Lung cancer prognostics

Abstract

A method of providing a prognosis of lung cancer is conducted by analyzing the expression-of a group of genes. Gene expression profiles in a variety of medium such as microarrays are included as are kits that contain them.

Description

REFERENCE TO SEQUENCE LISTING, OR A COMPUTER PROGRAM LISTING COMPACT DISK APPENDIX

Reference to a “Sequence Listing,” a table, or a computer program listing appendix submitted on a compact disc and an incorporation by reference of the material on the compact disc including duplicates and the files on each compact disc shall be specified.

BACKGROUND

This application claims the benefit of U.S. Patent Application No. 60/632,053, filed Nov. 30, 2005 which is incorporated herein by reference.

This invention relates to prognostics for lung cancer based on the gene expression profiles of biological samples.

Lung cancer is the leading cause of cancer deaths in developed countries killing about 1 million people worldwide each year. An estimated 171,900 new cases are expected in 2003 in the US, accounting for about 13% of all cancer diagnoses. Non-small cell lung cancer (NSCLC) represents the majority (˜75%) of bronchogenic carcinomas while the remainder is small cell lung carcinomas (SCLC). NSCLC is comprised of three main subtypes: 40% adenocarcinoma, 40% squamous, and 20% large cell cancer. Adenocarcinoma has replaced squamous cell carcinoma as the most frequent histological subtype over the last 25 years, peaking the early 1990's. This may be associated with the use of “low tar” cigarettes resulting in deeper inhalation of cigarette smoke. Wingo et al. (1999). The overall 10-year survival rate of patients with NSCLC is a dismal 8-10%.

Approximately 25-30% of patients with NSCLC have stage I disease and of these 35-50% will relapse within 5 years after surgical treatment. Depending upon stage, adenocarcinoma has a higher relapse rate than squamous cell carcinoma with approximately 65% and 55% of SCC and adenocarcinoma patients surviving at 5 years, respectively. Mountain et al. (1987). Currently, it is not possible to identify those patients with a high risk of relapse. The ability to identify high-risk patients among the stage I disease group will allow for the consideration of additional therapeutic intervention leading to the potential for improved survival. Indeed, recent clinical trials have shown that adjuvant therapy following resection of lung tumors can lead to improved survival. Kato et al. (2004). Specifically, Kato et al. demonstrated that adjuvant chemotherapy with uracil-tegafur improves survival among patients with completely resected pathological stage I adenocarcinoma, particularly T2 disease.

Microarray gene expression profiling has recently been utilized to define prognostic signatures in patients with lung adenocarcinomas, (Beer et al. (2002)) however, no large studies have investigated gene expression profiles of prognosis in the squamous cell carcinoma population. Here, we have profiled 134 SCC samples and 10 normal matched lung samples on the Affymetrix U133A chip. Hierarchical clustering and Cox modeling has identified genes that correlate with patient prognosis. These signatures can be used to identify patients who may benefit from adjuvant therapy following initial surgery.

SUMMARY OF THE INVENTION

The present invention provides a method of assessing lung cancer status by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of lung cancer status.

The present invention provides a method of staging lung cancer patients by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of the lung cancer stage.

The present invention provides a method of determining lung cancer patient treatment protocol by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below predetermined cut-off levels are sufficiently indicative of risk of recurrence to enable a physician to determine the degree and type of therapy recommended to prevent recurrence.

The present invention provides a method of treating a lung cancer patient by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicate a high risk of recurrence and; treating the patient with adjuvant therapy if they are a high risk patient.

The present invention provides a method of determining whether a lung cancer patient is high or low risk of mortality by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 4 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of mortality to enable a physician to determine the degree and type of therapy recommended.

The present invention provides a method of generating a lung cancer prognostic patient report by determining the results of any one of the methods described herein and preparing a report displaying the results and patient reports generated thereby.

The present invention provides a composition comprising at least one probe set selected from the group consisting of: Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

The present invention provides a kit for conducting an assay to determine lung cancer prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

The present invention provides articles for assessing lung cancer status comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

The present invention provides a microarray or gene chip for performing the method described herein.

The present invention provides a diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts hierarchical clustering of 129 lung SCC patients.

FIG. 2 depicts plots of AUC vs. number of genes.

FIG. 3 depicts error rates of LOOCV v various cutoffs in the 65-sample training set.

FIG. 4 depicts Kaplan Meier plots of the 50-gene signature in the testing set.

FIG. 5 depicts unsupervised clustering identifies epidermnal differentiation pathway as being down-regulated in high-risk patients. A. Clustering of patients based on top 121 showed two clusters of patients. The majority of genes in cluster I were down-regulated (green). B. List of 20 genes associated with epidermal differentiation pathway. C. Kaplan Meier curve of clustered patient groups defined by the-20 epidermal-related genes.

FIG. 6 depicts verification of gene expression data using real-time RT-PCR. Four genes (NTRK2, FGFR2, VEGF, KRT13) were selected for RT-PCR. Expression correlate very well with Affymetrix chip data (R=0.71-0.96).

DETAILED DESCRIPTION OF THE INVENTION

Non-small cell lung cancer (NSCLC) represents the majority (˜75%) of lung carcinomas and is comprised of three main subtypes: 40% squamous, 40% adenocarcinoma, and 20% large cell cancer. Approximately 25-30% of patients with NSCLC have stage I disease and of these 35-50% will relapse within 5 years after surgical treatment. Current histopathology and genetic biomarkers are insufficient for identifying patients who are at a high risk of relapse. As described in the present invention, 129 primary squamous cell lung carcinomas and 10 matched normal lung tissues were profiled using the Affymetrix U133A gene chip. Unsupervised hierarchical clustering identified two clusters of patients with lung carcinoma that had no correlation with stage of disease but had significantly different median overall survival (p=0.036). Cox proportional hazard models were then utilized to identify an optimal set of 50 genes (Table 1) in a 65 patient training set that significantly predicted survival in a 64 patient test set. This signature achieved 52% specificity and 82% sensitivity and provided an overall predictive value of 71%. Kaplan-Meier analysis showed clear significant stratification of high and low risk patients (p=0.0075). The identification of prognostic signatures allows identification of patients with high-risk squamous cell lung carcinoma who could benefit from adjuvant therapy following initial surgery.

TABLE 1
SEQ ID NO: Rank
228        1
284        2
76         3
124        4
281        5
86         6
303        7
311        8
443        9
287        10
13         11
378        12
362        13
18         14
79         15
230        16
416        17
409        18
78         19
420        20
58         21
53         22
254        23
91         24
270        25
446        26
4          27
310        28
42         29
10         30
80         31
12         32
440        33
75         34
60         35
63         36
283        37
29         38
221        39
279        40
280        41
267        42
189        43
103        44
194        45
268        46
252        47
461        48
372        49
414        50

A Biomarker is any indicia of the level of expression of an indicated Marker gene. The indicia can be direct or indirect and measure over- or under-expression of the gene given the physiologic parameters and in comparison to an internal control, normal tissue or another carcinoma. Biomarkers include, without limitation, nucleic acids (both over and under-expression and direct and indirect). Using nucleic acids as Biomarkers can include any method known in the art including, without limitation, measuring DNA amplification, RNA, micro RNA, loss of heterozygosity (LOH), single nucleotide polymorphisms (SNPs, Brookes (1999)), microsatellite DNA, DNA hypo- or hyper-methylation. Using proteins as Biomarkers can include any method known in the art including, without limitation, measuring amount, activity, modifications such as glycosylation, phosphorylation, ADP-ribosylation, ubiquitination, etc., imunohistochemistry (IHC). Other Biomarkers include imaging, cell count and apoptosis markers.

The indicated genes provided herein are those associated with a particular tumor or tissue type. Marker gene may be associated with numerous cancer types but provided that the expression of the gene is sufficiently associated with one tumor or tissue type to be identified using the algorithm described herein to be specific for a lung cancer cell, the gene can be using in the claimed invention to determine cancer status and prognosis. Numerous genes associated with one or more cancers are known in the art. The present invention provides preferred Marker genes and even more preferred Marker gene combinations. These are described herein in detail.

A Marker gene corresponds to the sequence designated by a SEQ ID NO when it contains that sequence. A gene segment or fragment corresponds to the sequence of such gene when it contains a portion of the referenced sequence or its complement sufficient to distinguish it as being the sequence of the gene. A gene expression product corresponds to such sequence when its RNA, mRNA, or cDNA hybridizes to the composition having such sequence (e.g. a probe) or, in the case of a peptide or protein, it is encoded by such mRNA. A segment or fragment of a gene expression product corresponds to the sequence of such gene or gene expression product when it contains a portion of the referenced gene expression product or its complement sufficient to distinguish it as being the sequence of the gene or gene expression product.

The inventive methods, compositions, articles, and kits of described and claimed in this specification include one or more Marker genes. “Marker” or “Marker gene” is used throughout this specification to refer to genes and gene expression products that correspond with any gene the over- or under-expression of which is associated with a tumor or tissue type. The preferred Marker genes are described in more detail in Table 8.

The present invention provides a method of assessing lung cancer status by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of lung cancer status.

The present invention provides a method of staging lung cancer patients by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of the lung cancer stage. The stage can correspond to any classification system, including, but not limited to the TNM system or to patients with similar gene expression profiles.

The present invention provides a method of determining lung cancer patient treatment protocol by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of recurrence to enable a physician to determine the degree and type of therapy recommended to prevent recurrence.

The present invention provides a method of treating a lung cancer patient by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicate a high risk of recurrence and; treating the patient with adjuvant therapy if they are a high risk patient.

The present invention provides a method of determining whether a lung cancer patient is high or low risk of mortality by obtaining a biological sample from a lung cancer patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 4 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of mortality to enable a physician to determine the degree and type of therapy recommended.

In the above methods, the sample can be prepared by any method known in the art including, but not limited to, bulk tissue preparation and laser capture microdissection. The bulk tissue preparation can be obtained for instance from a biopsy or a surgical specimen.

In the above methods, the gene expression measuring can also include measuring the expression level of at least one gene constitutively expressed in the sample.

In the above methods, the specificity is preferably at least about 40% and the sensitivity at least at least about 80%.

In the above methods, the pre-determined cut-off levels are at least about 1.5-fold over- or under-expression in the sample relative to benign cells or normal tissue.

In the above methods, the pre-determined cut-off levels have at least a statistically significant p-value over-expression in the sample having metastatic cells relative to benign cells or normal tissue, preferably the p-value is less than 0.05.

In the above methods, gene expression can be measured by any method known in the art, including, without limitation on a microarray or gene chip, nucleic acid amplification conducted by polymerase chain reaction (PCR) such as reverse transcription polymerase chain reaction (RT-PCR), measuring or detecting a protein encoded by the gene such as by an antibody specific to the protein or by measuring a characteristic of the gene such as DNA amplification, methylation, mutation and allelic variation. The microarray can be for instance, a cDNA array or an oligonucleotide array. All these methods and can further contain one or more internal control reagents.

The present invention provides a method of generating a lung cancer prognostic patient report by determining the results of any one of the methods described herein and preparing a report displaying the results and patient reports generated thereby. The report can further contain an assessment of patient outcome and/or probability of risk relative to the patient population.

The present invention provides a composition comprising at least one probe set selected from the group consisting of: Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

The present invention provides a kit for conducting an assay to determine lung cancer prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7. The kit can further comprise reagents for conducting a microarray analysis, and/or a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

The present invention provides articles for assessing lung cancer status comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7. The articles can further contain reagents for conducting a microarray analysis and/or a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

The present invention provides a microarray or gene chip for performing the method of claim 1, 2, 5, 6 or 7. The microarray can contain isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7. Preferably, the microarray is capable of measurement or characterization of at least 1.5-fold over- or under-expression. Preferably, the microarray provides a statistically significant p-value over- or under-expression. Preferably, the p-value is less than 0.05. The microarray can contain a cDNA array or an oligonucleotide array and/or one or more internal control reagents.

The present invention provides a diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7. Preferably, the portfolio is capable of measurement or characterization of at least 1.5-fold over- or under-expression. Preferably, the portfolio provides a statistically significant p-value over- or under-expression. Preferably, the p-value is less than 0.05.

The mere presence or absence of particular nucleic acid sequences in a tissue sample has only rarely been found to have diagnostic or prognostic value. Information about the expression of various proteins, peptides or mRNA, on the other hand, is increasingly viewed as important. The mere presence of nucleic acid sequences having the potential to express proteins, peptides, or mRNA (such sequences referred to as “genes”) within the genome by itself is not determinative of whether a protein, peptide, or mRNA is expressed in a given cell. Whether or not a given gene capable of expressing proteins, peptides, or mRNA does so and to what extent such expression occurs, if at all, is determined by a variety of complex factors. Irrespective of difficulties in understanding and assessing these factors, assaying gene expression can provide useful information about the occurrence of important events such as tumorogenesis, metastasis, apoptosis, and other clinically relevant phenomena. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles. The gene expression profiles of this invention are used to provide diagnosis, status, prognosis and treatment protocol for lung cancer patients.

Sample preparation requires the collection of patient samples. Patient samples used in the inventive method are those that are suspected of containing diseased cells such as cells taken from a nodule in a fine needle aspirate (FNA) of tissue. Bulk tissue preparation obtained from a biopsy or a surgical specimen and Laser Capture Microdissection (LCM) are also suitable for use. LCM technology is one way to select the cells to be studied, minimizing variability caused by cell type heterogeneity. Consequently, moderate or small changes in Marker gene expression between normal or benign and cancerous cells can be readily detected. Samples can also comprise circulating epithelial cells extracted from peripheral blood. These can be obtained according to a number of methods but the most preferred method is the magnetic separation technique described in U.S. Pat. No. 6,136,182. Once the sample containing the cells of interest has been obtained, a gene expression profile is obtained using a Biomarker, for genes in the appropriate portfolios.

Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcriptase PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in U.S. Patents such as: U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.

Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying effects such as the onset, arrest, or modulation of uncontrolled cell proliferation. Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells. A large number of such techniques are available and useful. Preferred methods for determining gene expression can be found in U.S. Pat. Nos. 6,271,002; 6,218,122; 6,218,114; and 6,004,755.

Analysis of the expression levels is conducted by comparing such signal intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a diseased tissue can be compared with the expression intensities generated from benign or normal tissue of the same type. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.

Gene expression profiles can also be displayed in a number of ways. The most common method is to arrange raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum. Commercially available computer software programs are available to display such data including “GENESPRING” from Silicon Genetics, Inc. and “DISCOVERY” and “INFER” software from Partek, Inc.

In the case of measuring protein levels to determine gene expression, any method known in the art is suitable provided it results in adequate specificity and sensitivity. For example, protein levels can be measured by binding to an antibody or antibody fragment specific for the protein and measuring the amount of antibody-bound protein. Antibodies can be labeled by radioactive, fluorescent or other detectable reagents to facilitate detection. Methods of detection include, without limitation, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques.

Modulated Markers used in the methods of the invention are described in the Examples. The genes that are differentially expressed are either up regulated or down regulated in patients with various lung cancer prognostics. Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline. In this case, the baseline is determined based on the algorithm. The genes of interest in the diseased cells are then either up- or down-regulated relative to the baseline level using the same measurement method.

Diseased, in this context, refers to an alteration of the state of a body that interrupts or disturbs, or has the potential to disturb, proper performance of bodily functions as occurs with the uncontrolled proliferation of cells. Someone is diagnosed with a disease when some aspect of that person's genotype or phenotype is consistent with the presence of the disease. However, the act of conducting a diagnosis or prognosis may include the determination of disease/status issues such as determining the likelihood of relapse, type of therapy and therapy monitoring. In therapy monitoring, clinical judgments are made regarding the effect of a given course of therapy by comparing the expression of genes over time to determine whether the gene expression profiles have changed or are changing to patterns more consistent with normal tissue.

Genes can be grouped so that information obtained about the set of genes in the group provides a sound basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice. These sets of genes make up the portfolios of the invention. As with most diagnostic markers, it is often desirable to use the fewest number of markers sufficient to make a correct medical judgment. This prevents a delay in treatment pending further analysis as well unproductive use of time and resources.

One method of establishing gene expression portfolios is through the use of optimization algorithms such as the mean variance algorithm widely used in establishing stock portfolios. This method is described in detail in US patent publication number 20030194734. Essentially, the method calls for the establishment of a set of inputs (stocks in financial applications, expression as measured by intensity here) that will optimize the return (e.g., signal that is generated) one receives for using it while minimizing the variability of the return. Many commercial software programs are available to conduct such operations. “Wagner Associates Mean-Variance Optimization Application,” referred to as “Wagner Software” throughout this specification, is preferred. This software uses functions from the “Wagner Associates Mean-Variance Optimization Library” to determine an efficient frontier and optimal portfolios in the Markowitz sense is one option. Use of this type of software requires that microarray data be transformed so that it can be treated as an input in the way stock return and risk measurements are used when the software is used for its intended financial analysis purposes.

The process of selecting a portfolio can also include the application of heuristic rules. Preferably, such rules are formulated based on biology and an understanding of the technology used to produce clinical results. More preferably, they are applied to output from the optimization method. For example, the mean variance method of portfolio selection can be applied to microarray data for a number of genes differentially expressed in subjects with cancer. Output from the method would be an optimized set of genes that could include some genes that are expressed in peripheral blood as well as in diseased tissue. If samples used in the testing method are obtained from peripheral blood and certain genes differentially expressed in instances of cancer could also be differentially expressed in peripheral blood, then a heuristic rule can be applied in which a portfolio is selected from the efficient frontier excluding those that are differentially expressed in peripheral blood. Of course, the rule can be applied prior to the formation of the efficient frontier by, for example, applying the rule during data pre-selection.

Other heuristic rules can be applied that are not necessarily related to the biology in question. For example, one can apply a rule that only a prescribed percentage of the portfolio can be represented by a particular gene or group of genes. Commercially available software such as the Wagner Software readily accommodates these types of heuristics. This can be useful, for example, when factors other than accuracy and precision (e.g., anticipated licensing fees) have an impact on the desirability of including one or more genes.

The gene expression profiles of this invention can also be used in conjunction with other non-genetic diagnostic methods useful in cancer diagnosis, prognosis, or treatment monitoring. For example, in some circumstances it is beneficial to combine the diagnostic power of the gene expression based methods described above with data from conventional markers such as serum protein markers (e.g., Cancer Antigen 27.29 (“CA 27.29”)). A range of such markers exists including such analytes as CA 27.29. In one such method, blood is periodically taken from a treated patient and then subjected to an enzyme immunoassay for one of the serum markers described above. When the concentration of the marker suggests the return of tumors or failure of therapy, a sample source amenable to gene expression analysis is taken. Where a suspicious mass exists, a fine needle aspirate (FNA) is taken and gene expression profiles of cells taken from the mass are then analyzed as described above. Alternatively, tissue samples may be taken from areas adjacent to the tissue from which a tumor was previously removed. This approach can be particularly useful when other testing produces ambiguous results.

Kits made according to the invention include formatted assays for determining the gene expression profiles. These can include all or some of the materials needed to conduct the assays such as reagents and instructions and a medium through which Biomarkers are assayed.

Articles of this invention include representations of the gene expression profiles useful for treating, diagnosing, prognosticating, and otherwise assessing diseases. These profile representations are reduced to a medium that can be automatically read by a machine such as computer readable media (magnetic, optical, and the like). The articles can also include instructions for assessing the gene expression profiles in such media. For example, the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above. The articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format. Clustering algorithms such as those incorporated in “DISCOVERY” and “INFER” software from Partek, Inc. mentioned above can best assist in the visualization of such data.

Different types of articles of manufacture according to the invention are media or formatted assays used to reveal gene expression profiles. These can comprise, for example, microarrays in which sequence complements or probes are affixed to a matrix to which the sequences indicative of the genes of interest combine creating a readable determinant of their presence. Alternatively, articles according to the invention can be fashioned into reagent kits for conducting hybridization, amplification, and signal generation indicative of the level of expression of the genes of interest for detecting cancer.

The invention is further illustrated by the following non-limiting examples. All references cited herein are hereby incorporated herein.

EXAMPLES

Genes analyzed according to this invention are typically related to full-length nucleic acid sequences that code for the production of a protein or peptide. One skilled in the art will recognize that identification of full-length sequences is not necessary from an analytical point of view. That is, portions of the sequences or ESTs can be selected according to well-known principles for which probes can be designed to assess gene expression for the corresponding gene.

Example 1

Methods

Patient Population

134 fresh frozen, surgically resected lung SCC and 10 matched normal lung samples from 133 individual patients (LS-71 and LS-136 were duplicate samples from different areas of the same tumor) from all stages of squamous cell lung carcinoma were evaluated in this study. These samples were collected from patients from the University of Michigan Hospital between October 1991 and July 2002 with patient consent and Institutional Review Board (IRB) approval. Portions of the resected lung carcinomas were sectioned and evaluated by the study pathologist by routine hematoxylin and eosin (H&E) staining. Samples chosen for analysis contained greater than 70% tumor cells. Approximately one third of patients (with equal proportions for each stage) received radiotherapy or chemotherapy following surgery. Seventy-seven patients were lymph node negative. Follow-up data were available for all patients. The mean patient age was 68±10 (range 42-91) with approximately 45% of patients 70 years or older. One patient (LS-3) likely died of surgery-related causes and was therefore not utilized in identifying prognostic signatures. Also, three specimens had mixed histology and were also not included in prognostic profiling (LS-76, LS-84, LS-112).

Microarray Analysis

For isolation of RNA, 20 to 40 cryostat sections of 30 μm were cut from each sample, in total corresponding to approximately 100 mg of tissue. Before, in between, and after cutting the sections for RNA isolation, 5 μm sections were cut for hematoxylin and eosin staining to confirm the presence of tumor cells. Total RNA was isolated with RNAzol B (Campro Scientific, Veenendaal, Netherlands), and dissolved in DEPC (0.1%)-treated H₂O. About 2 ng of total RNA was resuspended in 10 μl of water and 2 rounds of the T7 RNA polymerase based amplification were performed to yield about 50 μg of amplified RNA. Quality of RNA was checked using the Agilent Bioanalyzer. The mean ribosomal ratio (28s/18s) for all samples was 1.5 (range: 1.0-2.1). Four micrograms of total RNA was amplified, labeled and aRNA was fragmented and hybridized to the Affymetrix U133A chip according to the manufacturer's instructions. Microarray data were extracted using the Affymetrix MAS 5 software. Global gene expression was scaled to an average intensity of 600 units. The data were then normalized using a spline quantile normalization method.

Statistical Analysis

Three complimentary statistical methods were performed to identify the optimal prognostic gene signature: Cox proportional-hazard regression modeling, bootstrapping, and a leave 20 percent out cross validation (L20OCV).

Univariate Cox proportional-hazard regression modeling was performed to identify genes that were significantly associated with overall survival. The Cox score was defined as the sum of the selected gene's log2-based chip signals multiplied by their z scores from the Cox regression. Similarly, Cox scores were calculated for patients in the testing set with the same selected genes from the training set. A series of cutoffs (percentile of risk index for the patients in the training set) was applied to predict the clinical outcome of patients in the testing set by comparing the patients° Cox score in the testing set with a cutoff for the risk index. If a patient's Cox score was higher than the cutoff, the patient was classified as “high risk”, otherwise, it is put in the “low risk” group. Kaplan-Meier analysis was performed to explore the survival characteristics of high-risk and low-risk patients. A cutoff of 3-year survival was employed since the majority of patients who will relapse in this population will have this occur within 3 years. Kiernan et al. (1993). Also many of these patients die due to non-cancer related illnesses after 3 years. Kiernan et al. (1993). This rationale was also employed when performing Cox modeling.

The bootstrap method was also employed to provide a more stringent means of defining prognostic genes. Using the same training and testing sets created above, 65 samples were selected, with replacement from the training set, and then Cox regression was performed on these samples. Each gene's P value and z score were recorded. This step was repeated 400 times thus giving 400 P values and z scores for each gene. For each gene, the top and bottom 5% of P values were removed and then the mean P value and the rank of each gene (based on the mean P value) were defined. Similarly, the top and bottom 5% z scores for each gene in the training set were removed and the sum of the remaining ones was calculated. Various numbers of top genes based on the mean P value were defined, their log2-based chip signal were multiplied with the sum of their z scores. This equated their Cox scores, namely, the risk index. The patients' Cox scores in the testing set was also calculated in this manner. Receiver operator characteristic (ROC) curves were drawn for patients in the training and testing sets and the area under the curve (AUC) values for each gene classifier was recorded. The AUC values were then plotted versus various numbers of gene classifiers to determine the optimal gene number that provides steady AUC values in the training set.

A L20OCV was also performed to confirm the optimal gene number of the classifier. First samples were partitioned into 5 groups with the same or very close numbers of samples. Five pairs of training and testing sets was generated with the training set consisting of 80% of samples and the testing set consisting of the remaining 20%. Therefore each sample was chosen exactly once in a testing set. Cox regression modeling was performed to select the top prognostic genes (from 2 to 200) in the training set and the selected genes were tested in the corresponding testing set. ROC was performed to calculate the AUC. The mean AUC of the 5 testing sets for gene number from 2 to 200 was calculated. This was repeated 100 times and the mean of 100 AUC's for gene numbers from 2 to 200 was then calculated. The mean AUC versus gene number (2 to 200) was plotted and the optimal number of genes in the signature was selected.

Hierarchical clustering was performed with GeneSpring7.0 (Silicon Genetics) to identify major clusters of patients and investigate their association with patient co-variates. Prior to clustering genes that had a coefficient of variation (CV) smaller than 0.3 (arbitrarily chosen) were removed so as to reduce the impact of genes that displayed minimal change in expression across the dataset. Thus a dataset with 11,101 genes was created for clustering analysis. The signal intensity of each gene was divided by the median expression level of that gene from all patients. Samples were clustered using Pearson correlation as measurement of similarity. Genes were clustered in the same way.

Results

Microarray Profiling

141 of the 144 microarrays gave excellent data (% present>40, scaling factor<10) while the remaining 3 samples (LS76, LS78, LS82) gave acceptable results (% present>30, scaling factor<15). Table 2 shows the clinical-pathological staging of the 134 SCC samples analyzed by microarray. All samples were included in initial clustering analysis. Genes were filtered from the dataset if they were not called present in at least 10% of all samples (including normal). This left 14,597 genes for analysis.

TABLE 2
Patient samples by stage
	Clinical	Number	Pathological
	Stage	(%)	Stage	Number
	1a	28 (20)	T1 N0 M0	27
	1b	50 (35)	T2 N0 M0	48
	IIA	7 (5)	T1 N1 M0	6
	IIB	31 (22)	T1 N1 M0	30
	IIIA	19 (14)	T2 N2 M0	10
			T3 N0 M0	1
			T3 N1 M0	3
			T3 N2 M0	4
	IIIB	5 (4)	T4 N0 M0	1
			T4 N1 M0	3
			T4 N2 M0	1
	Note.
	One duplicate stage IIb, 77 lymph node negative samples

Unsupervised Hierarchical Clustering

For unsupervised clustering the dataset was further filtered by removing genes (CV<30%) that had low variation of expression across the entire dataset. The 134 SCC and 10 normal lung samples were initially clustered based on unsupervised k-means clustering of the remaining 11,101 genes. The normal lung samples had a distinct profile from the carcinomas and clustered together. The 2 duplicate SCC samples (LS-71 and LS-136) clustered together demonstrating the reproducibility of the microarray analysis. Of the 133 unique patient carcinomas four were removed from further analysis since the patient either died due to surgery (LS3) or the sample had mixed histology (LS-76, LS-84, LS-112). When the 129 samples were clustered using the 11,101 genes two major clusters were formed, one with 55 patients and the other with 74 patients (FIG. 1A). No significant association between tumor stage, differentiation, or patient gender and the two clusters was identified. There were approximately equal proportions of each stage present in both clusters (cluster I consists of 31 stage I, 15 stage II and 9 stage III patients; cluster 2 consists of 42 stage I, 18 stage II and 14 stage III patients). However, the patients in cluster I and 2 showed significantly separated survival curves (FIG. 1B, p=0.036), indicating that expression profiles, irrespective of stage, existed that were associated with overall survival (FIG. 1B).

Identification of Prognostic Gene Signatures

To identify genes that could further stratify early stage patients into good and poor prognostic groups several complimentary statistical analyses were performed. This included: 1) Cox modeling on a training set and validating prognostic signatures on a test set of samples; 2) bootstrapping; and 3) L20OCV.

First, the 129 SCC samples were split into training and test sets with equal number of stages represented in both groups. Both groups showed similar overall median survival times. The 65-patient training set was analyzed using a bootstrapping method (see Methods section) to determine the optimal number of genes to be used in the prognostic signature. When increasing numbers of genes was plotted versus the AUC from a receiver operator characteristic analysis it could be seen that the signature performance began to plateau at around 50 genes (FIG. 2A). A L20OCV procedure was used to confirm the optimal number of prognostic genes in the 65-patient training set. The result showed that a signature has a stable performance when the number of genes reaches 50. Therefore, the top ranked 50 genes would be used as the signature. The 50-gene classifier demonstrated overall predictive value of 70% when used in the 64-patient test set (FIG. 2B).

A LOOCV procedure was then used in the 65-patient training set to determine the optimal cutoff of the risk index. The error rates were calculated with various cutoffs. This indicated that cutoff at 58%ile gave the lowest error rate (FIG. 3). Therefore, the 58% ile of patients was used as the cutoff for determining survival. The performance of the prognostic signature was then examined in the testing set using this cutoff. The signature achieved 52.4% specificity and 81.8% sensitivity in the testing set (FIG. 3). Kaplan-Meier plot also showed good separation between predicted high-risk group of patients and low risk group of patients (p=0.0075). Multivariate analysis including sex, differentiation, stage, tumor size, age, and lymph node status was performed. None of the parameters except for the 50-gene signature had a significant p-value (Table 3). Kaplan-Meier analysis was also performed using the 50-gene signature and a risk cutoff of 58%. The high-risk group was well separated from the low risk group in all patients (p=0.0075, FIG. 4A) and when only those with stage 1 disease were tested (p 0.029; FIG. 4B).

TABLE 3
Multivariate Analysis
Co-variate        P-value
50 gene signature 0.01
Sex               0.24
Differentiation   0.66
Stage             0.41
T                 0.91
Age               0.35
N                 0.99

Example 2

Identification of a Robust Prognostic Signature

Although we used a bootstrap method to avoid random sampling issues in the training-testing method, a more robust prognostic signature might be identified if we use all 129 samples in the training set. Therefore, a gene signature was also selected by bootstrapping the entire 129-patient dataset. Genes were ranked based on their mean P value and the top 100 genes were identified (Table 4). Twenty-three of these genes were in common with the top 50 genes identified from the training-test method.

We had data on time to relapse (TTR) for 16 patients. The mean TTR was 21.7 months with 88% of patients relapsing within 3 years. Since the majority of patients who die after 3 years die from non-cancer related causes we chose a cutoff of 36 months for classifying patients who will have a lung cancer-related death. Our defined classifiers were tested with or without a 36-month cutoff. The signatures had a better performance in the testing set when a 3-year cutoff was employed. Therefore, a gene signature selected with the time limit is better than without the time limit.

TABLE 4
SEQ ID NO: Rank
452        1
191        2
303        3
378        4
270        5
79         6
409        7
76         8
450        9
413        10
365        11
135        12
18         13
460        14
393        15
375        16
396        17
86         18
190        19
204        20
65         21
433        22
439        23
471        24
124        25
107        26
77         27
13         28
461        29
91         30
225        31
290        32
252        33
194        34
21         35
206        36
161        37
36         38
207        39
37         40
315        41
87         42
288        43
369        44
235        45
337        46
383        47
228        48
248        49
423        50
200        51
234        52
58         53
386        54
120        55
305        56
302        57
16         58
432        59
381        60
269        61
75         62
209        63
293        64
20         65
83         66
408        67
388        68
443        69
372        70
286        71
289        72
57         73
215        74
144        75
89         76
158        77
149        78
98         79
29         80
35         81
311        82
310        83
279        84
384        85
298        86
48         87
222        88
425        89
56         90
398        91
453        92
470        93
261        94
462        95
162        96
131        97
284        98
326        99
114        100

Example 3

Identification of a High-Risk Sub-Group of SCC Patients

The unsupervised hierarchical clustering described above identified two main groups of patients that differed significantly in their overall survival. A bootstrap analysis performed on the two patient groups found 121 genes (non-unique) whose expression levels were significantly different between the high- and low-risk groups (p <0.001, mean difference>3-fold; Table 5). Interestingly, the majority of these genes (118) were down-regulated in the high risk group (FIG. 5A, cluster 1). Pathway analysis demonstrated that genes involved in epidermal development functions, including keratins and small-proline rich proteins, were significantly enriched for in this dataset. These data, shown in Table 6, indicate that there are two major subtypes of SCC one of which has a gene expression profile consistent with poor differentiation and as such tends to be more aggressive. When the genes only involved in epidermal differentiation (FIG. 5B) were used to cluster the patient samples the two prognostically differentiated groups were maintained (FIG. 5C). These data indicate that there are two major subtypes of SCC one of which has a gene expression profile consistent with poor differentiation and as such tends to be more aggressive. The lack of expression of epidermal differentiation genes may be associated with a subgroup of tumors that are de-differentiated and therefore more aggressive.

TABLE 5
121 genes significantly different between low- and high-risk clusters
           Dunn-Sidak p-
SEQ ID NO: value
47         4.069E−08
52         0.001779787
61         4.78438E−06
64         3.94295E−08
70         6.14897E−11
71         5.40462E−10
72         4.99526E−07
91         1.17801E−09
92         0
93         1.51307E−07
94         0.00024053
97         3.25762E−06
101        0.000715044
102        4.042E−05
105        1.28648E−05
111        4.10746E−07
112        0.000129644
115        7.6587E−08
118        4.67009E−05
121        7.48718E−09
123        1.61815E−11
125        4.82759E−08
126        1.80901E−05
128        1.45634E−11
132        0.000571137
134        3.42792E−07
138        2.83176E−10
140        4.93018E−08
141        9.06164E−11
142        1.73482E−08
145        0
146        8.6277E−05
148        1.68459E−07
156        8.93603E−05
159        0
160        7.24383E−06
166        4.46788E−05
167        1.61815E−12
168        3.2363E−12
170        5.27808E−08
171        0
172        0
173        0
174        0
175        3.70691E−07
177        0.000964585
179        0.00023307
181        2.10853E−07
184        0.000261
185        1.22494E−09
186        0
188        8.3147E−08
192        0
193        1.33552E−06
194        0
195        8.04368E−07
196        0
198        1.78886E−07
213        0
214        0
216        1.77997E−11
219        1.44447E−07
223        6.79057E−08
229        2.21201E−09
231        0.000127662
232        0.000670091
233        0.000334014
236        0.000371339
237        5.35608E−10
238        0
243        0
245        1.5392E−07
246        3.77172E−06
251        9.51746E−06
253        1.61815E−12
257        7.19348E−07
259        3.2363E−12
260        0
262        0
263        1.61815E−12
278        3.2363E−12
285        3.95638E−09
313        3.06803E−07
318        0
320        1.10983E−05
321        2.86717E−06
322        0
323        1.46054E−05
324        2.65922E−05
331        0
332        1.77997E−10
333        0
341        3.60669E−08
348        0.001219264
349        4.42435E−08
353        0
357        9.21286E−05
358        2.91267E−09
360        1.67317E−09
366        0
367        1.06791E−07
371        0
373        0.000736609
397        1.53724E−10
402        0.001640004
405        1.89887E−05
407        0
418        7.28168E−11
419        1.13076E−08
424        2.83902E−05
426        0.001696015
429        2.33385E−05
435        2.53251E−06
445        8.59804E−08
457        0
458        0
459        0
463        9.60372E−09
468        4.52017E−06

TABLE 6
List of significantly enriched pathways
				GO.
	Gene.		Gene.#.On	Cate-
GO.ID	Count	GO.Class	.U133a	gory	p.value
8544	17	epidermal	56	P	7.31E−12
		differentiation
6325	3	chromatin architecture	12	P	2.75E−04
7586	3	digestion	15	P	7.08E−04
7156	4	homophilic cell	39	P	0.004886
		adhesion
7148	3	cell shape and cell	28	P	0.007914
		size control
7565	3	pregnancy	28	P	0.007914
165	2	MAPKKKcascade	15	P	0.008242
6805	2	xenobiotic metabolism	15	P	0.008242
7169	3	receptor tyrosine	41	P	0.029293
		kinase signaling
6832	2	small molecule	29	P	0.049333
		transport

Example 4

Gene Expression Signatures for Prognosis of Lung Cancer.

Methods

Real-Time Quantitative RT-PCR

Total RNA samples were normalized by OD₂₆₀. Quality testing included analysis by capillary electrophoresis using a Bioanalyzer (Agilent). For aRNA, the Ribobeast™ 1-Round Aminoallyl-aRNA amplification kit (Epicentre) was used. All first-strand cDNA synthesis, second-strand cDNA synthesis, in vitro transcription of aRNA, DNase treatment, purification and other steps were performed according to the manufacturer's protocol. For each sample aRNA was reverse transcribed into first-stand cDNA and used for real-time quantitative RT-PCR. The first-strand cDNA synthesis reaction contained, 100 ng of aRNA, 1 μl of 50 ng/μl T7-Oligo(dT) primer, 0.25 μl of 10 mM dNTPs, 1 μl of 5× Superscript™ III Reverse Transcriptase Buffer, 0.25 μl of 200 U/μl Superscript™ III Reverse Transcriptase (Invitrogen Corp), 0.25 μl of 100 mM DTT and 0.25 μl of 0.3 U/μl RNase Inhibitor (Epicentre) in a total reaction volume of 5 μl.

Teal-time quantitative RT-PCR analyses were performed on the ABI Prism 7900HT sequence detection system (Applied Biosystems). Each reaction contained 10 μl of 2× TaqMan® Universal PCR Master Mix (Applied Biosystems), 5 μl of cDNA template, and 1 μl of 20× Assays-on-Demand Gene Expression Assay Mix (Applied Biosystems) in a total reaction volume of 20 μl. The PCR consisted of an UNG activation step at 50° C. for 2 min and initial enzyme activation step at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 sec, 60° C. for 1 min.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on tissue microarrays containing 60 lung squamous cell carcinomas. Areas of the tumor that best represented the overall morphology were selected for generating a tissue microarray (TMA) block as previously described by Kononen et al. (1998). All controls stained negative for background.

Pathway Analysis

Pathway analysis was performed by first mapping the genes on the Affy U133A chip to the Biological Process categories of Gene Ontology (GO). The categories that had at least 10 genes on the U133A chip were used for subsequent pathway analyses. Genes that were selected from data analysis were mapped to the GO Biological Process categories. Then the hypergeometric distribution probability of the genes was calculated for each category. A category that had a p-value less than 0.05 and had at least two genes was considered over-represented in the selected gene list.

Identification of Core Set of Prognostic Genes

Briefly, 400 random training sets of 65 patients were selected from the 129 lung SCC patients. For each training set, Cox regression was performed to identify significant genes at the 5% significance level (i.e. P<0.05). 331 genes that are significant in more than 40% of the training sets are used as the core gene sets. These 331 genes are shown in Table 7.

Microarray Results Verification

To confirm the microarray results we initially performed TaqMan® quantitative RT-PCR on4 genes (FGFR2, KRT13, NTRK2, and VEGF). The correlation between the platforms ranged from 0.71 to 0.96 indicating the expression data were reproducible.

Immunohistochemistry was then performed on tissue microarrays to confirm expression of several of these proteins within the tumor cells. Various levels of expression of several keratins in addition to the tyrosine kinase proteins FGFR2 and NTKR2 in SCC cells was demonstrated.

Identification of a Core Set of Prognostic Genes

In the previous analysis a set of 50 genes was identified from a single training set of 65 patients. One problem with this approach is that the genes identified as predictors of prognosis can be unstable since the molecular signature strongly depends on the selection of patients in the training sets. The use of validation by repeated random sampling can avoid this instability. We therefore generated 400 random training sets of 65 patients from the 129 lung SCC patients and performed Cox regression to identify significant genes at the 5% significance level (i.e. P<0.05). 331 genes that were significant in more than 40% of the training sets were identified as a core set of prognostic genes in squamous cell lung cancer. These genes are SEQ ID NOs: in Table 7.

TABLE 7
331 Core genes
1	2	3	5	6	7	8	9	11
13	14	15	16	17	18	20	21	22
23	24	25	26	27	28	29	30	31
32	33	34	35	36	37	38	39	40
41	42	43	44	45	46	48	49	50
51	54	55	56	57	58	59	62	65
66	67	68	69	73	74	75	76	77
79	80	81	82	83	84	85	86	87
88	89	90	91	92	95	96	98	99
100	104	106	107	108	109	110	113	114
116	117	119	120	122	124	127	129	130
133	134	135	136	137	139	141	143	147
149	150	151	152	153	154	155	157	159
161	163	164	165	166	169	176	178	180
182	183	187	190	191	194	197	199	200
201	202	203	204	205	206	207	208	209
210	211	212	215	217	218	220	222	224
225	226	227	228	234	235	239	240	241
242	244	247	248	249	250	252	254	255
256	258	261	263	264	265	266	269	270
271	272	274	275	276	282	283	284	286
288	289	290	291	292	293	294	295	296
297	298	299	300	301	302	303	304	305
306	307	308	309	310	311	312	314	315
316	317	319	325	327	328	329	330	334
335	336	337	338	339	340	342	343	344
345	346	347	350	351	352	354	355	356
359	361	363	364	365	368	369	370	372
374	375	376	377	378	379	380	381	382
383	384	385	386	387	388	389	390	391
392	393	394	395	396	398	399	400	401
403	404	406	409	410	411	412	413	415
417	420	421	422	423	425	427	428	430
431	432	433	434	436	437	438	439	441
442	443	444	447	448	449	450	451	452
453	454	455	456	460	461	462	464	465
466	467	469	470	471	472	473

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

TABLE 8
SEQ ID NOs: and gene descriptions
1	1255_g_at	guanylate cyclase activator 1A (retina)	GUCA1A	L36861
2	200619_at	splicing factor 3b, subunit 2	SF3B2	NM_006842
3	200650_s_at	lactate dehydrogenase A	LDHA	NM_005566
4	200727_s_at	ARP2 actin-related protein 2 homolog	ACTR2	AA699583
5	200728_at	ARP2 actin-related protein 2 homolog	ACTR2	BE566290
6	200737_at	phosphoglycerate kinase 1	PGK1	NM_000291
7	200795_at	SPARC-like 1 (mast9, hevin)	SPARCL1	NM_004684
8	200810_s_at	cold inducible RNA binding protein	CIRBP	NM_001280
9	200811_at	cold inducible RNA binding protein	CIRBP	NM_001280
10	200824_at	glutathione S-transferase pi	GSTP1	NM_000852
11	200836_s_at	microtubule-associated protein 4	MAP4	NM_002375
12	200840_at	lysyl-tRNA synthetase	KARS	NM_005548
13	200863_s_at	RAB11A, member RAS oncogene family	RAB11A	AI215102
14	200893_at	splicing factor, arginine/serine-rich 10	SFRS10	NM_004593
15	200951_s_at	cyclin D2	CCND2	AW026491
16	200970_s_at	stress-associated endoplasmic reticulum protein 1	SERP1	AL136807
17	200993_at	importin 7	IPO7	AA939270
18	201003_x_at	ubiquitin-conjugating enzyme E2 variant 1	UBE2V1	NM_003349
19	201033_x_at	ribosomal protein, large, P0	RPLP0	NM_001002
20	201047_x_at	RAB6A, member RAS oncogene family	RAB6A	BC003617
21	201067_at	proteasome (prosome, macropain) 26S subunit,	PSMC2	BF215487
		ATPase, 2
22	201125_s_at	integrin, beta 5	ITGB5	NM_002213
23	201151_s_at	muscleblind-like	MBNL1	BF512200
24	201152_s_at	muscleblind-like	MBNL1	N31913
25	201154_x_at	ribosomal protein L4	RPL4	NM_000968
26	201170_s_at	basic helix-loop-helix domain containing, class B, 2	BHLHB2	NM_003670
27	201175_at	thioredoxin-related transmembrane protein 2	TMX2	NM_015959
28	201236_s_at	BTG family, member 2	BTG2	NM_006763
29	201251_at	pyruvate kinase, muscle	PKM2	NM_002654
30	201286_at	syndecan 1	SDC1	Z48199
31	201287_s_at	syndecan 1	SDC1	NM_002997
32	201351_s_at	YME1-like 1	YME1L1	AF070656
33	201353_s_at	bromodomain adjacent to zinc finger domain, 2A	BAZ2A	AI653126
34	201361_at	hypothetical protein MGC5508	MGC5508	NM_024092
35	201447_at	TIA1 cytotoxic granule-associated RNA binding	TIA1	H96549
36	201448_at	TIA1 cytotoxic granule-associated RNA binding	TIA1	AL046419
		transcript variant 1
37	201449_at	TIA1 cytotoxic granule-associated RNA binding	TIA1	AL567227
		transcript variant 1
38	201545_s_at	poly(A) binding protein, nuclear 1	PABPN1	NM_004643
39	201623_s_at	aspartyl-tRNA synthetase	DARS	BC000629
40	201667_at	gap junction protein, alpha 1	GJA1	NM_000165
41	201683_x_at	chromosome 14 open reading frame 92	C14orf92	BE783632
42	201718_s_at	erythrocyte membrane protein band 4.1-like 2	EPB41L2	BF511685
43	201725_at	chromosome 10 open reading frame 7	C10orf7	NM_006023
44	201779_s_at	ring finger protein 13	RNF13	AF070558
45	201780_s_at	ring finger protein 13	RNF13	NM_007282
46	201801_s_at	solute carrier family 29 (nucleoside transporters),	SLC29A1	AF079117
		mem 1
47	201820_at	keratin 5	KRT5	NM_000424
48	201892_s_at	IMP (inosine monophosphate) dehydrogenase 2	IMPDH2	NM_000884
49	202006_at	protein tyrosine phosphatase, non-receptor type 12	PTPN12	NM_002835
50	202170_s_at	aminoadipate-semialdehyde dehydrogenase-	AASDHPPT	AF151057
		phosphopantetheinyl transferase
51	202181_at	KIAA0247	KIAA0247	NM_014734
52	202219_at	solute carrier family 6, member 8	SLC6A8	NM_005629
53	202223_at	integral membrane protein 1	ITM1	NM_002219
54	202253_s_at	dynamin 2	DNM2	NM_004945
55	202288_at	FK506 binding protein 12-rapamycin assoc. pro 1	FRAP1	U88966
56	202349_at	torsin family 1, member A (torsin A)	TOR1A	NM_000113
57	202364_at	MAX interactor 1	MXI1	NM_005962
58	202397_at	nuclear transport factor 2	NUTF2	NM_005796
59	202418_at	Yip1 interacting factor homolog	YIF1	NM_020470
60	202471_s_at	isocitrate dehydrogenase 3 (NAD+) gamma	IDH3G	NM_004135
61	202489_s_at	FXYD domain-containing ion transport regulator 3	FXYD3	BC005238
62	202496_at	autoantigen	RCD-8	NM_014329
63	202503_s_at	KIAA0101 gene product	KIAA0101	NM_014736
64	202504_at	ataxia-telangiectasia group D-associated protein	TRIM29	NM_012101
65	202530_at	mitogen-activated protein kinase 14	MAPK14	NM_001315
66	202602_s_at	HIV TAT specific factor 1	HTATSF1	NM_014500
67	202746_at	integral membrane protein 2A	ITM2A	AL021786
68	202747_s_at	integral membrane protein 2A	ITM2A	NM_004867
69	202753_at	proteasome regulatory particle subunit p44S10	P44S10	NM_014814
70	202755_s_at	glypican 1	GPC1	AI354864
71	202756_s_at	glypican 1	GPC1	NM_002081
72	202831_at	glutathione peroxidase 2	GPX2	NM_002083
73	202887_s_at	DNA-damage-inducible transcript 4	DDIT4	NM_019058
74	202935_s_at	SRY-box 9	SOX9	AI382146
75	202990_at	phosphorylase, glycogen; liver	PYGL	NM_002863
76	203040_s_at	hydroxymethylbilane synthase	HMBS	NM_000190
77	203082_at	BMS1-like, ribosome assembly protein (yeast)	BMS1L	NM_014753
78	203190_at	NADH dehydrogenase (ubiquinone) Fe—S protein 8	NDUFS8	NM_002496
79	203196_at	ATP-binding cassette, sub-fam C (CFTR/MRP),	ABCC4	AI948503
		mem 4
80	203211_s_at	myotubularin related protein 2	MTMR2	AK027038
81	203368_at	cysteine-rich with EGF-like domains 1	CRELD1	NM_015513
82	203372_s_at	suppressor of cytokine signaling 2	SOCS2	AB004903
83	203378_at	pre-mRNA cleavage complex II protein Pcf11	PCF11	AB020631
84	203491_s_at	translokin	PIG8	AI123527
85	203494_s_at	translokin	PIG8	NM_014679
86	203545_at	asparagine-linked glycosylation 8 homolog	ALG8	NM_024079
87	203555_at	protein tyrosine phosphatase, non-receptor type 18	PTPN18	NM_014369
88	203573_s_at	Rab geranylgeranyltransferase, alpha subunit	RABGGTA	NM_004581
89	203589_s_at	transcription factor Dp-2	TFDP2	NM_006286
90	203611_at	telomeric repeat binding factor 2	TERF2	NM_005652
91	203638_s_at	fibroblast growth factor receptor 2	FGFR2	NM_022969
92	203639_s_at	fibroblast growth factor receptor 2	FGFR2	M80634
93	203691_at	protease inhibitor 3, skin-derived	PI3	NM_002638
94	203726_s_at	laminin, alpha 3	LAMA3	NM_000227
95	203759_at	ST3 beta-galactoside alpha-2,3-sialyltransferase 4	ST3GAL4	NM_006278
96	203787_at	single-stranded DNA binding protein 2	SSBP2	NM_012446
97	203798_s_at	visinin-like 1	VSNL1	NM_003385
98	203809_s_at	v-akt murine thymoma viral oncogene homolog 2	AKT2	AA769075
99	203853_s_at	GRB2-associated binding protein 2	GAB2	NM_012296
100	203885_at	RAB21, member RAS oncogene family	RAB21	NM_014999
101	203924_at	glutathione S-transferase A2	GSTA1	NM_000846
102	203953_s_at	Claudin 3	CLDN3	BE791251
103	203964_at	N-myc (and STAT) interactor	NMI	NM_004688
104	203974_at	haloacid dehalogenase-like hydrolase domain	HDHD1A	NM_012080
		containing 1A
105	204014_at	dual specificity phosphatase 4	DUSP4	NM_001394
106	204036_at	endothelial differentiation, lysophosphatidic acid	EDG2	AW269335
		G-protein-coupled receptor, 2
107	204037_at		EDG2	BF055366
108	204038_s_at		EDG2	NM_001401
109	204047_s_at	phosphatase and actin regulator 2	PHACTR2	AW295193
110	204049_s_at		PHACTR2	NM_014721
111	204136_at	collagen, type VII, alpha 1	COL7A1	NM_000094
112	204151_x_at	aldo-keto reductase family 1, member C1	AKR1C1	NM_001353
113	204154_at	cysteine dioxygenase, type I	CDO1	NM_001801
114	204206_at	MAX binding protein	MNT	NM_020310
115	204268_at	S100 calcium-binding protein A2	S100A2	NM_005978
116	204326_x_at	metallothionein 1X	MT1X	NM_002450
117	204367_at	Sp2 transcription factor	SP2	D28588
118	204379_s_at	fibroblast growth factor receptor 3	FGFR3	NM_000142
119	204385_at	kynureninase (L-kynurenine hydrolase)	KYNU	NM_003937
120	204388_s_at	monoamine oxidase A	MAOA	NM_000240
121	204455_at	bullous pemphigoid antigen 1	BPAG1	NM_001723
122	204460_s_at	RAD1 homolog	RAD1	AF074717
123	204469_at	protein tyrosine phosphatase, receptor-type, Z	PTPRZ1	NM_002851
		polypep 1
124	204493_at	BH3 interacting domain death agonist	BID	NM_001196
125	204532_x_at	UDP glycosyltransferase 1 family, polypep A9	UGT1A9	NM_021027
126	204542_at	sialyltransferase	SIAT7B	NM_006456
127	204547_at	RAB40B, member RAS oncogene family	RAB40B	NM_006822
128	204614_at	serine (or cysteine) proteinase inhibitor, clade B,	SERPINB2	NM_002575
		mem 2
129	204621_s_at	nuclear receptor subfamily 4, group A, member 2	NR4A2	AI935096
130	204622_x_at		NR4A2	NM_006186
131	204633_s_at	nuclear mitogen- and stress-activated protein	RPS6KA5	AF074393
		kinase-1
132	204636_at	collagen, type XVII, alpha 1	COL17A1	NM_000494
133	204672_s_at	ankyrin repeat domain 6	ANKRD6	NM_014942
134	204734_at	keratin 15	KRT15	NM_002275
135	204753_s_at	hepatic leukemia factor	HLF	AI810712
136	204754_at	hepatic leukemia factor	HLF	W60800
137	204755_x_at	hepatic leukemia factor	HLF	M95585
138	204855_at	serine (or cysteine) proteinase inhibitor, clade B,	SERPINB5	NM_002639
		mem 5
139	204887_s_at	polo-like kinase 4	PLK4	NM_014264
140	204952_at	GPI-anchored metastasis-associated protein	C4.4A	NM_014400
		homolog
141	204971_at	cystatin A (stefin A)	CSTA	NM_005213
142	205014_at	heparin-binding growth factor binding protein	FGFBP1	NM_005130
143	205022_s_at	checkpoint suppressor 1	CHES1	NM_005197
144	205054_at	nebulin	NEB	NM_004543
145	205064_at	small proline-rich protein 1B	SPRR1B	NM_003125
146	205081_at	cysteine-rich protein 1	CRIP1	NM_001311
147	205141_at	angiogenin, ribonuclease, RNase A family, 5	ANG	NM_001145
148	205157_s_at	keratin 17	KRT17	NM_000422
149	205176_s_at	integrin beta 3 binding protein (beta3-endonexin)	ITGB3BP	NM_014288
150	205206_at	Kallmann syndrome 1 sequence	KAL1	NM_000216
151	205219_s_at	galactokinase 2	GALK2	NM_002044
152	205267_at	POU domain, class 2, associating factor 1	POU2AF1	NM_006235
153	205367_at	adaptor protein with pleckstrin homology and src	APS	NM_020979
		homology 2 domains
154	205372_at	pleiomorphic adenoma gene 1	PLAG1	NM_002655
155	205450_at	phosphorylase kinase, alpha 1 (muscle)	PHKA1	NM_002637
156	205490_x_at	gap junction protein, beta 3	GJB3	BF060667
157	205569_at	lysosomal-associated membrane protein 3	LAMP3	NM_014398
158	205595_at	desmoglein 3	DSG3	NM_001944
159	205618_at	proline rich Gla (G-carboxyglutamic acid) 1	PRRG1	NM_000950
160	205623_at	aldehyde dehydrogenase 3	ALDH3A1	NM_000691
161	205624_at	carboxypeptidase A3 (mast cell)	CPA3	NM_001870
162	205789_at	CD1D antigen, d polypeptide	CD1D	NM_001766
163	205839_s_at	benzodiazapine receptor (peripheral) assoc pro 1	BZRAP1	NM_004758
164	205961_s_at	PC4 and SFRS1 interacting protein 1	PSIP1	NM_004682
165	205968_at	K+ voltage-gated channel, delayed-rectifier,	KCNS3	NM_002252
		subfamily S, member 3
166	205969_at	arylacetamide deacetylase (esterase)	AADAC	NM_001086
167	206032_at	desmocollin 3, transcript variant Dsc3a	DSC3	AI797281
168	206033_s_at	desmocollin 3, transcript variant Dsc3a	DSC3	AI797281
169	206068_s_at	acyl-Coenzyme A dehydrogenase, long chain	ACADL	AI367275
170	206094_x_at	UDP glycosyltransferase 1 family, polypeptide A6	UGT1A6	NM_001072
171	206122_at	SRY-box 20	SOX15	NM_006942
172	206164_at	chloride channel, calcium activated, family mem 2	CLCA2	NM_006536
173	206165_s_at	chloride channel, calcium activated, family mem 2	CLCA2	NM_006536
174	206166_s_at	calcium-activated chloride channel-2	CLCA2	NM_006536
175	206300_s_at	parathyroid hormone-like hormone	PTHLH	NM_002820
176	206331_at	calcitonin receptor-like	CALCRL	NM_005795
177	206400_at	lectin, galactoside-binding, soluble, 7	LGALS7	NM_002307
178	206461_x_at	metallothionein 1H	MT1H	NM_005951
179	206561_s_at	aldo-keto reductase family 1, member B10	AKR1B10	NM_020299
180	206566_at	solute carrier family 7 (cationic amino acid	SLC7A1	NM_003045
		transporter, y+ system), member 1
181	206581_at	basonuclin	BNC1	NM_001717
182	206641_at	tumor necrosis factor receptor superfamily, mem 17	TNFRSF17	NM_001192
183	206653_at	Polymerase (RNA) III (DNA directed) polypep G	POLR3G	BF062139
184	206658_at	hypothetical protein MGC10902	UPK3B	NM_030570
185	206756_at	carbohydrate (N-acetylglucosamine 6-O)	CHST7	NM_019886
		sulfotransferase 7
186	206912_at	forkhead box E1	FOXE1	NM_004473
187	207029_at	KIT ligand	KITLG	NM_000899
188	207126_x_at	UDP glycosyltransferase 1 family, polypep A1	UGT1A1 ///	NM_000463
189	207499_x_at	hypothetical protein FLJ10043	SMAP-1	NM_017979
190	207513_s_at	zinc finger protein 189	ZNF189	NM_003452
191	207620_s_at	calcium/calmodulin-dependent serine protein	CASK	NM_003688
		kinase
192	207935_s_at	keratin 13	KRT13	NM_002274
193	208153_s_at	FAT tumor suppressor homolog 2	FAT2	NM_001447
194	208228_s_at	fibroblast growth factor receptor 2	FGFR2	M87771
195	208502_s_at	paired-like homeodomain transcription factor 1	PITX1	NM_002653
196	208539_x_at	small proline-rich protein 2B	SPRR2A	NM_006945
197	208581_x_at	metallothionein 1X	MT1X	NM_005952
198	208596_s_at	UDP glycosyltransferase 1 family, polypep A3	UGT1A3	NM_019093
199	208657_s_at	septin 9	9-Sep	AF142408
200	208692_at	ribosomal protein S3	RPS3	U14990
201	208737_at	ATPase, H+ transporting, lysosomal 13 kDa, V1	ATP6V1G1	BC003564
		subunit G isoform 1
202	208758_at	5-aminoimidazole-4-carboxamide ribonucleotide	ATIC	D89976
		formyltransferase/IMP cyclohydrolase
203	208798_x_at	golgin-67	GOLGIN-	AF204231
			67
204	208856_x_at	ribosomal protein, large, P0	RPLP0	BC003655
205	208870_x_at	ATP synthase, H+ transporting, mitochondrial F1	ATP5C1	BC000931
		complex, gamma polypeptide 1
206	208933_s_at	lectin, galactoside-binding, soluble, 8	LGALS8	AI659005
207	208935_s_at	lectin, galactoside-binding, soluble, 8	LGALS8	L78132
208	208950_s_at	aldehyde dehydrogenase 7 family, mem A1	ALDH7A1	BC002515
209	209009_at	esterase D/formylglutathione hydrolase	ESD	BC001169
210	209041_s_at	ubiquitin-conjugating enzyme E2G 2	UBE2G2	BG395660
211	209117_at	WW domain binding protein 2	WBP2	U79458
212	209122_at	adipose differentiation-related protein	ADFP	BC005127
213	209125_at	keratin 6A	KRT6A	J00269
214	209126_x_at	keratin 6 isoform K6f	KRT6B	L42612
215	209204_at	LIM domain only 4	LMO4	AI824831
216	209212_s_at	transcription factor BTEB2	KLF5	AB030824
217	209215_at	tetracycline transporter-like protein	TETRAN	L11669
218	209220_at	glypican 3	GPC3	L47125
219	209260_at	stratifin	SFN	BC000329
220	209296_at	protein phosphatase 1B (formerly 2C), magnesium-	PPM1B	AF136972
		dependent, beta isoform
221	209309_at	zinc-alpha2-glycoprotein	AZGP1	D90427
222	209339_at	seven in absentia homolog 2	SIAH2	U76248
223	209351_at	keratin 14	KRT14	BC002690
224	209380_s_at	CFTR/MRP, member 5	ABCC5	AF146074
225	209411_s_at	Golgi associated, gamma adaptin ear containing,	GGA3	AW008018
		ARF binding protein 3
226	209446_s_at	Similar to hypothetical protein FLJ10803	—	BC001743
227	209457_at	dual specificity phosphatase 5	DUSP5	U16996
228	209509_s_at	dolichyl-phosphate	DPAGT1	BC000325
229	209587_at	hindlimb expressed homeobox protein backfoot	Bft	U70370
230	209647_s_at	IMAGE: 2972022	SOCS5	AW664421
231	209699_x_at	dihydrodiol dehydrogenase	AKR1C2	U05598
232	209719_x_at	squamous cell carcinoma antigen 1	SCCA1	U19556
233	209720_s_at	serine (or cysteine) proteinase inhibitor, clade B	SERPINB3	U19556
		(ovalbumin), member 3
234	209727_at	GM2 ganglioside activator	GM2A	M76477
235	209748_at	spastic paraplegia 4	SPG4	AB029006
236	209792_s_at	kallikrein 10	KLK10	BC002710
237	209800_at	keratin 16	KRT16	AF061812
238	209863_s_at	CUSP	TP73L	AF091627
239	209878_s_at	v-rel reticuloendotheliosis viral oncogene hom A,	RELA	M62399
240	209897_s_at	slit homolog 2 (Drosophila)	SLIT2	AF055585
241	209959_at	nuclear receptor subfamily 4, group A, member 3	NR4A3	U12767
242	209963_s_at	erythropoietin receptor	EPOR	M34986
243	210020_x_at	NB-1	CALML3	M58026
244	210052_s_at	TPX2, microtubule-associated protein homolog	TPX2	AF098158
245	210064_s_at	uroplakin 1B	UPK1B	NM_006952
246	210065_s_at	uroplakin Ib	UPK1B	NM_006952
247	210084_x_at	mast cell alpha II tryptase	—	AF206665
248	210133_at	chemokine (C—C motif) ligand 11	CCL11	D49372
249	210135_s_at	short stature homeobox 2	SHOX2	AF022654
250	210264_at	G protein-coupled receptor 35	GPR35	AF089087
251	210355_at	parathyroid-like protein	PTHLH	J03580
252	210406_s_at	RAB6A, member RAS oncogene family	RAB6A	AL136727
253	210505_at	alcohol dehydrogenase	ADH7	U07821
254	210512_s_at	vascular endothelial growth factor	VEGF	AF022375
255	210829_s_at	single-stranded DNA binding protein 2	SSBP2	AF077048
256	210876_at	annexin A2	ANXA2	M62896
257	211002_s_at	tripartite motif protein TRIM29 beta	TRIM29	AF230389
258	211105_s_at	nuclear factor of activated T-cells, cytoplasmic,	NFATC1	U80918
		calcineurin-dependent 1
259	211194_s_at	p73H	TP73L	AB010153
260	211195_s_at	p51 delta	TP73L	AB010153
261	211272_s_at	diacylglycerol kinase, alpha 80 kDa	DGKA	AF064771
262	211361_s_at	hurpin	hurpin	AJ001696
263	211401_s_at	fibroblast growth factor receptor 2	FGFR2	AB030078
264	211452_x_at	clone FLB4816 PRO1252	—	AF130054
265	211456_x_at	metallothionein 1H-like	—	AF333388
266	211474_s_at	serine (or cysteine) proteinase inhibitor, clade B	SERPINB6	BC004948
		(ovalbumin), member 6
267	211527_x_at	vascular permeability factor	VEGF	M27281
268	211547_s_at	Miller-Dieker lissencephaly protein	LIS1	L13387
269	211548_s_at	hydroxyprostaglandin dehydrogenase 15-(NAD)	HPGD	J05594
270	211596_s_at	leucine-rich repeats and immunoglobulin-like	LRIG1	AB050468
		domains 1
271	211634_x_at	immunoglobulin heavy constant mu	IGHM	M24669
272	211635_x_at	IgM rheumatoid factor RF-TT1, VH chain	—	M24670
273	211653_x_at	pseudo-chlordecone	AKR1C2	M33376
274	211689_s_at	transmembrane protease, serine 2	TMPRSS2	AF270487
275	211721_s_at	zinc finger proteins 551	ZNF551	BC005868
276	211734_s_at	IgE Fc, high affinity I, receptor for α polypep	FCER1A	BC005912
277	211756_at	parathyroid hormone-like hormone	PTHLH	BC005961
278	211834_s_at	p73Lp63p51p40KET	TP73L	AB042841
279	212061_at	KIAA0332	SR140	AB002330
280	212092_at	KIAA1051	PEG10	BE858180
281	212094_at	KIAA1051	PEG10	BE858180
282	212162_at	FLJ12811	—	AK022873
283	212189_s_at	component of oligomeric Golgi complex 4	COG4	AK022874
284	212228_s_at	hypothetical protein DKFZp434K046	DKFZP434K046	AC004382
285	212236_x_at	cytokeratin 17	KRT17	Z19574
286	212252_at	Ca2+ calmodulin-dependent protein kinase kinase 2β	CAMKK2	AA181179
287	212255_s_at	FLJ10822 fis	FLJ10822	AK001684
288	212286_at	ankyrin repeat domain 12	ANKRD12	AW572909
289	212311_at	KIAA0746 protein	KIAA0746	AA522514
290	212314_at	KIAA0746 protein	KIAA0746	AB018289
291	212424_at	programmed cell death 11	PDCD11	AW026194
292	212441_at	KIAA0232	KIAA0232	D86985
293	212458_at	sprouty-related, EVH1 domain containing 2	SPRED2	H97931
294	212466_at	sprouty-related, EVH1 domain containing 2	SPRED2	AW138902
295	212570_at	KIAA0830 protein	KIAA0830	AL573201
296	212573_at	KIAA0830 protein	KIAA0830	AF131747
297	212595_s_at	DAZ associated protein 2	DAZAP2	AL534321
298	212599_at	autism susceptibility candidate 2	AUTS2	AK025298
299	212600_s_at	ubiquinol-cytochrome c reductase core protein II	UQCRC2	AV727381
300	212662_at	poliovirus receptor	PVR	BE615277
301	212680_x_at	protein phosphatase 1, regulatory (inhibitor)	PPP1R14B	BE305165
		subunit 14B
302	212836_at	polymerase (DNA-directed), delta 3, accessory	POLD3	D26018
		subunit
303	212841_s_at	PTPRF interacting protein, binding protein 2	PPFIBP2	AI692180
304	212864_at	CDP-diacylglycerol synthase (phosphatidate	CDS2	Y16521
		cytidylyltransferase) 2
305	212914_at	chromobox homolog 7	CBX7	AV648364
306	212980_at	AHA1, activator of heat shock 90 kDa protein	AHSA2	AL050376
		ATPase homolog 2
307	213023_at	utrophin	UTRN	NM_007124
308	213034_at	KIAA0999 protein	KIAA0999	AB023216
309	213093_at	protein kinase C, alpha	PRKCA	AI471375
310	213199_at	DKFZP586P0123 protein	DKFZP586P0123	AL080220
311	213325_at	poliovirus receptor-related 3	PVRL3	AA129716
312	213366_x_at	ATP synthase, H+ transporting, mitochondrial F1	ATP5C1	AV711183
		complex, gamma polypeptide 1
313	213425_at	wingless-type MMTV integration site family,	WNT5A	AI968085
		member 5A
314	213440_at	RAB1A, member RAS oncogene family	RAB1A	AL530264
315	213471_at	nephronophthisis 4	NPHP4	AB014573
316	213490_s_at	mitogen-activated protein kinase kinase 2	MAP2K2	AI762811
317	213518_at	protein kinase C, iota	PRKCI	AI689429
318	213680_at	keratin 6A	KRT6B	AI831452
319	213700_s_at	Pyruvate kinase, muscle	PKM2	AA554945
320	213721_at	SRY-box 2	SOX2	L07335
321	213722_at	SRY-box 2	SOX2	AW007161
322	213796_at	Small proline-rich protein SPRK	SPRR1A	AI923984
323	213808_at	23688 clone	ADAM23	BE674466
324	213843_x_at	accessory proteins BAP31BAP29	SLC6A8	AW276522
325	213880_at	leucine-rich repeat-containing G protein-coupled	LGR5	AL524520
		receptor 5
326	213913_s_at	KIAA0984 protein	KIAA0984	AW134976
327	214073_at	cortactin	CTTN	BG475299
328	214100_x_at	IMAGE: 1964520		AI284845
329	214260_at	COP9 constitutive photomorphogenic homolog	COPS8	AI079287
		subunit 8
330	214441_at	syntaxin 6	STX6	NM_005819
331	214549_x_at	small proline-rich protein 1A	SPRR1A	NM_005987
332	214580_x_at	keratin 6B	KRT6B	AL569511
333	214680_at	neurotrophic tyrosine kinase, receptor, type 2	NTRK2	BF674712
334	214688_at	transducin-like enhancer of split 4	TLE4	BF217301
335	214735_at	phosphoinositide-binding protein PIP3-E	PIP3-E	AW166711
336	214812_s_at	KIAA0184	KIAA0184	D80006
337	214829_at	aminoadipate-semialdehyde synthase	AASS	AK023446
338	214965_at	hypothetical protein MGC26885	MGC26885	AF070574
339	215011_at	RNA, U17D small nucleolar	RNU17D	AJ006835
340	215030_at	G-rich RNA sequence binding factor 1	GRSF1	AK023187
341	215125_s_at	UDP glycosyltransferase 1 family, polypep A9	UGT1A9	AV691323
342	215189_at	keratin, hair, basic, 6 (monilethrix)	KRTHB6	X99142
343	215354_s_at	proline-, glutamic acid-, leucine-rich protein 1	PELP1	BC002875
344	215372_x_at	Hypothetical protein LOC151878	LOC151878	AU146794
345	215382_x_at	mast cell alpha II tryptase	—	AF206666
346	215561_s_at	interleukin 1 receptor, type I	IL1R1	AK026803
347	215786_at	Hepatitis B virus x associated protein	HBXAP	AK022170
348	215812_s_at	creatine transporter	SLC6A10	U41163
349	216052_x_at	Artemin	ARTN	AF115765
350	216147_at	Septin 11	11-Sep	AL353942
351	216221_s_at	pumilio homolog 2	PUM2	D87078
352	216248_s_at	nuclear receptor subfamily 4, group A, member 2	NR4A2	S77154
353	216258_s_at	UV-B repressed sequence, HUR 7		BE148534
354	216263_s_at	chromosome 14 open reading frame 120	C14orf120	AK022215
355	216288_at	cysteinyl leukotriene receptor 1	CYSLTR1	AU159276
356	216412_x_at	IgG to Puumala virus G2, light chain V region	—	AF043584
357	216594_x_at	aldo-keto reductase family 1, member C1	AKR1C1	S68290
358	216603_at	solute carrier family 7, member 8	—	AL365343
359	216722_at	VENT-like homeobox 2 pseudogene 1	VENTX2P1	AF164963
360	216918_s_at	bullous pemphigoid antigen 1 isoforms 1 and 3	DST	AL096710
361	217003_s_at	tMDC II, isoform [d]	—	AJ132823
362	217097_s_at	hypothetical protein DKFZp564F013	PHTF2	AC004990
363	217165_x_at	metallothionein 1F (functional)	MT1F	M10943
364	217198_x_at	immunoglobulin heavy constant gamma 1	IGHG1	U80164
365	217227_x_at	immunoglobulin lambda locus	IGLVJC	X93006
366	217272_s_at	serine (or cysteine) proteinase inhibitor, clade B,	hurpin	AJ001698
		member 13
367	217312_s_at	collagen type VII intergenic region	COL7A1	L23982
368	217388_s_at	kynureninase (L-kynurenine hydrolase)	KYNU	D55639
369	217418_x_at	membrane-spanning 4-domains, subfam A, mem 1	MS4A1	X12530
370	217480_x_at	similar to Ig kappa chain	LOC339562	M20812
371	217528_at	chloride channel, calcium activated, family mem 2	CLCA2	BF003134
372	217622_at	chromosome 22 open reading frame 3	C22orf3	AA018187
373	217626_at	IMAGE: 3089210	AKR1C2 ///	BF508244
			AKR1C1
374	217746_s_at	programmed cell death 6 interacting protein	PDCD6IP	NM_013374
375	217783_s_at	yippee-like	YPEL5	NM_016061
376	217786_at	SKB1 homolog	SKB1	NM_006109
377	217811_at	selenoprotein T	SELT	NM_016275
378	217841_s_at	protein phosphatase methylesterase-1	PME-1	NM_016147
379	217860_at	NADH dehydrogenase (ubiquinone) 1 alpha	NDUFA10	NM_004544
		subcomplex, 10,
380	217922_at	Mannosidase, alpha, class 1A, member 2	MAN1A2	AL157902
381	217994_x_at	hypothetical protein FLJ20542	FLJ20542	NM_017871
382	218070_s_at	GDP-mannose pyrophosphorylase A	GMPPA	NM_013335
383	218092_s_at	HIV-1 Rev binding protein	HRB	NM_004504
384	218192_at	inositol hexaphosphate kinase 2	IHPK2	NM_016291
385	218236_s_at	protein kinase D3	PRKD3	NM_005813
386	218238_at	GTP binding protein 4	GTPBP4	NM_012341
387	218239_s_at	GTP binding protein 4	GTPBP4	NM_012341
388	218288_s_at	hypothetical protein MDS025	MDS025	NM_021825
389	218305_at	importin 4	IPO4	NM_024658
390	218331_s_at	chromosome 10 open reading frame 18	C10orf18	NM_017782
391	218355_at	kinesin family member 4A	KIF4A	NM_012310
392	218384_at	calcium regulated heat stable protein 1	CARHSP1	NM_014316
393	218460_at	hypothetical protein FLJ20397	FLJ20397	NM_017802
394	218483_s_at	hypothetical protein FLJ21827	FLJ21827	NM_020153
395	218507_at	hypoxia-inducible protein 2	HIG2	NM_013332
396	218546_at	hypothetical protein FLJ14146	FLJ14146	NM_024709
397	218657_at	Link guanine nucleotide exchange factor II	RAPGEFL1	NM_016339
398	218696_at	eukaryotic translation initiation factor 2-α kinase 3	EIF2AK3	NM_004836
399	218699_at	RAB7, member RAS oncogene family-like 1	RAB7L1	BG338251
400	218750_at	hypothetical protein MGC5306	MGC5306	NM_024116
401	218769_s_at	ankyrin repeat, family A (RFXANK-like), 2	ANKRA2	NM_023039
402	218796_at	hypothetical protein FLJ20116	C20orf42	NM_017671
403	218834_s_at	heat shock 70 kDa protein 5 (glucose-regulated	HSPA5BP1	NM_017870
		protein, 78 kDa) binding protein 1
404	218957_s_at	hypothetical protein FLJ11848	FLJ11848	NM_025155
405	218960_at	transmembrane protease, serine 4	TMPRSS4	NM_016425
406	218962_s_at	hypothetical protein FLJ13576	FLJ13576	NM_022484
407	218990_s_at	small proline-rich protein 3	SPRR3	NM_005416
408	219129_s_at	hypothetical protein FLJ11526	SAP30L	NM_024632
409	219132_at	pellino homolog 2	PELI2	NM_021255
410	219154_at	Ras homolog gene family, member F	RHOF	NM_024714
411	219155_at	phosphatidylinositol transfer protein, cytoplasmic 1	PITPNC1	NM_012417
412	219201_s_at	twisted gastrulation homolog 1	TWSG1	NM_020648
413	219217_at	hypothetical protein FLJ23441	FLJ23441	NM_024678
414	219241_x_at	hypothetical protein FLJ20515	SSH3	NM_017857
415	219245_s_at	hypothetical protein FLJ13491	FLJ13491	AI309636
416	219250_s_at	fibronectin leucine rich transmem protein 3	FLRT3	NM_013281
417	219347_at	nudix (nucleoside diphosphate linked moiety X)-	NUDT15	NM_018283
		type motif 15
418	219389_at	hypothetical protein FLJ10052	FLJ10052	NM_017982
419	219554_at	Rh type C glycoprotein	RHCG	NM_016321
420	219582_at	opioid growth factor receptor-like 1	OGFRL1	NM_024576
421	219704_at	germ cell specific Y-box binding protein	YBX2	NM_015982
422	219732_at	plasticity related gene 3	PRG-3	NM_017753
423	219741_x_at	zinc finger protein 552	ZNF552	NM_024762
424	219756_s_at	hypothetical protein FLJ22792	POF1B	NM_024921
425	219854_at	zinc finger protein 14 (KOX 6)	ZNF14	NM_021030
426	219936_s_at	G protein-coupled receptor 87	GPR87	NM_023915
427	219959_at	molybdenum cofactor sulfurase	MOCOS	NM_017947
428	219962_at	angiotensin I converting enzyme (peptidyl-	ACE2	NM_021804
		dipeptidase A) 2
429	219995_s_at	hypothetical protein FLJ13841	FLJ13841	NM_024702
430	219997_s_at	COP9 constitutive photomorphogenic hom sub 7B	COPS7B	NM_022730
431	220046_s_at	cyclin L1	CCNL1	NM_020307
432	220177_s_at	transmembrane protease, serine 3	TMPRSS3	NM_024022
433	220285_at	chromosome 9 open reading frame 77	C9orf77	NM_016014
434	220466_at	hypothetical protein FLJ13215	FLJ13215	NM_025004
435	220664_at	small proline-rich protein 2C	SPRR2C	NM_006518
436	220668_s_at	DNA (cytosine-5-)-methyltransferase 3 beta	DNMT3B	NM_006892
437	221004_s_at	integral membrane protein 2C	ITM2C	NM_030926
438	221045_s_at	period homolog 3	PER3	NM_016831
439	221047_s_at	MAP/microtubule affinity-regulating kinase 1	MARK1	NM_018650
440	221050_s_at	GTP binding protein 2	GTPBP2	NM_019096
441	221064_s_at	chromosome 16 open reading frame 28	C16orf28	NM_023076
442	221096_s_at	hypothetical protein PRO1580	PRO1580	NM_018502
443	221234_s_at	BTB and CNC homology 1, basic leucine zipper	BACH2	NM_021813
		transcription factor 2
444	221286_s_at	proapoptotic caspase adaptor protein	PACAP	NM_016459
445	221305_s_at	UDP glycosyltransferase 1 family, polypep A8	UGT1A8	NM_019076
446	221326_s_at	delta-tubulin	TUBD1	NM_016261
447	221480_at	heterogeneous nuclear ribonucleoprotein D	HNRPD	BG180941
448	221513_s_at	UTP14, U3 small nucleolar ribonucleoprotein,	UTP14C/	BC001149
		homolog C/homolog A	UTP14A
449	221514_at	U3 small nucleolar ribonucleoprotein, hom A	UTP14A	BC001149
450	221580_s_at	hypothetical protein MGC5306	MGC5306	BC001972
451	221597_s_at	HSPC171 protein	HSPC171	BC003080
452	221622_s_at	uncharacterized hypothalamus protein HT007	HT007	AF246240
453	221649_s_at	peter pan homolog	PPAN	BC000535
454	221679_s_at	abhydrolase domain containing 6	ABHD6	AF225418
455	221770_at	ribulose-5-phosphate-3-epimerase	RPE	BE964473
456	221790_s_at	LDL receptor adaptor protein	ARH	AL545035
457	221795_at	Similar to hypothetical protein FLJ20093		AI346341
458	221796_at	Similar to hypothetical protein FLJ20093		AA707199
459	221854_at	ESTs	PKP1	AI378979
460	221884_at	ecotropic viral integration site 1	EVI1	BE466525
461	243_g_at	microtubule-associated protein 4	MAP4	M64571
462	31846_at	ras homolog gene family, member D	RHOD	AW003733
463	33323_r_at	stratifin	SFN	X57348
464	33850_at	microtubule-associated protein 4	MAP4	W28892
465	34858_at	potassium channel tetramerisation domain	KCTD2	D79998
		containing 2
466	37512_at	3-hydroxysteroid epimerase	RODH	U89281
467	41037_at	TEA domain family member 4	TEAD4	U63824
468	41469_at	elafin	PI3	L10343
469	44111_at	vacuolar protein sorting 33B	VPS33B	AI672363
470	49049_at	deltex 3 homolog	DTX3	N92708
471	49077_at	protein phosphatase methylesterase-1	PME-1	AL040538
472	59625_at	nucleolar protein 3	NOL3	AI912351
473	65438_at	KIAA1609 protein	KIAA1609	AA195124

REFERENCES

• Beer et al. (2002) “Gene-expression profiles predict survival of patients with lung adenocarcinoma” Nat Med 8:816-824
• Brookes (1999) “The essence of SNPs” Gene 23:177-186
• Kato et al. (2004) “A Randomized Trial of Adjuvant Chemotherapy with Uracil-Tegafur for Adenocarcinoma of the Lung” N Engl J Med 350:1713-1721
• Kiernan et al. (1993) “Stage I non-small cell cancer of the lung results of surgical resection at Fairfax Hospital” Va Med Q 120:146-149
• Kononen et al. (1998) “Tissue microarrays for high-throughput molecular profiling of tumor specimens” Nat Med 4:844-847
• Mountain et al. (1987) “Lung cancer classification: the relationship of disease extent and cell type to survival in a clinical trials population” J Surg Oncol 35:147-156
• Wingo et al. (1999) “Annual Report to the Nation on the Status of Cancer, 1973-1996, With a Special Section on Lung Cancer and Tobacco Smoking “J Natl Cancer Inst 91:675-690

Claims

1. A method of assessing lung cancer status comprising the steps of a. obtaining a biological sample from a lung cancer patient; and b. measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 wherein the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of lung cancer status.

2. A method of staging lung cancer patients comprising the steps of a. obtaining a biological sample from a lung cancer patient; and b. measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 wherein the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of the lung cancer stage.

3. The method of claim 2 wherein the stage corresponds to classification by the TNM system.

4. The method of claim 2 wherein the stage corresponds to patients with similar gene expression profiles.

5. A method of determining lung cancer patient treatment protocol comprising the steps of a. obtaining a biological sample from a lung cancer patient; and b. measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 wherein the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of recurrence to enable a physician to determine the degree and type of therapy recommended to prevent recurrence.

6. A method of treating a lung cancer patient comprising the steps of: a. obtaining a biological sample from a lung cancer patient; and b. measuring Biomarkers associated with Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7 wherein the expression levels of the Marker genes above or below pre-determined cut-off levels are indicate a high risk of recurrence and; c. treating the patient with adjuvant therapy if they are a high risk patient.

7. A method of determining whether a lung cancer patient is high or low risk of mortality comprising the steps of a. obtaining a biological sample from a lung cancer patient; and b. measuring Biomarkers associated with Marker genes corresponding to those selected from Table 4 wherein the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of mortality to enable a physician to determine the degree and type of therapy recommended.

8. The method of claim 1, 2, 5, 6 or 7 wherein the sample is prepared by a method are selected from the group consisting of bulk tissue preparation and laser capture microdissection.

9. The method of claim 8 wherein the bulk tissue preparation is obtained from a biopsy or a surgical specimen.

10. The method of claim 1, 2, 5, 6 or 7 further comprising measuring the expression level of at least one gene constitutively expressed in the sample.

11. The method of claim 1, 2, 5, 6 or 7 wherein the sample is obtained from a primary tumor.

12. The method of claim 1, 2, 5, 6 or 7 wherein the specificity is at least about 40%.

13. The method of claim 1, 2, 5, 6 or 7 wherein the sensitivity is at least at least about 80%.

14. The method of claim 1, 2, 5, 6 or 7 wherein the pre-determined cut-off levels are at least 1.5-fold over- or under-expression in the sample relative to benign cells or normal tissue.

15. The method of claim 1, 2, 5, 6 or 7 wherein the pre-determined cut-off levels have at least a statistically significant p-value over-expression in the sample having metastatic cells relative to benign cells or normal tissue.

16. The method of claim 28 wherein the p-value is less than 0.05.

17. The method of claim 1, 2, 5, 6 or 7 wherein gene expression is measured on a microarray or gene chip.

18. The method of claim 17 wherein the microarray is a cDNA array or an oligonucleotide array.

19. The method of claim 17 wherein the microarray or gene chip further comprises one or more internal control reagents.

18. The method of claim 1, 2, 5, 6 or 7 wherein gene expression is determined by nucleic acid amplification conducted by polymerase chain reaction (PCR) of RNA extracted from the sample.

20. The method of claim 18 wherein said PCR is reverse transcription polymerase chain reaction (RT-PCR).

21. The method of claim 20, wherein the RT-PCR further comprises one or more internal control reagents.

22. The method of claim 1, 2, 5, 6 or 7 wherein gene expression is detected by measuring or detecting a protein encoded by the gene.

23. The method of claim 22 wherein the protein is detected by an antibody specific to the protein.

24. The method of claim 1, 2, 5, 6 or 7 wherein gene expression is detected by measuring a characteristic of the gene.

25. The method of claim 24 wherein the characteristic measured is selected from the group consisting of DNA amplification, methylation, mutation and allelic variation.

26. A method of generating a lung cancer prognostic patient report comprising the steps of: determining the results of any one of claims 1, 2, 5, 6 or 7; and preparing a report displaying the results.

27. The method of claim 26 wherein the report contains an assessment of patient outcome and/or probability of risk relative to the patient population.

28. A patient report generated by the method according to claim 26.

29. A composition comprising at least one probe set selected from the group consisting of: Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

30. A kit for conducting an assay to determine lung cancer prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table I, Table 4, Table 5 or Table 7.

31. The kit of claim 30 further comprising reagents for conducting a microarray analysis.

32. The kit of claim 30 further comprising a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

33. Articles for assessing lung cancer status comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

34. The articles of claim 33 further comprising reagents for conducting a microarray analysis.

35. The articles of claim 34 further comprising a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.

36. A microarray or gene chip for performing the method of claim 1, 2, 5, 6 or 7.

37. The microarray of claim 36 comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

38. The microarray of claim 37 wherein the measurement or characterization is at least 1.5-fold over- or under-expression.

39. The microarray of claim 37 wherein the measurement provides a statistically significant p-value over- or under-expression.

40. The microarray of claim 39 wherein the p-value is less than 0.05.

41. The microarray of claim 37 comprising a cDNA array or an oligonucleotide array.

42. The microarray of claim 37 further comprising or more internal control reagents.

43. A diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 1, Table 4, Table 5 or Table 7.

44. The portfolio of claim 43 wherein the measurement or characterization is at least 1.5-fold over- or under-expression.

45. The portfolio of claim 44 wherein the measurement provides a statistically significant p-value over- or under-expression.

46. The portfolio of claim 44 wherein the p-value is less than 0.05.