WO2004111197A2

WO2004111197A2 - Gene expression signatures, methods and compositions for diagnosing disorders of the lung

Info

Publication number: WO2004111197A2
Application number: PCT/US2004/018492
Authority: WO
Inventors: Jerome S. Brody; Avrum Spira; Nila Shah; John F. Palma
Original assignee: Trustees Of Boston University; Affymetrix, Inc.
Priority date: 2003-06-10
Filing date: 2004-06-10
Publication date: 2004-12-23
Also published as: WO2004111197A3

Abstract

A minimally invasive sample procurement method for obtaining airway epithelial cell RNA that can be analyzed by expression profiling, e.g., by array-based gene expression profiling, is disclosed. These methods can be used to identify patterns of gene expression that are diagnostic of lung disorders, e.g., cancer, to identify subjects at risk for developing lung disorders and to custom design an array, e.g., a microarray, for the diagnosis or prediction of lung disorders or susceptibility to lung disorders. Arrays and informative genes are also disclosed for this purpose.

Description

GENE EXPRESSION SIGNATURES, METHODS AND COMPOSITIONS FOR DIAGNOSING DISORDERS OF THE LUNG

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/477,218, filed June 10, 2003, U.S. Provisional Application No. 60/483,387, filed June 27, 2003, and U.S. Provisional Application No. 60/497,599, filed August 25, 2003.

The entire teachings of the above applications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant ES00354 from The Katherine and Roland Douglass Fund (American Cancer Society) and by grant HL07035 from the National Institute of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Lung cancer claims more than 150,000 lives every year in the United States, exceeding the combined mortality from breast, prostate and colorectal cancers. Cigarette smoking is the most predominant cause of lung cancer. Presently, 25% of the U.S. population smokes, but only 10% to 15% of heavy smokers develop lung cancer. Former smokers remain at risk for developing cancer and now constitute a large reservoir of new lung cancer cases. Approximately 85% of all subjects with lung cancer die within three years of diagnosis. Unfortunately survival rates have not changed substantially in the past several decades, in large part because there are no effective methods for identifying smokers who are at highest risk for developing lung cancer and no effective tools for early diagnosis. One major hurdle in developing an early detection screen for lung cancer is that present methods for diagnosis require removal of tissue from inside the lung. Moreover, while it appears that a subset of smokers are more susceptible to the carcinogenic effects of cigarette smoke and are more likely to develop lung cancer, particular risk factors, and particularly genetic risk factors, for individuals have gone largely unidentified.

SUMMARY OF THE INVENTION

Work described herein demonstrates the utility of a minimally invasive sample procurement method and gene expression-based tools for the diagnosis of diseases of the lung, particularly lung cancer. Work described herein is further based on the identification of unique sets of expressed genes associated with smokers and non-smokers that constitute expression signatures. Epithelial cell gene expression profiles obtained from relatively accessible sites can provide important diagnostic and therapeutic information which can be applied to diagnose and treat lung disorders. For example, the gene expression profiles or signatures disclosed herein can be used to distinguish between, e.g., non-smokers and smokers, smokers and smokers with cancer, non-smokers and smokers with cancer, or a combination thereof.

In particular, work described herein provides a minimally invasive sample procurement method for obtaining airway epithelial cell RNA that can be analyzed by expression profiling, e.g., by array-based gene expression profiling. These methods can be used to determine how airway epithelial cell gene expression profiles are affected by cigarette smoke and how these profiles differ in smokers with and without lung cancer. These methods can also be used to identify patterns of gene expression that are diagnostic of lung disorders, e.g., cancer, and to identify subjects at risk for developing lung disorders. All or a subset of the genes identified according to the methods described herein (e.g., the genes disclosed in FIGS. IAIN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E) can be used to design an array, e.g., a microarray, specifically intended for the diagnosis or prediction of lung disorders or susceptibility to lung disorders, optionally in combination with probes intended for the diagnosis or prediction of other disorders (e.g., in combination with probes intended for the diagnosis or prediction of other types of cancer), based on the gene expression signatures exhibited by smokers and non- smokers as shown in FIGS. 6A-6R. For example, an array can be designed which has probes for one or more of the genes disclosed herein immobilized thereon: The efficacy of such custom-designed arrays can be further tested, for example, in a large clinical trial of smokers.

In one embodiment, the invention relates to a method of distinguishing a biological sample from a non-smoker and a biological sample from a smoker, and/or a biological sample from a smoker and a biological sample from a smoker with cancer, and/or a biological sample from a non-smoker and a biological sample from a smoker with cancer, wherein the method comprises obtaining a biological sample from an individual to be diagnosed; determining the level of expression of one or more informative genes in said sample, and comparing the determined level of expression with the corresponding level of expression in an appropriate control (e.g., one or more of a non-smoker, a smoker and a smoker with cancer). In a preferred embodiment, the biological sample is obtained from an airway epithelial cell. In one embodiment, the airway epithelial cell is obtained from a bronchoscopy or buccal mucosal scraping. In a preferred embodiment the informative genes are selected from the group consisting of the genes in FIGS. 1 A-IN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A- 7L and 8A-8E. Preferably the expressions of two or more, five or more, ten or more, fifteen or more, twenty or more, fifty or more, one hundred or more, or one thousand or more informative genes are determined. hi a particular embodiment, the biological sample is a nucleic acid sample, and in a preferred embodiment the level of expression of one or more informative genes is determined using oligonucleotides which hybridize to the one or more informative genes. In one embodiment the oligonucleotides are immobilized on a substrate, hi a particular embodiment, the nucleic acid sample is RNA. In another particular embodiment, the biological sample comprises a gene expression product, and in a preferred embodiment the level of expression of one or more informative genes is determined using polymers (e.g., polypeptides) which bind to gene expression products encoded by the one or more informative genes. In a particular embodiment the polymers are immobilized on a substrate.

In one embodiment, the invention relates to a method of diagnosing a disease of the lung, in particular lung cancer, comprising obtaining a biological sample from an individual to be diagnosed; and determining the expression of one or more informative genes in said sample, wherein increased expression of an informative gene whose expression is increased in individuals having a disease of the lung, or decreased expression of an informative gene whose expression is decreased in individuals having a disease of the lung, is indicative of a disease of the lung in the individual. In one embodiment, individual to be diagnosed is an individual who has been exposed to tobacco smoke, an individual who has smoked, or an individual who smokes. In a preferred embodiment, the biological sample is obtained from an airway epithelial cell. In one embodiment, the airway epithelial cell is obtained from a bronchoscopy or buccal mucosal scraping.

In a preferred embodiment of the method, the informative genes are selected from the group consisting of the genes shown in FIGS. 1 A-IN, 2A-2F, 3A-3D, 4A- 4P, 5A-5F, 6A-6R, 7A- 7L and 8A-8E. Preferably the expressions of two or more, five or more, ten or more, fifteen or more, twenty or more, fifty or more, one hundred or more, or one thousand or more informative genes are determined. In one embodiment, the disease of the lung is selected from the group consisting of asthma, chronic bronchitis, emphysema, primary pulmonary hypertension, acute respiratory distress syndrome, hypersensitivity pneumonitis, eosinophilic pneumonia, persistent fungal infection, pulmonary fibrosis, systemic sclerosis, ideopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, and lung cancer, such as adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and benign neoplasms^xof the lung (e.g., bronchial adenomas and hamartomas).

In a particular embodiment, the biological sample is a nucleic acid sample, and in a preferred embodiment the level of expression of one or more informative genes is determined using oligonucleotides which hybridize to the one or more informative genes. In one embodiment the oligonucleotides are immobilized on a substrate. In a particular embodiment, the biological sample is RNA. In a preferred embodiment, the expression is determined using a microarry having one or more probes (e.g., oligonucleotides) for said one or more genes immobilized thereon. In another particular embodiment, the biological sample comprises a gene expression product, and in a preferred embodiment the level of expression of one or more informative genes is determined using polymers (e.g., polypeptides) which bind to gene expression products encoded by the one or more informative genes. In a particular embodiment the polymers are immobilized on a substrate. The invention further relates to a method of obtaining a biological sample for use in expression analysis comprising obtaining an airway epithelial cell sample from an individual. The method may further comprise rendering nucleic acid molecules in said cell sample available for hybridization, and/or rendering gene expression products in said cell sample available for binding to polymers. The invention also relates to a method of treating a disease of the lung, in particular lung cancer, comprising administering to an individual in need thereof an effective amount of an agent which increases the expression of an informative gene whose expression is decreased in said individual as compared with a normal individual. The invention further relates to a method of treating a disease of the lung, in particular lung cancer, comprising administering to an individual in need thereof an effective amount of an agent which decreases the expression of an informative gene whose expression is increased in said individual as compared with a normal individual. The invention also relates to a method of treating a disease of the lung, in particular lung cancer, comprising administering to an individual in need thereof an effective amount of an agent which increases the activity of an expression product of an informative gene whose activity is decreased in said individual as compared with a normal individual. The invention also relates to a method of treating a disease of the lung, in particular lung cancer, comprising administering to an individual in need thereof an effective amount of an agent which decreases the activity of an expression product of an informative gene whose activity is increased in said individual as compared with a normal individual.

The invention also provides a substrate for use in expression analyses having immobilized thereon a plurality of polymers (e.g., oligonucleotides, polypeptides, etc.) which bind specifically to one or more informative genes disclosed herein or expression products of said genes, hi a particular embodiment, the substrate is an array (e.g., a microarray) having immobilized thereon a plurality of oligonucleotides. In one embodiment, less than 1000 oligonucleotides or polymers are immobilized on the array, hi another embodiment, the oligonucleotides hybridize specifically to one allelic form of one or more informative genes disclosed herein, hi a particular embodiment, the informative genes are selected from the group consisting essentially of the genes shown in FIGS. 1 A-IN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A- 6R, 7A-7L and 8A-8E. In one embodiment the substrate has immobilized thereon only polymers which bind specifically to one or more of the informative genes disclosed herein or expression products of said genes. In other embodiments the substrate has additional polymers immobilized thereon, such as polymers which bind specifically to informative genes for disorders such as types of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings. FIGS. IA- IN is a table of genes which are differentially expressed in smokers and smokers with cancer. Column one is the probe set designation for the gene; Column two is the common name(s) for the gene; Column three is the GenBank accession number for the gene sequence; Column four is the map (chromosomal) position of the gene; and Column five is descriptive information for the gene. The genes which overlap between FIGS. 1 A-IN and FIGS. 2A-2F are indicated with an "^A" (21 genes). The genes which overlap between FIGS. 1A-1N and FIGS. 3A-3D are indicated with an "*" (5 genes). FIGS. 2A-2F is a table of genes which are differentially expressed in smokers with cancer and non- smokers. Column one is the probe set designation for the gene; Column two is the common name(s) for the gene; Column three is the GenBank accession number for the gene sequence; Column four is the map (chromosomal) position of the gene; and Column five is descriptive information for the gene. The genes which overlap between FIGS. IA- IN and FIGS. 2A-2F are indicated with an "^Λ" (21 genes). The genes which overlap between FIGS. 2A-2F and FIGS. 3A-3D are indicated with an "#" (23 genes).

FIGS. 3A-3D is a table of genes which are differentially expressed in smokers and non- smokers. Column one is the probe set designation for the gene; Column two is the common name(s) for the gene; Column three is the GenBank accession number for the gene sequence; Column four is the map (chromosomal) position of the gene; and Column five is descriptive information for the gene. The genes which overlap between FIGS. 3A-3D and FIGS. 1 A-IN are indicated with an "*" (5 genes). The genes which overlap between FIGS. 3 A-3D and FIGS. 2A-2F are indicated with an "#" (23 genes).

FIGS. 4A-4P is a table of 131 genes which are differentially expressed and can be used to distinguish between non-smokers, smokers and smokers with cancer. Column one is the fold change in expression between non-smokers and smokers; Column two shows how the expression differs in smokers as compared to non- smokers; Column three is the fold change in expression between non-smokers and smokers with cancer; Column four shows how the expression differs in smokers with cancer as compared to non-smokers; Column five is the fold change in expression between smokers and smokers with cancer; Column six shows how the expression differs in smokers with cancer as compared to smokers; Column seven is the common name(s) for the gene; Column eight is the GenBank accession number for the gene sequence; Column nine is the map position of the gene; Column ten is the chromosomal position of the gene; Column 11 is the description of the gene.

FIGS. 5A-5F is a table of data similar to the data shown in FIGS. 4A-4P for 131 genes which are differentially expressed and can be used to distinguish between non-smokers, smokers and smokers with cancer. Column one is the probe set designation for the gene; Column two is the common name(s) for the gene; Column three is the GenBank accession number for the gene sequence; Column four is the map (chromosomal) position of the gene; and Column five is descriptive information for the gene. FIGS. 6A-6R is a table of genes which are differentially expressed in smokers and non-smokers. Column one is the probe set designation for the gene; Column two is the common name(s) for the gene; Column three is the GenBank accession number for the gene sequence; Column four is the p value for the smoker/non-smoker comparison; Column five shows whether the gene is up or down regulated; Column six shows the fold change in expression; Column seven is the GenBank description of the gene; Column eight is the functional category of the gene as indicated in GenBank; Column nine is the map (chromosomal) position of the gene; Column ten is the link locus; Column eleven is the Unigene reference for the gene; and Column twelve is OMIM reference for the gene. On FIG. 6R, five probe sets are shown which resulted in redundant results.

FIGS. 7A-7L is a table of genes which are differentially expressed in samples from smokers (S), nonsmokers (NS) and smokers with cancer (C). Column one is the probe set designation for the gene; Column two is the gene title; Column three is the gene symbol; Column four is the chromosomal location of the gene; Column five is the locus link.

FIGS. 8A-8E is a table of genes which are differentially expressed in samples from smokers (S) and smokers with cancer (C). Column one is the probe set designation for the gene; Column two is the gene title; Column three is the gene symbol; Column four is the chromosomal location of the gene; Column five is the locus link.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows. Lung cancer in smokers involves histopathological and molecular progression from normal to premalignant to cancer. Gene expression arrays of lung tumors have been used to characterize expression profiles of lung cancers, and to show the progression of molecular changes from non-malignant lung tissue to lung cancer. The ability to determine which individuals, particularly among smokers, have molecular changes in their airway epithelial cells and how these changes relate to premalignant and malignant changes that have been described in the lung has great potential for determining risk and for diagnosing cancer at a stage when treatment can be more effective, thus reducing the mortality and morbidity rates of lung cancer. The relative ease with which airway epithelial cells can be obtained (from bronchoscopy and buccal mucosal scrapings) suggests that this approach will have wide clinical applicability and has the potential for becoming a standard clinical screening tool for the large number of subjects at risk for developing disorders of the lung.

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof. An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.

Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome

Analysis: A Laboratory Manual Series (VoIs. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3^rd Ed., W.H. Freeman Pub., New York, NY and Berg et al. (2002) Biochemistry, 5^th Ed., W.H. Freeman Pub., New York, NY, all of which are herein incorporated in their entirety by reference for all purposes. The present invention can employ solid substrates, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S.S.N 09/536,841, WO 00/58516, U.S. Patents Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication Number WO 99/36760) and PCT/USOl/04285 (International Publication Number WO 01/58593), which are all incorporated herein by reference in their entirety for all purposes.

The term "array" as used herein refers to an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports. Additionally, the term "array" is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (for example, from 1 to about 1000 nucleotide monomers in length) onto a substrate.

The term "solid support", "support", and "substrate" as used herein are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Patent No. 5,744,305 for exemplary substrates.

Patents that describe synthesis techniques in specific embodiments include U.S. Patents Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays.

Nucleic acid arrays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, CA) under the brand name GeneChip®. The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring, and profiling methods can be shown in U.S. Patents Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are shown in U.S.S.N. 60/319,253, 10/013,598 (U.S. Patent Application Publication No.: 20030036069), and U.S. Patents Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Patents Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

The present invention also contemplates sample preparation methods in certain preferred embodiments. Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila et al, Nucleic Acids Res. 19, 4967 (1991); Eckert et al, PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al, IRL Press, Oxford); and U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S Patent No. 6,300,070 and U.S. Patent Application No. 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al, Science 241, 1077 (1988) and Barringer et al Gene 89:117 (1990)), transcription amplification (Kwoh et al, Proc. Natl. Acad. ScI USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al, Proc. Nat. Acad. ScI USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (CP- PCR) (U.S. Patent No 4,437,975), arbitrarily primed polymerase chain reaction (AP- PCR) (U.S. Patent No 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). {See, U.S. Patents Nos. 5,409,818, 5,554,517, and

6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.S.N. 09/854,317, each of which is incorporated herein by reference. Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al, Genome Research 11, 1418 (2001), in U.S. Patent Nos. 6,361,947, 6,391,592 and U.S. Patent Application Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication No. 20030096235), 09/910,292 (U.S. Patent Application Publication No. 20030082543, U.S. Patent No. 6,632,611), and 10/013,598 (U.S. Patent Application Publication No. 20030036069). Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2^nd Ed. Cold Spring Harbor, N. Y, 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, CA₅ 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Patent Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623, each of which are incorporated herein by reference.

The present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Patent Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent Application No. 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Patents Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent Application No. 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes. The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard- disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et ah, Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rasbidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2^nd ed., 2001).

The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Patent Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Patent Application Nos. 10/063,559 (U.S. Patent Application Publication No. 20020183936), 60/349,546, 60/376,003, 60/394,574, 60/403,381.

Further methods for practicing the present invention are also exemplified in Spira et ah, Chest 125(5 Suppl): 115S (2004) ; Spira et al, Biotechniques

36(3)AU-7 (2004) ; and Powell et al.,Am. J. Respir. Cell. MoI. Biol. 29(2): 157-62 (2003), the teachings of all of which are incorporated herein in their entirety. As used herein, an informative gene is a gene which is differentially expressed in sample populations with different phenotypes (e.g., presence of or susceptibility to disorders of the lung, particularly lung cancer) or behavioral traits (e.g., smoking), e.g., differentially expressed in smokers as compared with non- smokers, in smokers with cancer as compared to smokers without cancer, in smokers likely to develop cancer as compared with smokers unlikely to develop cancer, etc. In one embodiment, an informative gene displays at least a fold change shown in one of the rows of Column six of FIGS. 6A-6R. In a preferred embodiment, an informative gene displays at least a two-fold, more preferably at least a five-fold, and even more preferably at least a ten-fold difference in expression between two sample populations. Examples of informative genes are shown in FIGS. 1 A-IN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, IA-Ih and 8A-8E; other informative genes can be identified using methods described herein. As used herein, gene expression products are proteins, peptides, or nucleic acid molecules (e.g., mRNA, tRNA, rRNA, or cRNA) that are involved in transcription or translation. The nucleic acid molecule levels measured can be derived directly from the gene or, alternatively, from a corresponding regulatory gene. All forms of gene expression products can be measured, including, for example, spliced variants. Similarly, gene expression can be measured by assessing the level of protein or derivative thereof translated from mRNA. In this embodiment the determination of the gene expression profile can be made using techniques for protein detection and quantitation known in the art. For example, antibodies specific for the protein or polypeptide can be obtained using methods which are routine in the art, and the specific binding of such antibodies to protein or polypeptide gene expression products can be detected and measured. Alternatively, polymers immobilized on a substrate (e.g., protein arrays) can be used.

In the embodiment wherein arrays of nucleic acids are immobilized on a surface, the number of nucleic acid sequences may be selected for different applications, and may be, for example, about 100 or more, or, e.g., in some embodiments, more than 10⁵ or 10⁸. In one embodiment, the surface comprises at least 100 probe nucleic acids each preferably having a different sequence, each probe contained in an area of less than about 0.1 cm², or, for example, between about 1 um² and 10,000 um², and each probe nucleic acid having a defined sequence and location on the surface. In one embodiment, at least 1,000 different nucleic acids are provided on the surface, wherein each nucleic acid is contained within an area less than about 10^"3 cm², as described, for example, in U.S. Patent No. 5,510,270, the disclosure of which is incorporated herein.

Arrays of nucleic acids for use in gene expression monitoring are described in PCT WO 97/10365, the disclosure of which is incorporated herein. In one embodiment, arrays of nucleic acid probes are immobilized on a surface, wherein the array comprises more than 100 different nucleic acids and wherein each different nucleic acid is localized in a predetermined area of the surface, and the density of the different nucleic acids is greater than about 60 different nucleic acids per 1 cm².

Arrays of nucleic acids immobilized on a surface which may be used also are described in detail in U.S. Patent No. 5,744,305, the disclosure of which is incorporated herein. As disclosed therein, on a substrate, nucleic acids with different sequences are immobilized each in a predefined area on a surface. For example, 10, 50, 60, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ different monomer sequences may be provided on the substrate. The nucleic acids of a particular sequence are provided within a predefined region of a substrate, having a surface area, for example, of about 1 cm² to 10^"10 cm². In some embodiments, the regions have areas of less than about 10^"1, 10^'2, 10^"3, 10^"4, 10^"5, 10^'6, 10^"7, 10^"8, 10^"9, or 10^'10 cm². For example, in one embodiment, there is provided a planar, non-porous support having at least a first surface, and a plurality of different nucleic acids attached to the first surface at a density exceeding about 400 different nucleic acids/cm², wherein each of the different nucleic acids is attached to the surface of the solid support in a different predefined region, has a different determinable sequence, and is, for example, at least 4 nucleotides in length. The nucleic acids may be, for example, about 4 to 20 nucleotides in length. The number of different nucleic acids may be, for example, 1000 or more. In the embodiment where polynucleotides of a known chemical sequence are synthesized at known locations on a substrate, and binding of a complementary nucleotide is detected, and wherein a fluorescent label is detected, detection may be implemented by directing light to relatively small and precisely known locations on the substrate. For example, the substrate is placed in a microscope detection apparatus for identification of locations where binding takes place. The microscope detection apparatus includes a monochromatic or polychromatic light source for directing light at the substrate, means for detecting fluoresced light from the substrate, and 'means for determining a location of the fluoresced light. The means for detecting light fluoresced on the substrate may in some embodiments include a photon counter. The means for determining a location of the fluoresced light may include an x/y translation table for the substrate. Translation of the substrate and data collection are recorded and managed by an appropriately programmed digital computer, as described in U.S. Patent No. 5,510,270, the disclosure of which is incorporated herein.

The sample to be assessed can be any sample that contains a gene expression product. Suitable sources of gene expression products, i.e., samples, can include cells, lysed cells, cellular material for determining gene expression, or material containing gene expression products. Examples of such samples are blood, plasma, lymph, urine, tissue, mucus, sputum, saliva or other cell samples. Methods of , obtaining such samples are known in the art. In a preferred embodiment, the sample is an airway epithelial cell.

Disorders which may be diagnosed or treated by methods described herein include, but are not limited to, asthma, chronic bronchitis, emphysema, bronchietasis, primary pulmonary hypertension and acute respiratory distress syndrome. The methods described herein may also be used to diagnose or treat lung ' disorders that involve the immune system including, hypersensitivity pneumonitis, eosinophilic pneumonias and persistent fungal infections. Other disorders include pulmonary fibrosis, systemic sclerosis, ideopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, cancers of the lung such as adenocarcinoma, squamous cell carcinoma, small cell and large cell carcinomas, and benign neoplasms of the lung including bronchial adenomas and hamartomas.

The present invention also provides methods for monitoring the effect of a treatment regimen in an individual by monitoring the expression for one or more informative genes. For example, a baseline expression level for one or more informative genes from the individual can be determined, and repeated gene expression levels can be determined at time points during treatment. A shift in gene expression from a profile correlated with the disorder or with poor treatment outcome to a profile correlated with lack of the disorder or improved treatment outcome is evidence of an effective therapeutic regimen, while a repeated profile correlated with the disorder or with poor treatment outcome is evidence of an ineffective therapeutic regimen. It is also clear that the present invention can be used to generate databases comprising informative genes which will have many applications in medicine, research and industry and which can be used as a point of comparison in many of the analyses described herein. The present invention has many preferred embodiments and relies on many patents, applications and other references for details known to those of the art. Therefore, when a patent, application, or other reference is cited herein, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.

EXAMPLES Example 1 Methods

Samples of epithelial cells, obtained by brushing airway surfaces, were obtained from intra- and extra-pulmonary airways in 11 normal non-smokers (NS), 15 smokers without lung cancer (S), and 9 smokers with lung cancer (SC). 5-10 μg of RNA was extracted using standard trizol-based methods, quality of RNA was assayed in gels, and the RNA was processed using standard protocols developed by Affymetrix for the Ul 33 human array. Expression profiles, predictive algorithms, and identification of critical genes are made using bioinformatic methods. In particular, "Significance analysis of microarrays" (SAM) software (Tusher et ah, PNAS, 98:5116-21, 2001) and "Prediction analysis for microarrays" (PAM) software (Tibshirani et al, PNAS, 99:6561-12, 2002) (both Stanford University Proteomics Center) were used.

For the analysis shown in FIGS. 4A-4P, samples were obtained as described above from 6 smokers with lung cancer (SC), 9 smokers without lung cancer (S) and 5 normal non-smokers (NS). Results

FIGS. IA- IN show genes which are differentially expressed in smokers and smokers with cancer; FIGS. 2A-2F show genes which are differentially expressed in smokers with cancer and non- smokers; and FIGS. 3A-3D show genes which are differentially expressed in smokers and non- smokers.

FIGS. 4A-4P and 5A-5F show 131 genes which are differentially expressed and can be used to distinguish between non-smokers, smokers and smokers with cancer.

FIGS. 6A-6R show genes which are differentially expressed in smokers and non-smokers.

FIGS. IA-TL is a table of genes which are differentially expressed in samples from smokers (S), nonsmokers (NS) and smokers with cancer (C).

FIGS. 8A-8E is a table of genes which are differentially expressed in samples from smokers (S) and smokers with cancer (C).

The contents of all, references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAMSWhat is claimed is:

1. A method of diagnosing a lung cancer comprising: a) obtaining a nucleic acid sample from an individual to be diagnosed;

b) determining the expression of one or more informative genes in said sample, wherein the informative genes are selected from the group consisting of the genes shown in Figures 1 A-IN, 2A-2F, 3A-3D, 4A- 4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E, wherein increased expression of an informative gene whose expression is increased in individuals having a lung cancer, or decreased expression of an informative gene whose expression is decreased in individuals having a lung cancer, is indicative of a lung cancer in the individual.

2. A method according to Claim 1, wherein the lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and benign neoplasms of the lung.

3. A method according to Claim 2, wherein the lung cancer is a benign neoplasm selected from the group consisting of bronchial adenomas and hamartomas.

4. A method according to Claim 1, wherein the nucleic acid sample is RNA.

5. A method according to Claim 1, wherein the individual to be diagnosed is an individual who has been exposed to tobacco smoke.

6. A method according to Claim 1, wherein the individual to be diagnosed is an individual who has smoked.

7. A method according to Claim 1, wherein the individual to be diagnosed is an individual who smokes.

8. A method according to Claim 1, wherein the expression of two or more informative genes is determined.

9. A method according to Claim 1, wherein the expression of five or more informative genes is determined.

10. A method according to Claim 1, wherein the expression often or more informative genes is determined.

11. A method according to Claim 1 , wherein the expression of fifteen or more informative genes is determined.

12. A method according to Claim 1 , wherein the expression of twenty or more informative genes is determined.

13. A method according to Claim 1 , wherein the expression of fifty or more informative genes is determined.

14. A method according to Claim 1 , wherein the expression of one hundred or more informative genes is determined.

15. A method according to Claim 1, wherein the nucleic acid sample is obtained from an airway epithelial cell.

16. A method according to Claim 15, wherein the airway epithelial cell is obtained from a bronchoscopy or buccal mucosal scraping.

17. A method according to Claim 1, wherein the expression is determined using a microarry having one or more probes for said one or more genes immobilized thereon.

18. A method of treating a lung cancer comprising administering to an individual in need thereof an effective amount of an agent which increases the expression of an informative gene whose expression is decreased in said individual as compared with a normal individual, wherein the informative gene is selected from the group consisting of the genes shown in Figures IAIN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E.

19. A method of treating a lung cancer comprising administering to an individual in need thereof an effective amount of an agent which decreases the expression of an informative gene whose ^"expression is increased in said individual as compared with a normal individual, wherein the informative gene is selected from the group consisting of the genes shown in Figures IA- IN, 2A-2F, 3 A-3D, 4A-4P, 5 A-5F, 6A-6R, 7A-7L and 8 A-8E.

20. A method of treating a lung cancer comprising administering to an individual in need thereof an effective amount of an agent which increases the activity of an expression product of an informative gene whose activity is decreased in said individual as compared with a normal individual, wherein the informative gene is selected from the group consisting of the genes shown in

Figures 1A-1N, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E.

21. A method of treating a lung cancer comprising administering to an individual in need thereof an effective amount of an agent which decreases the activity of an expression product of an informative gene whose activity is increased in said individual as compared with a normal individual, wherein the informative gene is selected from the group consisting of the genes shown in Figures 1 A-IN, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E.

22. A substrate having immobilized thereon a plurality of less than one thousand polymers which bind specifically to an expression product of one or more genes selected from the group consisting essentially of the genes shown in Figures 1A-1N, 2A-2F, 3A-3D, 4A-4P, 5A-5F, 6A-6R, 7A-7L and 8A-8E.