 2   NM_006291   TNFAIP2   14       mutL homolog 1, colon cancer, nonpolyposis   NM_000249   MLH1   15       type 2 ( E. coli )       thymidylate synthetase   NM_001071   TYMS   16       intercellular adhesion molecule 1 (CD54), human   NM_000201   ICAM1   17       rhinovirus receptor       general transcription factor IIA, 2, 12 kDa   NM_004492   GTF2A2   18       Rho-associated, coiled-coil containing protein   NM_004850   ROCK2   19       kinase 2       ATP binding protein associated with cell differentiation   NM_005783   APACD   20       metastais-associated gene family, member 2   NM_004739   MTA2   23       chemokine (C—X—C motif) ligand 10   NM_001565   CXCL10   35       splicing factor, arginine/serine-rich 6   NM_006275   SFRS6   43       protein kinase C binding protein 1   NM_012408   PRKCBP1   57           NM_183047       124       hepatocellular carcinoma-associated antigen   NM_018487   HCA112   89       112       hypothetical protein FLJ20618   NM_017903   FLJ20618   102       SET translocation (myeloid leukaemia-   NM_003011.1   SET   103       associated)       ATPase, class II, type 9a   Xm_030577.9   ATP9a   104                    
or from
 
         [0000]                                                  TABLE 3                           SEQ ID       Gene name   Ref seq   Gene symbol   NO.:                                heterogeneous nuclear ribonucleoprotein L   NM_001533   HNRPL   11       NCK adaptor protein 2   NM_003581   NCK2   21       phytanoyl-CoA hydroxylase (Refsum disease)   NM_006214   PHYH   22       metastais-associated gene family, member 2   NM_004739   MTA2   23       amiloride binding protein 1 (amine oxidase   NM_001091   ABP1   24       (copper-containing))       biliverdin reductase A   NM_000712   BLVRA   25       phospholipase C, beta 4   NM_000933   PLCB4   26       chemokine (C—X—C motif) ligand 9   NM_002416   CXCL9   27       purine-rich element binding protein A   NM_005859   PURA   28       quinolinate phosphoribosyltransferase (nicotinate-   NM_014298   QPRT   29       nucleotide pyrophosphorylase (carboxylating))       retinoic acid receptor responder (tazarotene   NM_004585   RARRES3   30       induced) 3       chemokine (C-C motif) ligand 4   NM_002984   CCL4   31       forkhead box O3A   NM_001455   FOXO3A   32       metallothionein 1X   NM_005952   MT1X   55       interferon, alpha-inducible protein (clone IFI-6-   NM_002038   G1P3   34       16)   NM_022873       123       chemokine (C—X—C motif) ligand 10   NM_001565   CXCL10   35       metallothionein 1G   NM_005950,   MT1G   36           NM_005950       tumor necrosis factor receptor superfamily,   NM_000043   TNFRSF6   37       member 6   NM_152877       133           NM_152876       132           NM_152875       134           NM_152872       130           NM_152873       33           NM_152871       129           NM_152874       131       endothelial cell growth factor 1 (platelet-   NM_001953   ECGF1   38       derived)       SCO cytochrome oxidase deficient homolog 2   NM_005138   SCO2   39       (yeast)       chemokine (C—X—C motif) ligand 13 (B-cell   NM_006419   CXCL13   40       chemoattractant)       Granulysin   NM_006433   GNLY   41       splicing factor, arginine/serine-rich 6   NM_006275   SFRS6   43       protein kinase C binding protein 1   NM_012408   PRKCBP1   57           NM_183047       124       hepatocellular carcinoma-associated antigen   NM_018487   HCA112   89       112       hypothetical protein FLJ20618   NM_017903   FLJ20618   102       SET translocation (myeloid leukaemia-   NM_003011.1   SET   103       associated)       ATPase, class II, type 9a   Xm_030577.9   ATP9a   104                    
or from
 
         [0000]                                                  TABLE 4                           SEQ ID       Gene name   Ref seq   Gene symbol   NO.:                                heterogeneous nuclear ribonucleoprotein L   NM_001533   HNRPL   11       metastais-associated gene family, member 2   NM_004739   MTA2   23       chemokine (C—X—C motif) ligand 10   NM_001565   CXCL10   35       CD2 antigen (p50), sheep red blood cell receptor   NM_001767   CD2   42       splicing factor, arginine/serine-rich 6   NM_006275   SFRS6   43       teratocarcinoma-derived growth factor 1   NM_003212   TDGF1   44       metallothionein 1H   NM_005951   MT1H   45       cytochrome P450, family 2, subfamily B, poly-   NM_000767   CYP2B6   46       peptide 6       tumor necrosis factor (ligand) superfamily,   NM_003811   TNFSF9   47       member 9       RNA binding motif protein 12   NM_006047,   RBM12   48           NM_006047       heat shock 105 kDa/110 kDa protein 1   NM_006644   HSPH1   49       staufen, RNA binding protein ( Drosophila )   NM_004602   STAU   50           NM_017452       125           NM_017453       126       lymphocyte antigen 6 complex, locus G6D   NM_021246   LY6G6D   51       calcium binding protein P22   NM_007236   CHP   52       CDC14 cell division cycle 14 homolog B ( S. cerevisiae )   NM_003671   CDC14B   53           NM_033331       115       epiplakin 1   XM_372063   EPPK1   54       metallothionein 1X   NM_005952   MT1X   55       transforming growth factor, beta receptor II   NM_003242   TGFBR2   56       (70/80 kDa)       protein kinase C binding protein 1   NM_012408   PRKCBP1   57           NM_183047       129       transmembrane 4 superfamily member 6   NM_003270   TM4SF6   58       pleckstrin homology domain containing, family   NM_021200   PLEKHB1   59       B (evectins) member 1       apolipoprotein L, 1   NM_003661   APOL1   60           NM_145343       125       indoleamine-pyrrole 2,3 dioxygenase   NM_002164   INDO   61       forkhead box A2   NM_021784   FOXA2   62           NM_021784       hepatocellular carcinoma-associated antigen   NM_018487   HCA112   89       112       mitochondrial solute carrier protein   NM_016612   MSCP   101           NM_016612       hypothetical protein FLJ20618   NM_017903   FLJ20618   102       SET translocation (myeloid leukaemia-   NM_003011.1   SET   103       associated)       ATPasa, class II, type 9a   Xm_030577.9   ATP9a   104                    
or from
 
         [0000]                                                  TABLE 5                           SEQ ID       Gene name   Ref seq   Gene symbol   NO.:                                heterogeneous nuclear ribonucleoprotein L   NM_001533   HNRPL   11       metastais-associatad gene family, member 2   NM_004739   MTA2   23       chemokine (C—X—C motif) ligand 10   NM_001565   CXCL10   35       splicing factor, arginine/serine-rich 6   NM_006275   SFRS6   43       protein kinase C binding protein 1   NM_012408   PRKCBP1   57           NM_183047       124       granzyme H (cathepsin G-like 2, protein h-   NM_033423   GZMH   63       CCPX)       baculoviral IAP repeat-containing 3   NM_001165   BIRC3   64           NM_001165         Homo sapiens  metallothionein 1H-like protein       AF333388   135               (Hs 382039)       KIAA0182 protein   NM_014615   KIAA0182   117       G protein-coupled receptor 56   NM_005682   GPR56   65           NM_301524       116       metallothionein 2A   NM_005953   MT2A   66       F-box only protein 21   NM_015002   FBXO21   67       erythrocyte membrane protein band 4.1-like 1   NM_012156   EPB41L1   68       hypothetical protein MGC21416   NM_173834   MGC21416   69       protein O-fucosyltranaferase 1   NM_015352   POFUT1   70       metallothionein 1E (functional)   NM_175617   MT1E   71       troponin T1, skeletal, slow   NM_003283   TNNT1   72       chimerin (chimaerin) 2   NM_004067   CHN2   73       heterogeneous nuclear ribonucleoprotein H1   NM_005520   HNRPH1   74       (H)       ATP synthase, H+ transporting, mitochondrial   NM_004046   ATP5A1   75       F1 complex, alpha subunit, isoform 1, cardiac       muscle       eukaryotic translation initiation factor 5A   NM_001970   EIF5A   76       perforin 1 (pore forming protein)   NM_005041   PRF1   77       OGT(O-Glc-NAc transferase)-interacting protein   NM_014965   OIP106   78       106 KDa       DEAD (Asp-Glu-Ala-Asp) box polypeptide 27   NM_017895   DDX27   79       hepatocellular carcinoma-associated antigen   NM_018487   HCA112   89       112       hypothetical protein FLJ20232   NM_019008   FLJ20232   99       apolipoprotein L, 2   NM_030882,   APOL2   100           NM_145343       120       hypothetical protein FLJ20618   NM_017903   FLJ20618   102       SET translocation (myeloid leukaemia-   NM_003011.1   SET   103       associated)       ATPase, class II, type 9a   Xm_030577.9   ATP9a   104                    
or from
 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                 SEQ ID 
               
               
                 Gene name 
                 Ref seq 
                 Gene symbol 
                 NO.: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 heterogeneous nuclear ribonucleoprotein L 
                 NM_001533 
                 HNRPL 
                 11 
               
               
                 metastais-associated gene family, member 2 
                 NM_004739 
                 MTA2 
                 23 
               
               
                 chemokine (C—X—C motif) ligand 10 
                 NM_001565 
                 CXCL10 
                 35 
               
               
                 metallothionein 1G 
                 NM_005950 
                 MT1G 
                 36 
               
               
                 splicing factor, arginine/serine-rich 6 
                 NM_006275 
                 SFRS6 
                 43 
               
               
                 protein kinase C binding protein 1 
                 NM_012408 
                 PRKCBP1 
                 57 
               
               
                   
                 NM_183047 
                   
                 129 
               
               
                 vacuolar protein sorting 35 (yeast) 
                 NM_018206 
                 VPS35 
                 80 
               
               
                 tripartite motif-containing 44 
                 NM_017583 
                 TRIM44 
                 81 
               
               
                 transmembrane, prostate androgen induced 
                 NM_020182 
                 TMEPAI 
                 82 
               
               
                 RNA 
                 NM_199169 
                   
                 127 
               
               
                   
                 NM_199170 
                   
                 128 
               
               
                 dynein, cytoplasmic, light polypeptide 2A 
                 NM_014183 
                 DNCL2A 
                 83 
               
               
                   
                 NM_177953 
                   
                 122 
               
               
                 leucine aminopeptidase 3 
                 NM_015907 
                 LAP3 
                 84 
               
               
                 chromosome 20 open reading frame 35 
                 NM_018478 
                 C20orf35 
                 85 
               
               
                   
                 NM_033542 
                   
                 118 
               
               
                 solute carrier family 38, member 1 
                 NM_030674 
                 SLC38A1 
                 86 
               
               
                 CGI-85 protein 
                 NM_016028 
                 CGI-85 
                 87 
               
               
                 death associated transcription factor 1 
                 NM_022105, 
                 DATF1 
                 88 
               
               
                   
                 NM_080796 
                   
                 121 
               
               
                 hepatocellular carcinoma-associated antigen 
                 NM_018487 
                 HCA112 
                 89 
               
               
                 112 
               
               
                 sestrin 1 
                 NM_014454 
                 SESN1 
                 90 
               
               
                 hypothetical protein FLJ20315 
                 NM_017763 
                 FLJ20315 
                 91 
               
               
                 hypothetical protein FLJ20647 
                 NM_017918 
                 FLJ20647 
                 92 
               
               
                 membrane protein expressed in epithelial-like 
                 NM_024792 
                 CT120 
                 93 
               
               
                 lung adenocarcinoma 
               
               
                 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 
                 NM_014314 
                 RIG-I 
                 94 
               
               
                 keratin 23 (histone deacetylase inducible) 
                 NM_015515 
                 KRT23 
                 95 
               
               
                 UDP-N-acetyl-alpha-D- 
                 NM_007210 
                 GALNT6 
                 96 
               
               
                 galactosamine:polypeptide N- 
               
               
                 acetylgalactosaminyltransferase 6 (GalNAc-T6) 
               
               
                 aryl hydrocarbon receptor nuclear translocator- 
                 NM_020183 
                 ARNTL2 
                 97 
               
               
                 like 2 
               
               
                 apobec-1 complementation factor 
                 NM_014576 
                 ACF 
                 98 
               
               
                   
                 NM_138932 
                   
                 119 
               
               
                 hypothetical protein FLJ20618 
                 NM_017903 
                 FLJ20618 
                 102 
               
               
                 SET translocation (myeloid leukaemia- 
                 NM_003011.1 
                 SET 
                 103 
               
               
                 associated) 
               
               
                 ATPase, class II, type 9a 
                 Xm_030577.9 
                 ATP9a 
                 104 
               
               
                   
               
             
          
         
       
     
         [0078]    Another embodiment of the invention concerning the determination of microsatellite status is based on the expression pattern of at least 2 genes, such as at least 3 genes, such as at least 4 genes, such as at least 5 genes, such as at least 6 genes, such as at least 7 genes, such as at least 8 genes, such as at least 9 genes selected from the group of genes listed in Table 7 below. 
         [0079]    RNA purification Colon specimens were obtained fresh from surgery and were immediately snap frozen in liquid nitrogen either as was, in OCD-compound or in a SDS/guadinium thiocyanate solution. Total RNA was isolated using RNAzol (WAK-Chemie Medical) or spin column technology (Sigma) following the manufactures&#39; instructions. 
         [0080]    Gene expression analysis These procedures were performed at described in detail elsewhere (Dyrskødt et al). Briefly, ten μg of total RNA was used as starting material for the target preparation as described. First and second strand cDNA synthesis was performed using the SuperScript II System (Invitrogen) according to the manufacturers&#39; instructions except using an oligo-dT primer containing a T7 RNA polymerase promoter site. Labelled aRNA was prepared using the BioArray High Yield RNA Transcript Labelling Kit (Enzo) using Biotin labelled CTP and UTP (Enzo) in the reaction together with unlabeled NTP&#39;s. Unincorporated nucleotides were removed using RNeasy columns (Qiagen). Fifteen μg of cRNA was fragmented, loading onto the Affymetrix HG_U133A probe array cartridge and hybridized for 16 h. The arrays were washed and stained in the Affymetrix Fluidics Station and scanned using a confocal laser-scanning microscope (Hewlett Packard GeneArray Scanner G2500A). The readings from the quantitative scanning were analyzed by the Affymetrix Gene Expression Analysis Software (MAS 5.0) and normalized using RMA (robust multi array normalisation, Irizarry et al. 2002) in the statistical application R. Redundant probesets (as defined form Unigene build 168) with high correlation (&gt;0.5) over all samples were removed, which reduced the dataset to approximately 14.400 probesets. This dataset was used a source for all further calculations in this manuscript. 
       Unsupervised Agglomerative Hierarchical Clustering 
       [0081]    For hierarchical cluster analysis 1239 genes with a variation across all samples greater than 0.5 were median-centred to a magnitude of 1. Samples and genes were then clustered using average linkage clustering with a modified Person correlation as similarity metric (Eisen et al., PNAS 95: 14863-14868, 1998). The cluster dendrogram was visualized with TreeView (Eisen). 
       Group Testing 
       [0082]    We make a statistical test where the p-value is evaluated through permutations. For each group and gene we calculate the average and the sum of squared deviations from the average. We then sum these over the genes and the groups: 
         [0000]    
       
         
           
             
               S 
               1 
             
             = 
             
               
                 ∑ 
                 groups 
               
                
               
                 
                   ∑ 
                   genes 
                 
                  
                 
                   
                     ( 
                     
                       
                         X 
                         ij 
                       
                       - 
                       
                         
                           X 
                           _ 
                         
                         
                           
                             gr 
                              
                             
                               ( 
                               i 
                               ) 
                             
                           
                            
                           j 
                         
                       
                     
                     ) 
                   
                   2 
                 
               
             
           
         
       
     
         [0083]    This expression is calculated for joining DK with SF and MSI with MSS such that we end up with two groups. The sum of squared deviations is denoted S 2 . As a test statistic we use S 1 /S 2 . A small value indicates that there is a real reduction in the deviations when going from 2 to 4 groups and thus the groups have a real significance. To judge if a value is significantly small we use permutations. For each of the four groups left when joining DK and SF we randomly allocate the members to a pseudo DK and pseudo SF in such a way that the number of members in each group are as in the original data. 
         [0084]    To get an understanding of this separation we performed a test to see if this is caused by few genes or if many genes are involved. For this test we calculated S 1 =Σ genes  S 1 (gene) and similarly with S 2 =Σ genes  S 2 (gene). For each gene j we used the test statistic S 1 (j)/S 2 (j) (Table 3). 
       Multidimentional Scaling 
       [0085]    We carried out multidimentional scaling on median-centered and normalized data using CMD—scale in the statistical application R and visualized in a two-dimensional plot. 
       Microsatellite Status Classifier 
       [0086]    The readings from the quantitative scanning were analyzed by the Affymetrix Gene Expression Analysis Software (MAS 5.0) and normalized using RMA (robust multi array normalisation, Irizarry et al. 2002) in the statistical application R. Redundant probesets (as defined form Unigene build 168) with high correlation (&gt;0.5) over all samples were removed, which reduced the dataset to approximately 14.400 probesets. 
         [0087]    The microsatellite instability status classifier was based on a dataset of 4.266 genes. These genes result from the removal of genes with a variance over all tumor samples smaller than 0.2 and genes that separate Danish from Finnish samples with a t-value numerically greater than 2. We used a normal distribution with the mean dependent on the gene and the group (MSI, MSS). For each gene, we calculated the variation between the groups and the variation within the groups to select genes with a high ratio between these. To classify a sample, we calculated the sum over the genes of the squared distance from the sample value to the group mean, standardized by the variance and assigned the sample to the nearest group. The sample to be classified was excluded when calculating group means and variances. 
       Estimation of Classifier Stability 
       [0088]    We validated the performance of the classifier by permutation. One hundred datasets consisting of 30 MSS samples and 25 MSI samples were randomly chosen by permutation for training of the classifier with the remaining samples in each case being assign to a testset. Averages over the 100 data sets of the number of errors in the cross-validation of the training set and in the test set were used as a measure of the precision of the classifier. 
         [0089]    Real-time PCR (RT-PCR). The procedures were as described (Birkenkamp-Demtroder) except that we used short LNA (Locked Nucleic Acid) enhanced probes from a Human Probe Library (Exiqon™). In short, cDNA was synthesized from single samples some of which were previously analyzed on GeneChips. Reverse transcription was performed using Superscript II RT (Invitrogen). Real-time PCR analysis was performed on selected genes using the primers (DNA Technology) and probes (Exiqon, DK) described in figure legend X. All samples were normalized to GAPDH as described previously (Birkenkamp-Demtroder et. al. Cancer Res., 62: 4352-4363, 2002). 
       Rebuilding of Classifier Based on Real-Time PCR 
       [0090]    The 79 tumors samples that were not analysed by real-time PCR were transformed into log ratios using one of the tumor samples as reference and used for training of the classifier. Then 23 samples of which 18 were also analyzed on arrays were equally transformed into log ratios using the same tumor sample as above as reference and tested. The idea behind this translation is that we expect the normalized PCR values to be proportional to the normalized array values, and on a log scale this becomes an additive difference. The difference is gene specific and is therefore estimated for each gene separately. The variation obtained from the microarray data, and used in the classifier, can be used directly on the PCR platform. 
       Results 
     Hierarchical Clustering 
       [0091]    The clinical specimens used in this study were collected in two different countries from 14 different clinics in the period 1994 to 2001. The samples were selected to keep a balanced representation of microsatellite instable (MSI) and microsatellite stable (MSS) tumors from both the right- and left-sided colon. The MSI class was represented both by sporadic MSI and hereditary MSI (HNPCC) tumors. Only Dukes&#39; B and Dukes&#39; C tumor samples were included were selected (table 19). Before any attempt to divide a diverse sample collection into distinct classes analyzed the data for systematic bias that may have been introduces during the experimental procedures. A fast and easy way to discover both true distinct classes as well as systematic biases in the data is to perform a hierarchical clustering. 
         [0092]    The phylogenetic tree resulting from hierarchical clustering on 1239 genes ( FIG. 6 ) reveals that the main separating factor is microsatellite status. On the upper trunk we find two clusters represented mainly by normal biopsies (14/21) and MSS tumors (18/25), respectively. The lower trunk is divided into a MSI cluster (30/36) and a second MSS cluster (MSS2-cluster) (34/37). A closer inspection of the two MSS clusters unveil that one is dominated by Danish samples (19/25) and one by Finnish samples (26/37 check). Also, it is worth to notice that the MSI cluster contains a vast majority of Finnish samples (32/36) and that the sporadic MSI samples are interspersed among the hereditary samples. The normal biopsies cluster tight together with a slight tendency to separation according to origin. Tree normal samples cluster within the MSI cluster indicating that resection of these samples may have been to close to the tumor lesion. 
         [0093]    Inspection of the gene cluster dendrogram shows that the two groups of MSS tumors are mainly separated by a large cluster of genes being upregulated in the Danish samples (data not shown) indicating that a systematic difference between Danish and Finnish samples. 
       Significance of Observed Groups 
       [0094]    Based on these observations, we performed a series of test to evaluate if the observed separation of tumors into MSS and MSI as well as DK and SF are significant. For these tests the tumor samples were grouped into four virtual tumor-groups labelled, i.e. Danish MSI (MSI-DK), Danish MSS (MSS-DK), Finnish MSI (MSI-SF) and Finnish MSS (MSS-SF). Based on 5082 genes with a variance above 0.2, we tested if all four groups are significant or if some of the groups can be joined. We considered the two possibilities of joining DK and SF, and of joining MSI and MSS and made a statistical test where the p-value is evaluated through permutations. In 100 permutations of each group combination our test value S1/S2 is considerably smaller than in all permutation (Table 20) demonstrating a very clear separation between DK and SF and between MSI and MSS. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Permutation test of groups 
               
             
          
           
               
                 Pseudo 
                   
                 Smaller values in 
                 Minimum in 100 
               
               
                 group 
                 S1/S2 from data 
                 100 permutations 
                 permutations 
               
               
                   
               
             
          
           
               
                 DK-SF 
                 0.9072795 
                 0 
                 0.962269 
               
               
                 I-S 
                 0.9166195 
                 0 
                 0.9583325 
               
               
                   
               
             
          
         
       
     
         [0095]    Such a clear distinction between groups may rely on a few highly separating genes or a general difference in the gene expression profile including many genes. For both the DK-SF and MSI-MSS the effect are caused by many genes even at very criteria, i.e. low test statistic S 1 (j)/S 2 (j) values (Table 21). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Permutation test of genes 
               
             
          
           
               
                   
                 S 1 (j)/S 2 (j) 
               
             
          
           
               
                 Pseudo group 
                 &lt;0.6 
                 &lt;0.7 
                 &lt;0.8 
                 &lt;0.9 
               
               
                   
               
             
          
           
               
                 DK-SF 
                 number of genes 
                 36 
                 136 
                 522 
                 1785 
               
               
                   
                 max in 100 permutations 
                 0 
                 0 
                 2 
                 225 
               
               
                 MSI-MSS 
                 number of genes 
                 17 
                 103 
                 399 
                 1507 
               
               
                   
                 max in 100 permutations 
                 0 
                 1 
                 8 
                 250 
               
               
                   
               
             
          
         
       
     
         [0096]    When a property is present that influences a large proportion of the genes this may obscure separation of clinical relevant features in unsupervised clustering. To visualize the effect of such properties, we calculated distances by multidimensional scaling between samples with and without of 816 genes separating DK from SF with a t-value numerically greater than 2 ( FIG. 7 ). We see an improved separation of MSI and MSS with Danish and Finnish cases mixed. The MSI-DK samples are not completely separated as they are found both between the MSI-SF and the MSS samples. (These plots are not entirely unsupervised since the groups have been used to remove gene). 
       Construction of an MSI-MSS Classifier 
       [0097]    For the construction of a classifier we used the expression profiles from 97 tumors for which no ambiguity had been identified in relation to microsatellite status. The 816 genes separating DK from SF were excluded, as these would be unreliable for MS classification. We built a maximum likelihood classifier in order to select a minimum of genes giving the largest possible separation of the two groups. We tested the performance of the classifier using 1-1000 genes and found that it was stable showing 3-6 errors when using 4-400 genes. Of these 106 genes were especially suited for discrimination of MSS from MSI (table 22). 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 22 
               
               
                   
               
               
                   
                   
                 LOCUS 
                   
                   
                   
               
               
                 AFFYID 
                 SYMBOL 
                 LINK 
                 OMIM 
                 REFSEQ 
                 GENENAME 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1405_i_at 
                 CCL5 
                 6352 
                 187011 
                 NM_002985 
                 chemokine (C-C motif) ligand 5 
               
               
                 200628_s_at 
                 WARS 
                 7453 
                 191050 
                 NM_004184 
                 tryptophanyl-tRNA synthetase 
               
               
                 200814_at 
                 PSME1 
                 5720 
                 600654 
                 NM_006263 
                 proteasome (prosome, macropain) activator subunit 
               
               
                   
                   
                   
                   
                   
                 1 (PA28 alpha) 
               
               
                 201641_at 
                 BST2 
                 684 
                 600534 
                 NM_004335 
                 bone marrow stromal cell antigen 2 
               
               
                 201649_at 
                 UBE2L6 
                 9246 
                 603890 
                 NM_004223 
                 ubiquitin-conjugating enzyme E2L 6 
               
               
                 201674_s_at 
                 AKAP1 
                 8165 
                 602449 
                 NM_003488 
                 A kinase PRKA anchor protein 1 
               
               
                 201762_s_at 
                 PSME2 
                 5721 
                 602161 
                 NM_002818 
                 proteasome (prosome, macropain) activator subunit 
               
               
                   
                   
                   
                   
                   
                 2 (PA28 beta) 
               
               
                 201884_at 
                 CEACAM5 
                 1048 
                 114890 
                 NM_004363 
                 carcinoembryonic antigen-related cell adhesion 
               
               
                   
                   
                   
                   
                   
                 molecule 5 
               
               
                 201910_at 
                 FARP1 
                 10160 
                 602654 
                 NM_005766 
                 FERM, RhoGEF (ARHGEF) and pleckstrin domain 
               
               
                   
                   
                   
                   
                   
                 protein 1 (chondrocyte-derived) 
               
               
                 201976_s_at 
                 MYO10 
                 4651 
                 601481 
                 NM_012334 
                 myosin X 
               
               
                 202072_at 
                 HNRPL 
                 3191 
                 603083 
                 NM_001533 
                 heterogeneous nuclear ribonucleoprotein L 
               
               
                 202203_s_at 
                 AMFR 
                 267 
                 603243 
                 NM_001144 
                 autocrine motility factor receptor 
               
               
                 202262_x_at 
                 DDAH2 
                 23564 
                 604744 
                 NM_013974 
                 dimethylarginine dimethylaminohydrolase 2 
               
               
                 202510_s_at 
                 TNFAIP2 
                 7127 
                 603300 
                 NM_006291 
                 tumor necrosis factor, alpha-induced protein 2 
               
               
                 202520_s_at 
                 MLH1 
                 4292 
                 120436 
                 NM_000249 
                 mutL homolog 1, colon cancer, nonpolyposis type 2 
               
               
                   
                   
                   
                   
                   
                 ( E. coli ) 
               
               
                 202589_at 
                 TYMS 
                 7298 
                 188350 
                 NM_001071 
                 thymidylate synthetase 
               
               
                 202637_s_at 
                 ICAM1 
                 3383 
                 147840 
                 NM_000201 
                 Intercellular adhesion molecule 1 (CD54), human 
               
               
                   
                   
                   
                   
                   
                 rhinovirus receptor 
               
               
                 202678_at 
                 GTF2A2 
                 2958 
                 600519 
                 NM_004492 
                 general transcription factor IIA, 2, 12 kDa 
               
               
                 202762_at 
                 ROCK2 
                 9475 
                 604002 
                 NM_004850 
                 Rho-associated, coiled-coil containing protein kinase 2 
               
               
                 203008_x_at 
                 APACD 
                 10190 
                   
                 NM_005783 
                 ATP binding protein associated with cell differentiation 
               
               
                 203315_at 
                 NCK2 
                 8440 
                 604930 
                 NM_003581 
                 NCK adaptor protein 2 
               
               
                 203335_at 
                 PHYH 
                 5264 
                 602026 
                 NM_006214 
                 phytanoyl-CoA hydroxylase (Refsum disease) 
               
               
                 203444_s_at 
                 MTA2 
                 9219 
                 603947 
                 NM_004739 
                 metastais-associated gene family, member 2 
               
               
                 203559_s_at 
                 ABP1 
                 26 
                 104610 
                 NM_001091 
                 amiloride binding protein 1 (amine oxidase (copper- 
               
               
                   
                   
                   
                   
                   
                 containing)) 
               
               
                 203773_x_at 
                 BLVRA 
                 644 
                 109750 
                 NM_000712 
                 biliverdin reductase A 
               
               
                 203896_s_at 
                 PLCB4 
                 5332 
                 600810 
                 NM_000933 
                 phospholipase C, beta 4 
               
               
                 203915_at 
                 CXCL9 
                 4283 
                 601704 
                 NM_002416 
                 chemokine (C—X—C motif) ligand 9 
               
               
                 204020_at 
                 PURA 
                 5813 
                 600473 
                 NM_005859 
                 purine-rich element binding protein A 
               
               
                 204044_at 
                 QPRT 
                 23475 
                 606248 
                 NM_014298 
                 quinolinate phosphoribosyltransfarase (nicotinate- 
               
               
                   
                   
                   
                   
                   
                 nucleotide pyrophosphorylase (carboxylating)) 
               
               
                 204070_at 
                 RARRES3 
                 5920 
                 605092 
                 NM_004585 
                 retinoic acid receptor responder (tazarotene induced) 3 
               
               
                 204103_at 
                 CCL4 
                 6351 
                 182284 
                 NM_002984 
                 chemokine (C-C motif) ligand 4 
               
               
                 204131_s_at 
                 FOXO3A 
                 2309 
                 602681 
                 NM_001455 
                 forkhead box O3A 
               
               
                 204326_x_at 
                 MT1X 
                 4501 
                 156359 
                 NM_005952 
                 metallothionein 1X 
               
               
                 204415_at 
                 G1P3 
                 2537 
                 147572 
                 NM_002038, 
                 interferon, alpha-inducible protein (clone IFI-6-16) 
               
               
                   
                   
                   
                   
                 NM_022873 
               
               
                 204533_at 
                 CXCL10 
                 3627 
                 147310 
                 NM_001565 
                 chemokine (C—X—C motif) ligand 10 
               
               
                 204745_x_at 
                 MT1G 
                 4495 
                 156353 
                 NM_005950, 
                 metallothionein 1G 
               
               
                   
                   
                   
                   
                 NM_005950 
               
               
                 204780_s_at 
                 TNFRSF6 
                 355 
                 134637 
                 NM_000043, 
                 tumor necrosis factor receptor superfamily, member 6 
               
               
                   
                   
                   
                   
                 NM_152877, 
               
               
                   
                   
                   
                   
                 NM_152876, 
               
               
                   
                   
                   
                   
                 NM_152875, 
               
               
                   
                   
                   
                   
                 NM_152872, 
               
               
                   
                   
                   
                   
                 NM_152873, 
               
               
                   
                   
                   
                   
                 NM_152871 
               
               
                 204858_s_at 
                 ECGF1 
                 1890 
                 131222 
                 NM_001953 
                 endothelial cell growth factor 1 (platelet-derived) 
               
               
                 205241_at 
                 SCO2 
                 9997 
                 604272 
                 NM_005138 
                 SCO cytochrome oxidase deficient homolog 2 
               
               
                   
                   
                   
                   
                   
                 (yeast) 
               
               
                 205242_at 
                 CXCL13 
                 10563 
                 605149 
                 NM_006419 
                 chemokine (C—X—C motif) ligand 13 (B-cell chemoat- 
               
               
                   
                   
                   
                   
                   
                 tractant) 
               
               
                 205495_s_at 
                 GNLY 
                 10578 
                 188855 
                 NM_006433, 
                 granulysin 
               
               
                   
                   
                   
                   
                 NM_006433 
               
               
                 205831_at 
                 CD2 
                 914 
                 186990 
                 NM_001767 
                 CD2 antigen (p50), sheep red blood cell receptor 
               
               
                 206108_s_at 
                 SFRS6 
                 6431 
                 601944 
                 NM_006275 
                 splicing factor, arginine/serine-rich 6 
               
               
                 206286_s_at 
                 TDGF1 
                 6997 
                 187395 
                 NM_003212 
                 teratocarcinoma-derived growth factor 1 
               
               
                 206461_x_at 
                 MT1H 
                 4496 
                 156354 
                 NM_005951 
                 metallothionein 1H 
               
               
                 206754_s_at 
                 CYP2B6 
                 1555 
                 123930 
                 NM_000767 
                 cytochrome P450, family 2, subfamily B, polypeptide 6 
               
               
                 206907_at 
                 TNFSF9 
                 8744 
                 606182 
                 NM_003811 
                 tumor necrosis factor (ligand) superfamily, member 9 
               
               
                 206918_s_at 
                 RBM12 
                 10137 
                 607179 
                 NM_006047, 
                 RNA binding motif protein 12 
               
               
                   
                   
                   
                   
                 NM_006047 
               
               
                 206976_s_at 
                 HSPH1 
                 10808 
                   
                 NM_006644 
                 heat shock 105 kDa/110 kDa protein 1 
               
               
                 207320_x_at 
                 STAU 
                 6780 
                 601716 
                 NM_004602, 
                 staufen, RNA binding protein ( Drosophila ) 
               
               
                   
                   
                   
                   
                 NM_004602, 
               
               
                   
                   
                   
                   
                 NM_017452, 
               
               
                   
                   
                   
                   
                 NM_017453 
               
               
                 207457_s_at 
                 LY6G6D 
                 58530 
                 606038 
                 NM_021246 
                 lymphocyte antigen 6 complex, locus G6D 
               
               
                 207993_s_at 
                 CHP 
                 11261 
                 606988 
                 NM_007236 
                 calcium binding protein P22 
               
               
                 208022_s_at 
                 CDC14B 
                 8555 
                 603505 
                 NM_003671, 
                 CDC14 cell division cycle 14 homolog B ( S. cerevisiae ) 
               
               
                   
                   
                   
                   
                 NM_003671, 
               
               
                   
                   
                   
                   
                 NM_033331 
               
               
                 208156_x_at 
                 EPPK1 
                 83481 
                   
                   
                 epiplakin 1 
               
               
                 208581_x_at 
                 MT1X 
                 4501 
                 156359 
                 NM_005952 
                 metallothionein 1X 
               
               
                 208944_at 
                 TGFBR2 
                 7048 
                 190182 
                 NM_003242 
                 transforming growth factor, beta receptor II 
               
               
                   
                   
                   
                   
                   
                 (70/80 kDa) 
               
               
                 209048_s_at 
                 PRKCBP1 
                 23613 
                   
                 NM_012408, 
                 protein kinase C binding protein 1 
               
               
                   
                   
                   
                   
                 NM_012408, 
               
               
                   
                   
                   
                   
                 NM_183047 
               
               
                 209108_at 
                 TM4SF6 
                 7105 
                 300191 
                 NM_003270 
                 transmembrane 4 superfamily member 6 
               
               
                 209504_s_at 
                 PLEKHB1 
                 58473 
                 607651 
                 NM_021200 
                 pleckstrin homology domain containing, family B 
               
               
                   
                   
                   
                   
                   
                 (evectins) member 1 
               
               
                 209546_s_at 
                 APOL1 
                 8542 
                 603743 
                 NM_003661, 
                 apolipoprotein L, 1 
               
               
                   
                   
                   
                   
                 NM_003661, 
               
               
                   
                   
                   
                   
                 NM_145343 
               
               
                 210029_at 
                 INDO 
                 3620 
                 147435 
                 NM_002164 
                 indoleamine-pyrrole 2,3 dioxygenase 
               
               
                 210103_s_at 
                 FOXA2 
                 3170 
                 600288 
                 NM_021784, 
                 forkhead box A2 
               
               
                   
                   
                   
                   
                 NM_021784 
               
               
                 210321_at 
                 GZMH 
                 2999 
                 116831 
                 NM_033423 
                 granzyme H (cathepsin G-like 2, protein h-CCPX) 
               
               
                 210538_s_at 
                 BIRC3 
                 330 
                 601721 
                 NM_001165, 
                 baculoviral IAP repeat-containing 3 
               
               
                   
                   
                   
                   
                 NM_001165 
               
               
                 211456_x_at 
                 AF333388 
               
               
                 212057_at 
                 KIAA0182 
                 23199 
                   
                 XM_050495 
                 KIAA0182 protein 
               
               
                 212070_at 
                 GPR56 
                 9289 
                 604110 
                 NM_005682 
                 G protein-coupled receptor 56 
               
               
                 212185_x_at 
                 MT2A 
                 4502 
                 156360 
                 NM_005953 
                 metallothionein 2A 
               
               
                 212229_s_at 
                 FBXO21 
                 23014 
                   
                 NM_015002, 
                 F-box only protein 21 
               
               
                   
                   
                   
                   
                 NM_015002 
               
               
                 212336_at 
                 EPB41L1 
                 2036 
                 602879 
                 NM_012156, 
                 erythrocyte membrane protein band 4,1-like 1 
               
               
                   
                   
                   
                   
                 NM_012156 
               
               
                 212341_at 
                 MGC21416 
                 286451 
                   
                 NM_173834 
                 hypothetical protain MGC21416 
               
               
                 212349_at 
                 POFUT1 
                 23509 
                 607491 
                 NM_015352, 
                 protein O-fucosyltransferase 1 
               
               
                   
                   
                   
                   
                 NM_015352 
               
               
                 212859_x_at 
                 MT1E 
                 4493 
                 156351 
                 NM_175617 
                 metallothionein 1E (functional) 
               
               
                 213201_s_at 
                 TNNT1 
                 7138 
                 191041 
                 NM_003283, 
                 troponin T1, skeletal, slow 
               
               
                   
                   
                   
                   
                 NM_003283, 
               
               
                   
                   
                   
                   
                 XM_352926 
               
               
                 213385_at 
                 CHN2 
                 1124 
                 602857 
                 NM_004067 
                 chimerin (chimaerin) 2 
               
               
                 213470_s_at 
                 HNRPH1 
                 3187 
                 601035 
                 NM_005520 
                 heterogeneous nuclear ribonucleoprotein H1 (H) 
               
               
                 213738_s_at 
                 ATP5A1 
                 498 
                 164360 
                 NM_004046 
                 ATP synthase, H+ transporting, mitochondrial F1 
               
               
                   
                   
                   
                   
                   
                 complex, alpha subunit, isoform 1, cardiac muscle 
               
               
                 213757_at 
                 EIF5A 
                 1984 
                 600187 
                 NM_001970 
                 eukaryotic translation initiation factor 5A 
               
               
                 214617_at 
                 PRF1 
                 5551 
                 170280 
                 NM_005041 
                 perforin 1 (pore forming protein) 
               
               
                 214924_s_at 
                 OIP106 
                 22906 
                 608112 
                 NM_014965 
                 OGT(O-Glc-NAc transferase)-interacting protein 106 KDa 
               
               
                 215693_x_at 
                 DDX27 
                 55661 
                   
                 NM_017895 
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 27 
               
               
                 215780_s_at 
                 Hs.382039 
               
               
                 216336_x_at 
                 AL031602 
               
               
                 217727_x_at 
                 VPS35 
                 55737 
                 606931 
                 NM_018206 
                 vacuolar protein sorting 35 (yeast) 
               
               
                 217759_at 
                 TRIM44 
                 54765 
                   
                 NM_017583 
                 tripartite motif-containing 44 
               
               
                 217875_s_at 
                 TMEPAI 
                 56937 
                 606564 
                 NM_020182, 
                 transmembrane, prostate androgen induced RNA 
               
               
                   
                   
                   
                   
                 NM_020182, 
               
               
                   
                   
                   
                   
                 NM_199169, 
               
               
                   
                   
                   
                   
                 NM_199170 
               
               
                 217917_s_at 
                 DNCL2A 
                 83658 
                 607167 
                 NM_014183, 
                 dynein, cytoplasmic, light polypeptide 2A 
               
               
                   
                   
                   
                   
                 NM_014183, 
               
               
                   
                   
                   
                   
                 NM_177953 
               
               
                 217933_s_at 
                 LAP3 
                 51056 
                 170250 
                 NM_015907 
                 leucine aminopeptidase 3 
               
               
                 218094_s_at 
                 C20orf35 
                 55861 
                   
                 NM_018478, 
                 chromosome 20 open reading frame 35 
               
               
                   
                   
                   
                   
                 NM_018478 
               
               
                 218237_s_at 
                 SLC38A1 
                 81539 
                   
                 NM_030674 
                 solute carrier family 38, member 1 
               
               
                 218242_s_at 
                 CGI-85 
                 51111 
                   
                 NM_016028, 
                 CGI-85 protein 
               
               
                   
                   
                   
                   
                 NM_016028 
               
               
                 218325_s_at 
                 DATF1 
                 11083 
                 604140 
                 NM_022105, 
                 death associated transcription factor 1 
               
               
                   
                   
                   
                   
                 NM_022105, 
               
               
                   
                   
                   
                   
                 NM_080796 
               
               
                 218345_at 
                 HCA112 
                 55365 
                   
                 NM_018487 
                 hepatocellular carcinoma-associated antigen 112 
               
               
                 218346_s_at 
                 SESN1 
                 27244 
                 606103 
                 NM_014454 
                 sestrin 1 
               
               
                 218704_at 
                 FLJ20315 
                 54894 
                   
                 NM_017763 
                 hypothetical protein FLJ20315 
               
               
                 218802_at 
                 FLJ20647 
                 55013 
                   
                 NM_017918 
                 hypothetical protein FLJ20647 
               
               
                 218898_at 
                 CT120 
                 79850 
                   
                 NM_024792 
                 membrane protein expressed in epithelial-like lung 
               
               
                   
                   
                   
                   
                   
                 adenocarcinoma 
               
               
                 218943_s_at 
                 RIG-I 
                 23586 
                   
                 NM_014314 
                 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 
               
               
                 218963_s_at 
                 KRT23 
                 25984 
                 606194 
                 NM_015515, 
                 keratin 23 (histone deacetylase inducible) 
               
               
                   
                   
                   
                   
                 NM_015515 
               
               
                 219956_at 
                 GALNT6 
                 11226 
                 605148 
                 NM_007210 
                 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 
               
               
                   
                   
                   
                   
                   
                 acetylgalactosaminyltransferase 6 (GalNAc-T6) 
               
               
                 220658_s_at 
                 ARNTL2 
                 56938 
                   
                 NM_020183 
                 aryl hydrocarbon receptor nuclear translocator-like 2 
               
               
                 220951_s_at 
                 ACF 
                 29974 
                   
                 NM_014576, 
                 apobec-1 complementation factor 
               
               
                   
                   
                   
                   
                 NM_014576, 
               
               
                   
                   
                   
                   
                 NM_138932 
               
               
                 221516_s_at 
                 FLJ20232 
                 54471 
                   
                 NM_019008 
                 hypothetical protein FLJ20232 
               
               
                 221653_x_at 
                 APOL2 
                 23780 
                 607252 
                 NM_030882, 
                 apolipoprotein L, 2 
               
               
                   
                   
                   
                   
                 NM_030882 
               
               
                 221920_s_at 
                 MSCP 
                 51312 
                   
                 NM_016612, 
                 mitochondrial solute carrier protein 
               
               
                   
                   
                   
                   
                 NM_016612 
               
               
                 222244_s_at 
                 FLJ20618 
                 55000 
                   
                 NM_017903 
                 hypothetical protein FLJ20618 
               
               
                   
               
             
          
         
       
     
         [0098]    The minimum of three errors was found even using only 7 genes (Table 23). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 23 
               
             
             
               
                   
               
               
                 Genes used for the classification of MSS vs MSI tumors 
               
             
          
           
               
                 Name 
                 Symbol 
                 Unigene 
                 MSS 
                 MSI 
               
               
                   
               
             
          
           
               
                 hepatocellular carcinoma- 
                 HCA112 
                 Hs.12126 
                 1261 
                 653 
               
               
                 associated antigen 112 
               
               
                 metastasis-associated 1-like 1 
                 MTA1L1 
                 Hs.173043 
                 45 
                 91 
               
               
                 chemokine (C—X—C motif) 
                 CXCL10 
                 Hs.2248 
                 104 
                 274 
               
               
                 ligand 10 
               
               
                 heterogeneous nuclear 
                 HNRPL 
                 Hs.2730 
                 194 
                 630 
               
               
                 ribonucleoprotein L 
               
               
                 hypothetical protein FLJ20618 
                 FLJ20618 
                 Hs.52184 
                 776 
                 388 
               
               
                 splicing factor, arginine/serine- 
                 SFRS6 
                 Hs.6891 
                 74 
                 446 
               
               
                 rich 6 
               
               
                 protein kinase C binding protein 1 
                 PRKCBP1 
                 Hs.75871 
                 294 
                 168 
               
               
                   
               
             
          
         
       
     
       Classification of Ambiguous Samples 
       [0099]    Application of the 7-gene classifier to the four samples showing ambiguity in the microsatellite analyses assigns all four to be microsatellite stable tumor class. Notably, all four showed expression levels of Tumor Growth Factor β induced protein (TFGBI), MLH1 and thymidylate synthase (TYMS) that are atypical for MSI tumors. Furthermore, these tumors were all from the left colon. Thus the misclassified tumors are clearly truly MSS or they belong to a yet undefined class of MSI tumors. 
       Stability of Classification 
       [0100]    To estimate the stability of the classifier based on all 97 tumor samples, we generated one hundred new classifiers based on randomly chosen datasets consisting of 30 MSS and 25 MSI samples. In each case the classifiers were tested with the remaining samples. The performance for each set was evaluated and averaged over all 100 training and test sets (Table 24). The mean error rate for MSS tumors was 0.52% and 1.38% for MSI tumors. The seven genes defined above were found to be those genes that were most frequently used in the crossvalidation loop. More than 50% of the errors were related to three tumors of which two were wrongly classified in all permutation and one in 94%. The remaining errors were mainly caused by four tumors with error rates of 40-47% showing that the former three samples are truly assigned contradictory to result from the microsatellite analysis and that four samples could not be assigned with confidence too any of the classes. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 24 
               
             
             
               
                   
               
               
                 Performance of the classifier 
               
             
          
           
               
                   
                 Trainings set 
                 Test set 
               
               
                   
                 Errors in crossvalidation 
                 Test errors 
               
               
                   
                   
               
             
          
           
               
                 MSI 
                 2.8% (n = 25, range 0-6) 
                 1.4% (n = 10, range 0-4) 
               
               
                 MSS 
                 0.70% (n = 30, range 0-3)  
                 0.52% (n = 29, range 0-2)  
               
               
                 All 
                 1.7% (n = 55, range 1-7) 
                 1.9% (n = 39, range 0-5) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 25 
               
               
                   
               
               
                 Sensitivity, Specificity, and Predictive Value of Test for MSS 
               
               
                 based on the eight gene Classifier 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Positive for MSS 
                 True = (0.9948 * 29) = 
                 False = (0.138 * 10) = 1.38 
               
               
                   
                 28,8492 
               
               
                 Negative for MSS 
                 False = (0.0052 * 29) = 
                 True = (0.962 * 10) = 9.62 
               
               
                   
                 0.1508 
               
               
                   
               
             
          
           
               
                   
                 Sensitivity 
                 28.9507/29 = 99.5% 
               
               
                   
                 Specificity 
                 9.62/10 = 96.2% 
               
               
                   
                 Positive predictive value 
                 28.8492/30.2292 = 95.4% 
               
               
                   
                 Negative predictive value 
                 9.62/9.7708 = 98.5% 
               
               
                   
                   
               
               
                   
                 *Based on a prevalence for MSS of 85% 
               
             
          
         
       
     
       Survival Classifier 
       [0101]    Using the same classification methods described above, we build classifiers for survival based on either all samples or the above defined groups of MSI-H and MSS. As seen in  FIG. 10  a distinction of patient with good prognosis (&gt;5 year survival) from patient with bad prognosis (&lt;5 years survival) can be achieved with higher precision and using only a fraction of the genes by first separating into MSI-H and MSS groups. 
       Construction of a Classifier for Sporadic Versus Hereditary Microsatellite Instable Tumors 
       [0102]    In order to identify a gene set for identification of hereditary microsatellite instable tumors we applied 19 sporadic microsatellite instable samples and 18 microsatellite instable samples to supervised classification as described above. We found ten genes we high scored for separation of sporadic MSI-H from hereditary MSI-H tumours (Table 26). In crossvalidation we found a minimum number of one error using two genes ( FIG. 9A ) and were used in at least 36 of the 37 crossvalidation loops. The genes were: the mismatch repair gene MLH1 that show a general downregulation in sporadic disease and PIWIL1 that is lower expressed in hereditary cases ( FIG. 9B ). Using these two genes only one error occurred: a sporadic microsatellite instable was classified as hereditary. Based on T-test we performed 500 permutations to test the significance of these two genes for marker genes and found both genes highly significant with p-values &lt;0.005. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 26 
               
               
                   
               
               
                 AFFYID 
                 SYMBOL 
                 LOCUSLINK 
                 OMIM 
                 REFSEQ 
                 AFFYDESCRIPTION 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 206194_at 
                 HOXC6 
                 3223 
                 142972 
                 NM_004503 
                 Homeo box C4 
               
               
                 214868_at 
                 PIWIL1 
                 9271 
                 605571 
                 NM_004764.2 
                 Piwi ( Drosophila )-like 1 
               
               
                 202520_s_at 
                 MLH1 
                 4292 
                 120436 
                 NM_000249.2 
                 MutL ( E. coli ) homolog 1 
               
               
                   
                   
                   
                   
                   
                 (colon cancer, nonpoly- 
               
               
                   
                   
                   
                   
                   
                 posis type 2) 
               
               
                 202517_at 
                 CRMP1 
                 1400 
                 602462 
                 NM_001313.2 
                 Collapsin response mediator 
               
               
                   
                   
                   
                   
                   
                 protein 1 
               
               
                 205453_at 
                 HOXB2 
                 3212 
                 142967 
                 NM_002145.2 
                 Homeo box B2 (HOXB2) 
               
               
                 217791_s_at 
                 PYCS/ADH18A1 
                 5832 
                 138250 
                 NM_002860.2 
                 Pyrroline-5-carboxylate 
               
               
                   
                   
                   
                   
                   
                 synthetase (glutamate 
               
               
                   
                   
                   
                   
                   
                 gamma-semialdehyde 
               
               
                   
                   
                   
                   
                   
                 synthetase) 
               
               
                   
                   
                   
                   
                   
                 (/PYCS/ADH18A1) 
               
               
                 202393_s_at 
                 TIEG 
                 7071 
                 601878 
                 NM_005655.1 
                 TGFB inducible early 
               
               
                   
                   
                   
                   
                   
                 growth response (TIEG) 
               
               
                 218803_at 
                 CHFR 
                 55743 
                 605209 
                 NM_018223.1 
                 Checkpoint with forkhead 
               
               
                   
                   
                   
                   
                   
                 and ring finger domains 
               
               
                   
                   
                   
                   
                   
                 (CHFR) 
               
               
                 219877_at 
                 FLJ13842 
                 79698 
                   
                 NM_024645.1 
                 Hypothetical protein 
               
               
                   
                   
                   
                   
                   
                 FLJ13842 (FLJ13842) 
               
               
                 202241_at 
                 C8FW 
                 10221 
                   
                 NM_025195.2 
                 Phosphoprotein regulated 
               
               
                   
                   
                   
                   
                   
                 by mitogenic pathways 
               
               
                   
                   
                   
                   
                   
                 (C8FW) 
               
               
                   
               
             
          
         
       
     
       Cross Platform Classification 
       [0103]    Real time PCR was applied both to verify the array data and examine if the 7-gene classifier would also perform on this platform. We chose 23 samples of which 18 were also analyzed on arrays. The correlation between the two platforms was high (data not shown). In order to test the performance of classification using PCR data we re-build our classifier with a 79 samples array dataset including only those tumors that were not analyzed with PCR. Two samples were classified in discordance with the microsatellite instability test of which one of them was ambiguously classified by the 7-gene array classifier. 
       Relation Between Microsatellite-Instability Status, Stage and Survival 
       [0104]    Based on the 7-gene classifier, classification of 36 patients with Dukes&#39; B tumors receiving no adjuvant chemotherapy, 18 were classified as MSI tumors and 18 as MSS tumors. The overall survival was highly significantly related to the classification since all nine patients that died within five years of follow-up were belonged to the MSS group (P=0.0014) ( FIG. 10A ). Thus, the 7-gene classifier clearly proved to be a strong predictor of survival in Dukes B and it can be used to select patients who need adjuvant chemotherapy, namely those classified as MSS. 
         [0105]    Among 65 patients with Dukes&#39; C tumors receiving adjuvant chemotherapy, 17 were classified as MSI tumors and as 48 MSS tumors. Of these, 6 MSI and 27 MSS patients died within five years of follow-up meaning no significant difference in overall survival between these groups (P=0.55) ( FIG. 10B ). A trend was that the MSI showed a poorer short-term survival than the MSS, contrary to Dukes B patients. This difference can be attributed to the fact that a recent large study has shown that chemotherapy only benefit the MSS tumor patients, thus improving their survival to a level comparable to that which is characteristic of MSI tumor patients. 
       Clinical Application of the Discovery 
       [0106]    In the clinic the 106 or less genes described can be used for predicting outcome of colorectal cancer when examined at the RNA level and also on the protein level as each gene identified is the project is transcribed to RNA that is further translated into protein. The genes can also be used determine which patient should be treated with chemotherapy as only non-microsatellite instable tumors will respond to 5-FU based therapy. Building classifiers can achieve a further stratification of patient with god and bad prognosis after stratification into microsatellite instable and stable tumors. The genes used to identify hereditary disease can be used to decide which patient should enter into sequencing analysis of mismatch repair genes. 
         [0107]    The RNA determination can be made in any form using any method that will quantify RNA. The proteins can be measured with any method quantification method that can determine the level of proteins. 
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         Birkenkamp-Demtroder K, Christensen L L, Olesen S H, Frederiksen C M, Laiho P, Aaltonen L A, Laurberg S, Sorensen F B, Hagemann R, ORntoft T F. Gene expression in colorectal cancer. Cancer Res. 2002 Aug. 1; 62(15):4352-63. 
         Boland C R, Thibodeau S N, Hamilton S R, Sidransky D, Eshleman J R, Burt R W, Meltzer S J, Rodriguez-Bigas M A, Fodde R, Ranzani G N, Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998 Nov. 15; 58(22):5248-57. Review. 
         Chapusot C, Martin L, Bouvier A M, Bonithon-Kopp C, Ecarnot-Laubriet A, Rageot D, Ponnelle T, Laurent Puig P, Faivre J, Piard F. Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas. Br J Cancer. 2002 Aug. 12; 87(4):400-4. 
         Dyrskjot L, Thykjaer T, Kruhoffer M, Jensen J L, Marcussen N, Hamilton-Dutoit S, Wolf H, Orntoft T F. Identifying distinct classes of bladder carcinoma using microarrays. Nat Genet. 2003 January; 33(1):90-6. 
         Frederiksen C M, Knudsen S, Laurberg S, Orntoft T F. Classification of Dukes&#39; B and C colorectal cancers using expression arrays. J Cancer Res Clin Oncol. 2003 May; 129(5):263-71. 
         Huang J, Qi R, Quackenbush J, Dauway E, Lazaridis E, Yeatman T. Effects of ischemia on gene expression. J Surg Res. 2001 August; 99(2):222-7. 
         Irizarry R A, Bolstad B M, Collin F, Cope L M, Hobbs B, Speed T P. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003 Feb. 15; 31 (4):e15. 
         Loukola A, Eklin K, Laiho P, Salovaara R, Kristo P, Jarvinen H, Mecklin J P, Launonen V, Aaltonen L A. Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC). Cancer Res. 2001 Jun. 1; 61(11):4545-9. 
         Markowitz S, Hines J D, Lutterbaugh J, Myeroff L, Mackay W, Gordon N, Rustum Y, Luna E, Kleinerman J. Mutant K-ras oncogenes in colon cancers Do not predict Patient&#39;s chemotherapy response or survival. Clin Cancer Res. 1995 April; 1(4):441-5. 
         Mori Y, Selaru F M, Sato F, Yin J, Simms L A, Xu Y, Olaru A, Deacu E, Wang S, Taylor J M, Young J, Leggett B, Jass J R, Abraham J M, Shibata D, Meltzer S J. The impact of microsatellite instability on the molecular phenotype of colorectal tumors. Cancer Res. 2003 Aug. 1; 63(15):4577-82. 
         Ribic C M, Sargent D J, Moore M J, Thibodeau S N, French A J, Goldberg R M, Hamilton S R, Laurent-Puig P, Gryfe R, Shepherd L E, Tu D, Redston M, Gallinger S. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med. 2003 Jul. 17; 349(3):247-57.