CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Application No. 61/783,983, filed March 14, 2013. The entire contents of this referenced application are incorporated by reference herein.

FIELD OF THE INVENTION

The invention generally relates to methods and compositions for assessing pulmonary disease risk using genomic information.

BACKGROUND OF INVENTION

Chronic obstructive pulmonary disease (COPD) is a group of lung conditions, including emphysema, chronic bronchitis, and bronchiectasis, which are characterized by largely irreversible airflow obstruction. COPD causes considerable morbidity and mortality. Currently, it represents the fourth leading cause of death in the world, and it is expected to increase both in prevalence and in mortality over the next decades.

SUMMARY OF INVENTION

Provided herein are methods for characterizing the COPD status of a subject. In some embodiments, methods provided herein are useful for establishing appropriate diagnostic intervention plans and/or treatment plans for subjects having or suspected of having COPD, and for aiding healthcare providers in establishing such plans. Accordingly, in some embodiments, methods provided herein are useful for diagnosing COPD in subjects and managing treatment of such subjects. In some embodiments, methods are provided that involve assessing the COPD status of a subject based on expression levels of informative-genes in a biological sample obtained from a subject during a routine cell or tissue sampling procedure. In some

embodiments, methods are provided that involve establishing COPD risk scores based on expression levels of informative-genes. In some embodiments, appropriate diagnostic intervention plans are established based at least in part on the COPD risk scores. In some embodiments, the methods assist health care providers with making early and accurate diagnoses. In some embodiments, the methods assist health care providers with establishing appropriate therapeutic interventions early on in patient clinical evaluations. In some embodiments, the methods involve evaluating biological samples obtained during bronchoscopic procedures. In some embodiments, the methods are beneficial because they enable health care providers to make informative decisions regarding patient diagnosis and/or treatment from otherwise uninformative bronchoscopies. In some embodiments, the risk assessment leads to appropriate surveillance for monitoring low risk lesions. In some embodiments, the risk assessment leads to faster diagnosis, and thus, faster treatment for COPD.

Certain methods described herein, alone or in combination with other methods, provide useful information for health care providers to assist them in making diagnostic and treatment decisions for a patient. Certain methods disclosed herein are employed in instances where other methods have failed to provide useful information regarding the COPD status of a patient.

Certain methods disclosed herein provide an alternative or complementary method for evaluating cell or tissue samples obtained during routine bronchoscopy procedures, and increase the likelihood that the procedures will result in useful information for managing a patient's care. The methods disclosed herein are highly sensitive, and produce information regarding the likelihood that a subject has COPD from cell or tissue samples (e.g., histologically normal tissue) that may be obtained from positions remote from diseased tissue. Certain methods described herein can be used to assess the likelihood that a subject has COPD by evaluating histologically normal cells or tissues obtained during a routine cell or tissue sampling procedure (e.g., standard ancillary bronchoscopic procedures such as brushing, biopsy, lavage, and needle- aspiration). However, it should be appreciated that any suitable tissue or cell sample can be used. Often the cells or tissues that are assessed by the methods appear histologically normal.

In some embodiments, the methods are useful for confirming that a subject has or does not have COPD. According to some aspects of the invention, methods are provided for evaluating the COPD status of a subject using gene expression information that involve one or more of the following acts: (a) obtaining a biological sample from the respiratory tract of a subject, (b) subjecting the biological sample to a gene expression analysis, in which the gene expression analysis comprises determining the expression levels of a plurality of informative- genes in the biological sample, (c) computing a COPD risk score based on the expression levels of the plurality of informative-genes, (d) determining that the subject is in need of a first diagnostic intervention to evaluate COPD status, if the level of the COPD risk score is beyond (e.g., above) a first threshold level, and (e) determining that the subject is in need of a second diagnostic intervention to evaluate COPD status, if the level of the COPD risk score is beyond (e.g., below) a second threshold level. In some embodiments, the subject has been identified as a candidate for bronchoscopy and/or as having a suspicious lesion in the respiratory tract. In some embodiments, the subject is not a candidate for bronchoscopy. For example, a subject may have such severe airway damage that bronchoscopy is not possible. In some embodiments, the methods further comprise (f) determining that the subject is in need of a third diagnostic intervention to evaluate COPD status, if the level of the COPD risk score is between the first threshold and the second threshold levels.

In some embodiments, a diagnostic intervention (e.g., first or second or third

intervention) comprises performing spirometry on the subject to evaluate pulmonary function. In some embodiments, spirometry is used to determine a forced expiratory volume in one second. Forced Expiratory Volume in one second (FEV is the amount of air which can be forcibly exhaled from the lungs of a subject in the first second of a forced exhalation. In some embodiments, spirometry is used to determine a forced vital capacity. Forced vital capacity (FVC) is the volume of air that can forcibly be blown out by a subject after full inspiration. In some embodiments, a diagnostic intervention (e.g., first or second or third intervention) comprises assessing whether a subject has irreversible airflow obstruction. In some

embodiments, irreversible airflow obstruction is assessed by determining a post-bronchodilator forced expiratory volume in one second to forced vital capacity ratio (FEVVFVC) . In some embodiments, a FEVVFVC of less than 0.7 or less than 0.75 is indicative of irreversible airflow obstruction. In some embodiments, a diagnostic intervention (e.g., first or second or third

intervention) comprises engaging in watchful waiting (or monitoring). In some embodiments, watchful waiting comprises periodically performing spirometry to evaluate lung function or imaging the respiratory tract to evaluate tissue. In some embodiments, watchful waiting comprises periodically performing spirometry to evaluate lung function or imaging the respiratory tract to evaluate a tissue for up to one year, two years, four years, five years or more. In some embodiments, watchful waiting comprises performing spirometry to evaluate lung function or imaging the respiratory tract to evaluate tissue at least once per year. In some embodiments, watchful waiting comprises performing spirometry to evaluate lung function or imaging the respiratory tract to evaluate tissue at least twice per year. In some embodiments, watchful waiting (or monitoring) comprises periodically repeating steps (a) to (e). In some embodiments, watchful waiting comprises periodically repeating steps (a) to (f). In some embodiments, the third diagnostic intervention comprises performing a bronchoscopy or spirometry or other procedure to evaluate lung function or health. In some embodiments, the third diagnostic intervention comprises repeating steps (a) to (e). In certain embodiments, the third diagnostic intervention comprises repeating steps (a) to (e) within six months of determining that the COPD risk score is between the first threshold and the second threshold levels. In certain embodiments, the third diagnostic intervention comprises repeating steps (a) to (e) within three months of determining that the COPD risk score is between the first threshold and the second threshold levels. In some embodiments, the third diagnostic intervention comprises repeating steps (a) to (e) within one month of determining that the COPD risk score is between the first threshold and the second threshold levels. Any one or more of a number of different treatment interventions may be established for a subject identified as having COPD or at risk of having COPD according to methods provided herein. In some embodiments, the intervention is aiding the subject in a smoking cessation program, which may or may not involve treatment with a therapeutic to minimize withdrawal side-effects or a nicotine replacement agent. In some embodiment, pharmacologic and/or non- pharmacologic therapy for COPD is used to reduce symptoms, reduce the frequency and severity of exacerbations, and/or improve health status and exercise tolerance. In some embodiment, a therapy for COPD is selected based on the results of the assessment or diagnostic methods disclosed herein. In some embodiments, a pharmacologic therapy for COPD is selected from: beta2-agonists that are short-acting, such as fenoterol, levalbuterol, salbutamol (albuterol), and terbutaline; beta2-agonists that are long-acting, such as formoterol, arformoterol, indacaterol, salmeterol and tulobuterol; anticholinergics that are short-acting, such as ipratropium bromide and oxitropium bromide; anticholinergics that are long-acting, such as aclidinium bromide, glycopyrronium bromide and tiotropium; a combination short-acting beta2- agonists plus anticholinergic in one inhaler, such as fenoterol/ipratropium and

salbutamol/ipratropium; methylxanthines such as aminophylline and theophylline; inhaled corticosteroids such as beclomethasone, budesonide and fluticasone; a combination long-acting beta2-agonists plus corticosteroids in one inhaler, such as formoterol/budesonide,

formoterol/mometasone and salmeterol/fluticasone; systemic corticosteroids such as prednisone and methyl-prednisolone; and phosphodiesterase-4 inhibitors such as roflumilast. In some embodiments, a non-pharmacologic therapy for COPD is selected from: pulmonary

rehabilitation (e.g., to reduce symptoms, improve quality of life, and/or increase physical and emotional participation in everyday activities); oxygen therapy (e.g., long-term administration of oxygen (>15 hours per day) to patients with chronic respiratory failure); ventilatory support; surgical treatment, such as lung volume reduction surgery (LVRS); bronchoscopic lung volume reduction (BLVR), lung transplantation, or bullectomy; a smoking cessation program; and physical activity.

In some embodiments, the plurality of informative genes comprises informative-mRNAs.

In some embodiments, the plurality of informative genes comprises at least two informative- mRNAs. In some embodiments, the plurality of informative genes is selected from the group of genes in Table 2. In some embodiments, the expression levels of a subset of these genes are evaluated and compared to reference expression levels (e.g., for normal patients that do not have COPD). In some embodiments, the subset includes a) genes for which an increase in expression is associated with COPD or an increased risk for COPD, b) genes for which a decrease in expression is associated with COPD or an increased risk for COPD, or both. In some

embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or about 50% of the genes in a subset have an increased level of expression in association with an increased risk for COPD. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or about 50% of the genes in a subset have a decreased level of expression in association with an increased risk for COPD. In some embodiments, an expression level is evaluated (e.g., assayed or otherwise interrogated) for each of 10-80 or more genes (e.g., 5-10, 10-20, 20-30, 30- 40, 40-50, 50-60, 60-70, 70-80, about 10, about 15, about 25, about 35, about 45, about 55, about 65, about 75, or more genes) selected from the genes in Table 2. In some embodiments, expression levels for one or more control genes also are evaluated (e.g., 1, 2, 3, 4, or 5 of the control genes). It should be appreciated that an assay can also include other genes, for example reference genes or other gene (regardless of how informative they are). However, if the expression profile for any of the informative gene subsets described herein is indicative of an increased risk for COPD, then an appropriate therapeutic or diagnostic recommendation can be made as described herein.

In some embodiments, the identification of changes in expression level of one or more subsets of genes from Table 2 can be provided to a physician or other health care professional in any suitable format. These gene expression profiles alone may be sufficient for making a diagnosis, providing a prognosis, or for recommending further diagnosis or a particular treatment. However, in some embodiments the gene expression profiles may assist in the diagnosis, prognosis, and/or treatment of a subject along with other information (e.g., other mRNA or miRNA expression information, and/or other physical or chemical information about the subject, including family history).

In some embodiments, a subject is identified as having a suspicious lesion in the respiratory tract by imaging the respiratory tract. In certain embodiments, imaging the respiratory tract comprises performing computer-aided tomography, magnetic resonance imaging, ultrasonography or a chest X-ray.

Methods are provided, in some embodiments, for obtaining biological samples from patients. Expression levels of informative-genes in these biological samples provide a basis for assessing the likelihood that the patient has COPD. Methods are provided for processing biological samples. In some embodiments, the processing methods ensure RNA quality and integrity to enable downstream analysis of informative-genes and ensure quality in the results obtained. Accordingly, various quality control steps (e.g. , RNA size analyses) may be employed in these methods. Methods are provided for packaging and storing biological samples. Methods are provided for shipping or transporting biological samples, e.g., to an assay laboratory where the biological sample may be processed and/or where a gene expression analysis may be performed. Methods are provided for performing gene expression analyses on biological samples to determine the expression levels of informative-genes in the samples. Methods are provided for analyzing and interpreting the results of gene expression analyses of informative- genes. Methods are provided for generating reports that summarize the results of gene expression analyses, and for transmitting or sending assay results and/or assay interpretations to a health care provider (e.g. , a physician). Furthermore, methods are provided for making treatment decisions based on the gene expression assay results, including making

recommendations for further treatment or invasive diagnostic procedures.

In some embodiments, aspects of the invention are based, at least in part, on the determination that the expression level of certain informative-genes in apparently histologically normal cells obtained from a first airway locus can be used to evaluate the likelihood of COPD at a second locus in the airway (for example, at a locus in the airway that is remote from the locus at which the histologically normal cells were sampled).

In some embodiments, aspects of the invention relate to determining the likelihood that a subject has COPD, by subjecting a biological sample obtained from a subject to a gene expression analysis, wherein the gene expression analysis comprises determining expression levels in the biological sample of at least one informative-genes (e.g. , at least two mRNAs selected from Table 2), and using the expression levels to assist in determining the likelihood that the subject has COPD.

In some embodiments, the step of determining comprises transforming the expression levels into a COPD risk-score that is indicative of the likelihood that the subject has COPD. In some embodiments, the COPD risk-score is the combination of weighted expression levels. In some embodiments, the COPD risk-score is the sum of weighted expression levels. In some embodiments, the expression levels are weighted by their relative contribution to predicting increased likelihood of having COPD.

In some embodiments, aspects of the invention relate to determining a treatment course for a subject, by subjecting a biological sample obtained from the subject to a gene expression analysis, wherein the gene expression analysis comprises determining the expression levels in the biological sample of at least two informative-genes (e.g. , at least two mRNAs selected from Table 2), and determining a treatment course for the subject based on the expression levels. In some embodiments, the treatment course is determined based on a COPD risk-score derived from the expression levels. In some embodiments, the subject is identified as a candidate for a COPD therapy based on a COPD risk-score that indicates the subject has a relatively high likelihood of having COPD. In some embodiments, the subject is identified as a candidate for an invasive lung procedure based on a COPD risk-score that indicates the subject has a relatively high likelihood of having COPD. In some embodiments, the invasive lung procedure is a transthoracic needle aspiration, mediastinoscopy or thoracotomy. In some embodiments, the subject is identified as not being a candidate for a COPD therapy or an invasive lung procedure based on a COPD risk-score that indicates the subject has a relatively low likelihood of having COPD. In some embodiments, a report summarizing the results of the gene expression analysis is created. In some embodiments, the report indicates the COPD risk-score.

In some embodiments, aspects of the invention relate to determining the likelihood that a subject has COPD by subjecting a biological sample obtained from a subject to a gene expression analysis, wherein the gene expression analysis comprises determining the expression levels in the biological sample of at least one informative-gene (e.g., at least one informative- mPvNA selected from Table 2), and determining the likelihood that the subject has COPD based at least in part on the expression levels.

In some embodiments, aspects of the invention relate to determining the likelihood that a subject has COPD, by subjecting a biological sample obtained from the respiratory epithelium of a subject to a gene expression analysis, wherein the gene expression analysis comprises determining the expression level in the biological sample of at least one informative-gene (e.g. , at least one informative-mRNA selected from Table 2), and determining the likelihood that the subject has COPD based at least in part on the expression level, wherein the biological sample comprises histologically normal tissue.

In some embodiments, aspects of the invention relate to a computer-implemented method for processing genomic information, by obtaining data representing expression levels in a biological sample of at least two informative-genes (e.g. , at least two informative-mRNAs from Table 2), wherein the biological sample was obtained of a subject, and using the expression levels to assist in determining the likelihood that the subject has COPD. A computer- implemented method can include inputting data via a user interface, computing (e.g. , calculating, comparing, or otherwise analyzing) using a processor, and/or outputting results via a display or other user interface.

In some embodiments, the step of determining comprises calculating a risk-score indicative of the likelihood that the subject has COPD. In some embodiments, computing the risk-score involves determining the combination of weighted expression levels, wherein the expression levels are weighted by their relative contribution to predicting increased likelihood of having COPD. In some embodiments, a computer-implemented method comprises generating a report that indicates the risk-score. In some embodiments, the report is transmitted to a health care provider of the subject.

It should be appreciated that in any embodiment or aspect described herein, a biological sample can be obtained from the respiratory epithelium of the subject. The respiratory epithelium can be of the mouth, nose, pharynx, trachea, bronchi, bronchioles, or alveoli.

However, other sources of respiratory epithelium also can be used. The biological sample can comprise histologically normal tissue. The biological sample can be obtained using bronchial brushings, broncho-alveolar lavage, or a bronchial biopsy. The subject can exhibit one or more symptoms of COPD and/or have a lesion that is observable by computer-aided tomography or chest X-ray.

In any of the embodiments or aspects described herein, the expression levels can be determined using a quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay, an oligonucleotide array assay, or other technique.

In some embodiments, aspects of the invention relate to a composition consisting essentially of at least one nucleic acid probe, wherein each of the at least one nucleic acids probe specifically hybridizes with an informative-gene (e.g., at least one informative-mRNA selected from Table 2).

In some embodiments, aspects of the invention relate to a composition comprising up to 5, up to 10, up to 25, up to 50, up to 100, or up to 200 nucleic acid probes, wherein each of at least two of the nucleic acid probes specifically hybridizes with an informative-gene (e.g. , at least one informative-mRNA selected from Table 2).

In some embodiments, nucleic acid probes are conjugated directly or indirectly to a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the nucleic acid probes are immobilized to a solid support. In some embodiments, the solid support is a glass, plastic or silicon chip.

In some embodiments, aspects of the invention relate to a kit comprising at least one container or package housing any nucleic acid probe composition described herein.

In some embodiments, expression levels are determined using a quantitative reverse transcription polymerase chain reaction.

According to some aspects of the invention, kits are provided that comprise primers for amplifying at least one informative-genes selected from Table 2. In some embodiments, the kits (e.g., gene arrays) comprise at least one primer for amplifying at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 informative- genes selected from Table 2. In some embodiments, the kits (e.g., gene arrays) comprise at least one primer for amplifying up to 5, up to 10, up to 25, up to 50, up to 75, up to 100 informative- genes selected from Table 2. In some embodiments, the kits comprise primers that consist essentially of primers for amplifying each of the informative-genes listed in Table 2. In some embodiments, the gene arrays comprise primers for amplifying one or more control genes, such as ACTB, GAPDH, YWHAZ, POLR2A, DDX3Y, or other control genes. In some

embodiments, ACTB, GAPDH, YWHAZ, and POLR2A are used as control genes for normalizing expression levels. In some embodiments, DDX3Y is a semi-identity control because it is a gender specific gene, which is generally more highly expressed in males than females. Thus, DDX3Y can be used in some embodiments to determine whether a sample is from a male or female subject. This information can be used to confirm accuracy of personal information about a subject and exclude samples during data analysis if the information is inconsistent with DDX3Y expression information. For example, if personal information indicates that a subject is female but DDX3Y is highly expressed in a sample (indicating a male subject), the sample can be excluded. Control genes can be used for normalization singularly or in any combination including with one or more additional control genes..

These and other aspects are described in more detail herein and are illustrated by the non- limiting figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ROC curve for a COPD gene-signature that differentiates subjects with and without COPD, resulting in an overall AUC of >0.80.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

In some embodiments, aspects of the invention relate to genes for which expression levels can be used to determine the likelihood that a subject (e.g., a human subject) has COPD. As used herein, "chronic obstructive pulmonary disease", or "COPD," is a disease characterized by a persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. In some embodiments, the chronic airflow limitation characteristic of COPD is caused by small airway disease (obstructive bronchiolitis) and/or parenchymal destruction (emphysema). In some embodiments, chronic inflammation causes structural changes and narrowing of the small airways and narrowing of the small airways. In some embodiments, destruction of the lung parenchyma, which also may be due to inflammatory processes, leads to the loss of alveolar attachments to the small airways and decreased lung elastic recoil. In some embodiments, these changes diminish the ability of the airways to remain open during expiration. In some embodiments, airflow limitation associated with COPD may be measured by spirometry, which is a widely available, reproducible test of lung function. In some embodiments, COPD is a disease classified by World Health Organization (WHO), according to the International

Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10), within the category of "COPD and allied conditions" (ICD-10 codes J42-46).

In some embodiments, methods provided herein comprise monitoring COPD progression and development of complications associated with COPD. In some of such embodiments, the methods may include, for example, monitoring expression of one or more of the informative- genes of Table 2; evaluating lung function by spirometry (e.g., at least once a year); evaluating smoking status and environment exposures (e.g., at least once per year); monitoring pharmacotherapy and other medical treatments for COPD; monitoring exacerbation history; and/or monitoring the presence or absence of comorbidities.

In some embodiments, COPD is diagnosed, at least in part, by using a pulmonary function test. In some embodiments, COPD is coincident with emphysema. In some

embodiments, a subject who has COPD has increased risk of developing lung cancer. In some embodiments, COPD it is indicative of a premalignant state.

In some embodiments, the expression levels (e.g., mRNA levels) of one or more genes described herein can be determined in airway samples (e.g., epithelial cells or other samples obtained during a bronchoscopy or from an appropriate bronchial lavage samples). In some embodiments, the patterns of increased and/or decreased mRNA expression levels for one or more subsets of useful genes (e.g., 1-5, 5-10, 10-15, 15-20, 20-25, 25-50, 50-80, or more genes) described herein can be determined and used for diagnostic, prognostic, and/or therapeutic purposes. It should be appreciated that one or more expression patterns described herein can be used alone, or can be helpful along with one or more additional patient-specific indicia or symptoms, to provide personalized diagnostic, prognostic, and/or therapeutic predictions or recommendations for a patient.

In some embodiments, provided herein are methods for establishing appropriate diagnostic intervention plans and/or treatment plans for subjects and for aiding healthcare providers in establishing appropriate diagnostic intervention plans and/or treatment plans. In some embodiments, methods are provided that involve making a risk assessment based on expression levels of informative-genes in a biological sample obtained from a subject during a routine cell or tissue sampling procedure. In some embodiments, methods are provided that involve establishing COPD risk scores based on expression levels of informative-genes. In some embodiments, appropriate diagnostic intervention plans are established based at least in part on the COPD risk scores. In some embodiments, methods provided herein assist health care providers with making early and accurate diagnoses. In some embodiments, methods provided herein assist health care providers with establishing appropriate therapeutic interventions early on in patients' clinical evaluations. In some embodiments, methods provided herein involve evaluating biological samples obtained during bronchoscopies procedure. In some embodiments, the methods are beneficial because they enable health care providers to make informative decisions regarding patient diagnosis and/or treatment from otherwise uninformative

bronchoscopies. In some embodiments, the risk assessment leads to appropriate surveillance for monitoring low risk lesions. Provided herein are methods for determining the likelihood that a subject has COPD. The methods alone or in combination with other methods provide useful information for health care providers to assist them in making diagnostic and therapeutic decisions for a patient. The methods disclosed herein are often employed in instances where other methods have failed to provide useful information regarding the COPD status of a patient. For example, approximately 50% of bronchoscopy procedures result in indeterminate or non-diagnostic information. There are multiple sources of indeterminate results, and may depend on the training and procedures available at different medical centers. However, in certain embodiments, molecular methods in combination with bronchoscopy are expected to improve COPD assessment accuracy.

Methods disclosed herein provide alternative or complementary approaches for evaluating cell or tissue samples obtained by bronchoscopy procedures (or other procedures for evaluating respiratory tissue), and increase the likelihood that the procedures will result in useful information for managing the patient' s care. The methods disclosed herein are highly sensitive, and produce information regarding the likelihood that a subject has COPD from cell or tissue samples (e.g. , bronchial brushings of airway epithelial cells) , which are often obtained from regions in the airway that are remote from malignant lung tissue. In general, the methods disclosed herein involve subjecting a biological sample obtained from a subject to a gene expression analysis to evaluate gene expression levels. However, in some embodiments, the likelihood that the subject has COPD is determined in further part based on the results of a histological examination of the biological sample or by considering other diagnostic indicia such as protein levels, mRNA levels, imaging results, chest X-ray exam results etc.

The term "subject," as used herein, generally refers to a mammal. Typically the subject is a human. However, the term embraces other species, e.g. , pigs, mice, rats, dogs, cats, or other primates. In certain embodiments, the subject is an experimental subject such as a mouse or rat. The subject may be a male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. The subject may be a smoker, a former smoker or a non-smoker. The subject may have a personal or family history of COPD or other lung disorder, including lung cancer. In some embodiments, the subject has one or more indicators that suggest COPD. In some embodiments, indicators that suggest COPD include dyspenea, which may be progressive (worsens over time), characteristically worse with exercise, and/or persistent;

chronic cough, which may be intermittent and/or unproductive; chronic sputum production; history of tobacco smoke; history of exposure to smoke from home cooking and/or heating fuels; history of exposure to occupational dusts and chemicals; and/or a family history of COPD. In some embodiments, the subject may exhibit one or more symptoms of COPD or other lung disorder. In some embodiments, the subject may have a new or persistent cough, worsening of an existing chronic cough, persistent bronchitis or repeated respiratory infections, chest pain, unexplained weight loss and/or fatigue, or breathing difficulties such as shortness of breath or wheezing. The subject may have a lesion, which may be observable by computer- aided tomography or chest X-ray. The subject may be a subject who has undergone a bronchoscopy or who has been identified as a candidate for bronchoscopy (e.g. , because of the presence of a detectable lesion or suspicious imaging result). A subject under the care of a physician or other health care provider may be referred to as a "patient."

Informative-genes

The expression levels of certain genes have been identified as providing useful information regarding the COPD status of a subject. These genes are referred to herein as "informative-genes." Informative-genes include protein coding genes and non-protein coding genes. It will be appreciated by the skilled artisan that the expression levels of informative-genes may be determined by evaluating the levels of appropriate gene products (e.g., mRNAs, miRNAs, proteins etc.) Accordingly, the expression levels of certain mRNAs have been identified as providing useful information regarding the lung cancer status of a subject. These mRNAs are referred to herein as "informative-mRNAs." Table 2 provide a listing of informative-genes that are differentially expressed in COPD.

Certain methods disclosed herein involve determining expression levels in the biological sample of at least one informative-gene. However, in some embodiments, the expression analysis involves determining the expression levels in the biological sample of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, least 80, or at least 90 informative-genes.

In some embodiments, the number of informative-genes for an expression analysis are sufficient to provide a level of confidence in a prediction outcome that is clinically useful. This level of confidence (e.g. , strength of a prediction model) may be assessed by a variety of performance parameters including, but not limited to, the accuracy, sensitivity specificity, and area under the curve (AUC) of the receiver operator characteristic (ROC). These parameters may be assessed with varying numbers of features (e.g. , number of genes, mRNAs) to determine an optimum number and set of informative-genes. An accuracy, sensitivity or specificity of at least 60%, 70%, 80%, 90%, may be useful when used alone or in combination with other information.

Any appropriate system or method may be used for determining expression levels of informative-genes. Gene expression levels may be determined through the use of a

hybridization-based assay. As used herein, the term, "hybridization-based assay" refers to any assay that involves nucleic acid hybridization. A hybridization-based assay may or may not involve amplification of nucleic acids. Hybridization-based assays are well known in the art and include, but are not limited to, array-based assays (e.g. , oligonucleotide arrays, microarrays), oligonucleotide conjugated bead assays (e.g., Multiplex Bead-based Luminex® Assays), molecular inversion probe assays, and quantitative RT-PCR assays. Multiplex systems, such as oligonucleotide arrays or bead-based nucleic acid assay systems are particularly useful for evaluating levels of a plurality of genes simultaneously. Other appropriate methods for determining levels of nucleic acids will be apparent to the skilled artisan.

As used herein, a "level" refers to a value indicative of the amount or occurrence of a substance, e.g. , an mRNA. A level may be an absolute value, e.g., a quantity of an mRNA in a sample, or a relative value, e.g. , a quantity of an mRNA in a sample relative to the quantity of the mRNA in a reference sample (control sample). The level may also be a binary value indicating the presence or absence of a substance. For example, a substance may be identified as being present in a sample when a measurement of the quantity of the substance in the sample, e.g. , a fluorescence measurement from a PCR reaction or microarray, exceeds a background value. Similarly, a substance may be identified as being absent from a sample (or undetectable in the sample) when a measurement of the quantity of the molecule in the sample is at or below background value. It should be appreciated that the level of a substance may be determined directly or indirectly.

Biological Samples

The methods generally involve obtaining a biological sample from a subject. As used herein, the phrase "obtaining a biological sample" refers to any process for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample may be obtained (e.g., at a point-of-care facility, a physician' s office, a hospital) by procuring a tissue or fluid sample from a subject. Alternatively, a biological sample may be obtained by receiving the sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject. The term "biological sample" refers to a sample derived from a subject, e.g., a patient. A biological sample typically comprises a tissue, cells and/or biomolecules. In some

embodiments, a biological sample is obtained on the basis that it is histologically normal, e.g., as determined by endoscopy, e.g., bronchoscopy. In some embodiments, the biological sample is a sample of respiratory epithelium. The respiratory epithelium may be of the mouth, nose, pharynx, trachea, bronchi, bronchioles, or alveoli of the subject. The biological sample may comprise epithelium of the bronchi. In some embodiments, the biological sample is free of detectable cancer cells, e.g., as determined by standard histological or cytological methods. In some embodiments, histologically normal samples are obtained for evaluation. Often biological samples are obtained by scrapings or brushings, e.g., bronchial brushings. However, it should be appreciated that other procedures may be used, including, for example, brushings, scrapings, broncho-alveolar lavage, a bronchial biopsy or a transbronchial needle aspiration.

It is to be understood that a biological sample may be processed in any appropriate manner to facilitate determining expression levels. For example, biochemical, mechanical and/or thermal processing methods may be appropriately used to isolate a biomolecule of interest, e.g., RNA, from a biological sample. Accordingly, a RNA or other molecules may be isolated from a biological sample by processing the sample using methods well known in the art.

COPD Assessment

Methods disclosed herein may involve comparing expression levels of informative- genes with one or more appropriate references. An "appropriate reference" is an expression level (or range of expression levels) of a particular informative-gene that is indicative of a known COPD status. An appropriate reference can be determined experimentally by a practitioner of the methods or can be a pre-existing value or range of values. An appropriate reference represents an expression level (or range of expression levels) indicative of COPD. For example, an appropriate reference may be representative of the expression level of an

informative-gene in a reference (control) biological sample obtained from a subject who is known to have COPD. When an appropriate reference is indicative of COPD, a lack of a detectable difference (e.g., lack of a statistically significant difference) between an expression level determined from a subject in need of characterization or diagnosis of COPD and the appropriate reference may be indicative of COPD in the subject. When an appropriate reference is indicative of COPD, a difference between an expression level determined from a subject in need of characterization or diagnosis of COPD and the appropriate reference may be indicative of the subject being free of COPD. In some embodiments, expression levels of informative- genes can be used to determine the severity of COPD in a subject.

Alternatively, an appropriate reference may be an expression level (or range of expression levels) of a gene that is indicative of a subject being free of COPD. For example, an appropriate reference may be representative of the expression level of a particular informative- gene in a reference (control) biological sample obtained from a subject who is known to be free of COPD. When an appropriate reference is indicative of a subject being free of COPD, a difference between an expression level determined from a subject in need of diagnosis of COPD and the appropriate reference may be indicative of COPD in the subject. Alternatively, when an appropriate reference is indicative of the subject being free of COPD, a lack of a detectable difference (e.g., lack of a statistically significant difference) between an expression level determined from a subject in need of diagnosis of COPD and the appropriate reference level may be indicative of the subject being free of COPD.

In some embodiments, the reference standard provides a threshold level of change, such that if the expression level of a gene in a sample is within a threshold level of change (increase or decrease depending on the particular marker) then the subject is identified as free of COPD, but if the levels are above the threshold then the subject is identified as being at risk of having COPD.

For example, increased expression of an mRNA that has a positive weight in the last column of Table 2, compared with the reference standard, is indicative of the subject having COPD. Furthermore, decreased expression of an mRNA that has a negative weight in the last column of Table 2, compared with the reference standard, is indicative of the subject having COPD.

The magnitude of difference between a expression level and an appropriate reference that is statistically significant may vary. For example, a significant difference that indicates

COPD may be detected when the expression level of an informative-gene in a biological sample is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000% higher, or lower, than an appropriate reference of that gene. Similarly, a significant difference may be detected when the expression level of informative- gene in a biological sample is at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or more higher, or lower, than the appropriate reference of that gene. In some embodiments, at least a 20% to 50% difference in expression between an informative-gene and appropriate reference is significant. Significant differences may be identified by using an appropriate statistical test. Tests for statistical significance are well known in the art and are exemplified in Applied Statistics for Engineers and Scientists by Petruccelli, Chen and Nandram 1999 Reprint Ed.

It is to be understood that a plurality of expression levels may be compared with plurality of appropriate reference levels, e.g., on a gene-by-gene basis. , in order to assess the COPD status of the subject. The comparison may be made as a vector difference. In such cases, Multivariate Tests, e.g., Hotelling's T test, may be used to evaluate the significance of observed differences. Such multivariate tests are well known in the art and are exemplified in Applied Multivariate Statistical Analysis by Richard Arnold Johnson and Dean W. Wichern Prentice Hall; 4 ^th edition (July 13, 1998).

Classification Methods

The methods may also involve comparing a set of expression levels (referred to as an expression pattern or profile) of informative-genes in a biological sample obtained from a subject with a plurality of sets of reference levels (referred to as reference patterns), each reference pattern being associated with a known COPD status, identifying the reference pattern that most closely resembles the expression pattern, and associating the known COPD status of the reference pattern with the expression pattern, thereby classifying (characterizing) the COPD status of the subject.

The methods may also involve building or constructing a prediction model, which may also be referred to as a classifier or predictor, that can be used to classify the disease status of a subject. As used herein, a "COPD-classifier" is a prediction model that characterizes the COPD status of a subject based on expression levels determined in a biological sample obtained from the subject. Typically the model is built using samples for which the classification (COPD) has already been ascertained. Once the model (classifier) is built, it may then be applied to expression levels obtained from a biological sample of a subject whose COPD is unknown in order to predict the COPD status of the subject. Thus, the methods may involve applying a COPD-classifier to the expression levels, such that the COPD-classifier characterizes the COPD status of a subject based on the expression levels. The subject may be further treated or evaluated, e.g., by a health care provider, based on the predicted COPD status.

The classification methods may involve transforming the expression levels into a COPD risk-score that is indicative of the likelihood that the subject has COPD. In some embodiments, such as, for example, when a linear discriminant classifier is used, the COPD risk-score may be obtained as the combination (e.g., sum, product) of weighted expression levels, in which the expression levels are weighted by their relative contribution to predicting increased likelihood of having COPD.

It should be appreciated that a variety of prediction models known in the art may be used as a COPD-classifier. For example, a COPD-classifier may comprises an algorithm selected from logistic regression, partial least squares, linear discriminant analysis, quadratic discriminant analysis, neural network, naive Bayes, C4.5 decision tree, k-nearest neighbor, random forest, and support vector machine or other appropriate method.

The COPD-classifier may be trained on a data set comprising expression levels of the plurality of informative-genes in biological samples obtained from a plurality of subjects identified as having COPD. For example, the COPD-classifier may be trained on a data set comprising expression levels of a plurality of informative-genes in biological samples obtained from a plurality of subjects identified as having COPD based histological findings. The training set will typically also comprise control subjects identified as not having COPD. As will be appreciated by the skilled artisan, the population of subjects of the training data set may have a variety of characteristics by design, e.g., the characteristics of the population may depend on the characteristics of the subjects for whom diagnostic methods that use the classifier may be useful. For example, the population may consist of all males, all females or may consist of both males and females. The population may consist of subjects with history of cancer, subjects without a history of cancer, or a subjects from both categories. The population may include subjects who are smokers, former smokers, and/or non-smokers. The population may include subjects who have lung cancer and/or subjects who not have lung cancer.

A class prediction strength can also be measured to determine the degree of confidence with which the model classifies a biological sample. This degree of confidence may serve as an estimate of the likelihood that the subject is of a particular class predicted by the model.

Accordingly, the prediction strength conveys the degree of confidence of the classification of the sample and evaluates when a sample cannot be classified. There may be instances in which a sample is tested, but does not belong, or cannot be reliably assigned to, a particular class. This may be accomplished, for example, by utilizing a threshold, or range, wherein a sample which scores above or below the determined threshold, or within the particular range, is not a sample that can be classified (e.g., a "no call"). Once a model is built, the validity of the model can be tested using methods known in the art. One way to test the validity of the model is by cross-validation of the dataset. To perform cross-validation, one, or a subset, of the samples is eliminated and the model is built, as described above, without the eliminated sample, forming a "cross-validation model." The eliminated sample is then classified according to the model, as described herein. This process is done with all the samples, or subsets, of the initial dataset and an error rate is determined. The accuracy the model is then assessed. This model classifies samples to be tested with high accuracy for classes that are known, or classes have been previously ascertained. Another way to validate the model is to apply the model to an independent data set, such as a new biological sample having an unknown COPD status.

As will be appreciated by the skilled artisan, the strength of the model may be assessed by a variety of parameters including, but not limited to, the accuracy, sensitivity and specificity. Methods for computing accuracy, sensitivity and specificity are known in the art and described herein (See, e.g., the Examples). The COPD-classifier may have an accuracy of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more. The COPD-classifier may have an accuracy in a range of about 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. The COPD-classifier may have a sensitivity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more. The COPD-classifier may have a sensitivity in a range of about 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. The COPD-classifier may have a specificity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more. The COPD-classifier may have a specificity in a range of about 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%. Clinical Treatment/Management

In certain aspects, methods are provided for determining a treatment course for a subject. The methods typically involve determining the expression levels in a biological sample obtained from the subject of one or more informative-genes, and determining a treatment course for the subject based on the expression levels. Often the treatment course is determined based on a COPD risk-score derived from the expression levels. The subject may be identified as a candidate for a COPD therapy based on a COPD risk-score that indicates the subject has a relatively high likelihood of having COPD. The subject may be identified as a candidate for an invasive lung procedure (e.g., transthoracic needle aspiration, mediastinoscopy, or thoracotomy) based on a COPD risk-score that indicates the subject has a relatively high likelihood of having COPD (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%). The subject may be identified as not being a candidate for a COPD therapy or an invasive lung procedure based on a COPD risk-score that indicates the subject has a relatively low likelihood (e.g., less than 50%, less than 40%, less than 30%, less than 20%) of having COPD. In some cases, an intermediate risk-score is obtained and the subject is not indicated as being in the high risk or the low risk categories. In some embodiments, a health care provider may engage in "watchful waiting" and repeat the analysis on biological samples taken at one or more later points in time, or undertake further diagnostics procedures to rule out COPD, or make a determination that COPD is present, soon after the risk determination was made. The methods may also involve creating a report that summarizes the results of the gene expression analysis. Typically the report would also include an indication of the COPD risk-score.

Computer Implemented Methods

Methods disclosed herein may be implemented in any of numerous ways. For example, certain embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. Such processors may be implemented as integrated circuits, with one or more processors in an integrated circuit component. Though, a processor may be implemented using circuitry in any suitable format.

Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone or any other suitable portable or fixed electronic device.

Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output.

Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format. Such computers may be interconnected by one or more networks in any suitable form, including as a local area network or a wide area network, such as an enterprise network or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

In this respect, aspects of the invention may be embodied as a computer readable medium (or multiple computer readable media) (e.g., a computer memory, one or more floppy discs, compact discs (CD), optical discs, digital video disks (DVD), magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other non-transitory, tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the invention discussed above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present invention as discussed above. As used herein, the term "non- transitory computer-readable storage medium" encompasses only a computer-readable medium that can be considered to be a manufacture (i.e. , article of manufacture) or a machine.

The terms "program" or "software" are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of the present invention as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present invention need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present invention.

As used herein, the term "database" generally refers to a collection of data arranged for ease and speed of search and retrieval. Further, a database typically comprises logical and physical data structures. Those skilled in the art will recognize the methods described herein may be used with any type of database including a relational database, an object-relational database and an XML-based database, where XML stands for "eXtensible-Markup-Language". For example, the gene expression information may be stored in and retrieved from a database. The gene expression information may be stored in or indexed in a manner that relates the gene expression information with a variety of other relevant information (e.g. , information relevant for creating a report or document that aids a physician in establishing treatment protocols and/or making diagnostic determinations, or information that aids in tracking patient samples). Such relevant information may include, for example, patient identification information, ordering physician identification information, information regarding an ordering physician's office (e.g. , address, telephone number), information regarding the origin of a biological sample (e.g. , tissue type, date of sampling), biological sample processing information, sample quality control information, biological sample storage information, gene annotation information, COPD risk classifier information, COPD risk factor information, payment information, order date information, etc.

Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments.

In some aspects of the invention, computer implemented methods for processing genomic information are provided. The methods generally involve obtaining data representing expression levels in a biological sample of one or more informative-genes and determining the likelihood that the subject has COPD based at least in part on the expression levels. Any of the statistical or classification methods disclosed herein may be incorporated into the computer implemented methods. In some embodiments, the methods involve calculating a risk-score indicative of the likelihood that the subject has COPD. Computing the risk-score may involve a determination of the combination (e.g. , sum, product or other combination) of weighted expression levels, in which the expression levels are weighted by their relative contribution to predicting increased likelihood of having COPD. The computer implemented methods may also involve generating a report that summarizes the results of the gene expression analysis, such as by specifying the risk-score. Such methods may also involve transmitting the report to a health care provider of the subject. Compositions and Kits

In some aspects, compositions and related methods are provided that are useful for determining expression levels of informative-genes. For example, compositions are provided that consist essentially of nucleic acid probes that specifically hybridize with informative-genes or with nucleic acids having sequences complementary to informative-genes. These

compositions may also include probes that specifically hybridize with control genes or nucleic acids complementary thereto. These compositions may also include appropriate buffers, salts or detection reagents. The nucleic acid probes may be fixed directly or indirectly to a solid support {e.g., a glass, plastic or silicon chip) or a bead {e.g., a magnetic bead). The nucleic acid probes may be customized for used in a bead-based nucleic acid detection assay.

In some embodiments, compositions are provided that comprise up to 5, up to 10, up to 25, up to 50, up to 100, or up to 200 nucleic acid probes. In some cases, each of the nucleic acid probes specifically hybridizes with an mRNA selected from Table 2 or with a nucleic acid having a sequence complementary to the mRNA. In some embodiments, probes that detect informative-mRNAs are also included. In some cases, each of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 of the nucleic acid probes specifically hybridizes with an mRNA selected from Table 2 or with a nucleic acid having a sequence complementary to the mRNA. The compositions may be prepared for detecting different genes in biochemically separate reactions, or for detecting multiple genes in the same biochemical reactions.

Also provided herein are oligonucleotide (nucleic acid) arrays that are useful in the methods for determining levels of multiple informative-genes simultaneously. Such arrays may be obtained or produced from commercial sources. Methods for producing nucleic acid arrays are also well known in the art. For example, nucleic acid arrays may be constructed by immobilizing to a solid support large numbers of oligonucleotides, polynucleotides, or cDNAs capable of hybridizing to nucleic acids corresponding to genes, or portions thereof. The skilled artisan is referred to Chapter 22 "Nucleic Acid Arrays" of Current Protocols In Molecular Biology (Eds. Ausubel et al. John Wiley and #38; Sons NY, 2000) or Liu CG, et ah, An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues.

Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9740-4, which provide non-limiting examples of methods relating to nucleic acid array construction and use in detection of nucleic acids of interest. In some embodiments, the arrays comprise, or consist essentially of, binding probes for at least 2, at least 5, at least 10, at least 20, at least 50, at least 60, at least 70 or more informative-genes. In some embodiments, the arrays comprise, or consist essentially of, binding probes for up to 2, up to 5, up to 10, up to 20, up to 50, up to 60, up to 70 or more informative- genes. In some embodiments, an array comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mRNAs selected from Table 2. In some embodiments, an array comprises or consists of 4, 5, or 6 of the mRNAs selected from Table 2. Kits comprising the oligonucleotide arrays are also provided. Kits may include nucleic acid labeling reagents and instructions for determining expression levels using the arrays.

The compositions described herein can be provided as a kit for determining and evaluating expression levels of informative-genes. The compositions may be assembled into diagnostic or research kits to facilitate their use in diagnostic or research applications. A kit may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more compositions described herein, along with instructions describing the intended application and the proper use of these compositions. Kits may contain the components in appropriate concentrations or quantities for running various experiments.

The kit may be designed to facilitate use of the methods described herein by researchers, health care providers, diagnostic laboratories, or other entities and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g. , in solution), or in solid form, (e.g. , a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable, for example, by the addition of a suitable solvent or other substance, which may or may not be provided with the kit. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g. , videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of diagnostic or biological products, which instructions can also reflect approval by the agency.

A kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.

The kit may include a container housing agents described herein. The components may be in the form of a liquid, gel or solid (e.g., powder). The components may be prepared sterilely and shipped refrigerated. Alternatively they may be housed in a vial or other container for storage. A second container may have other components prepared sterilely.

As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 1%, 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

All references described herein are incorporated by reference for the purposes described herein.

Exemplary embodiments of the invention will be described in more detail by the following examples. These embodiments are exemplary of the invention, which one skilled in the art will recognize is not limited to the exemplary embodiments. EXAMPLES

Example 1: Gene expression analysis of bronchoscopy samples.

Applicants collected several hundred clinical samples comprising bronchial epithelial cells obtained during routine bronchoscopy. Subjects were enrolled in the trial due to a suspicion of lung cancer who had been referred to a pulmonologist for bronchoscopy. A majority of the subjects were subsequently confirmed to have lung cancer by histological and pathological examination of cells taken from the lung either during bronchoscopy, or during some follow-up procedure. A minority of subjects were found to be cancer free at the time of presentation to the pulmonologist and up to 12 months following that date. Samples were utilized to develop a gene expression test to predict subjects with the highest risk of cancer in cases where bronchoscopy yields a non-positive result. The combination of false-negative cases (which occurs in 25-30% of the cancer cases) and the true-negative cases yield a combined set of non-positive bronchoscopy procedures, representing approximately 40-50% of the total cases referred to pulmonologists.

Applicants established a set of genes that are differentially expressed between subjects with and without cancer. Furthermore the expression intensities of these genes (representing both up- and down-regulated genes) were combined using multivariate classifier algorithms to yield a "score" which was used to predict subjects with and without cancer. The resulting test was shown to have high sensitivity and specificity and therefore add diagnostic value in cases where bronchoscopy is non-positive.

Applicants have developed methods to improve test specificity. Applicants have identified factors in the sample cohorts used to "train" and "test" the algorithm that influence test specificity. Two factors, which are related, were identified as subjects previously diagnosed with COPD and subjects indicated to be using inhaled medications (e.g., bronchodilators and corticosteroids). COPD is estimated to yield a 5-fold increase in lung cancer risk, compared to smokers without COPD.

Applicants have established sets of genes that distinguish smokers (current or former) with and without lung cancer based on airway field of injury. Specifically, RNA isolated from cytologically normal appearing cells collected from the bronchus of suspect lung cancer patients was used to generate a gene expression signature that predicts the risk of lung cancer with high accuracy. Applicants have determined that specificity of this gene signature differs between subjects with and without COPD (Table 1). Specifically, subjects with COPD but not cancer have significantly lower specificity than subjects with neither indication. There is less of a difference in prediction sensitivity of the signature between COPD positive and negative cases.

TABLE 1 - Cancer risk prediction specificity and sensitivity of subjects with and without

COPD

Example 2: Identification of differentially expressed genes associated with chronic obstructive pulmonary disease

Applicant sought to determine whether genes are differentially expressed between subjects with and without COPD (all of which are cancer-free). Applicants sought to determine if the expression levels of those genes could be combined in order to predict COPD cases, in subjects without lung cancer. COPD is a major risk of lung cancer and identification of subjects with COPD may be an effective means of identifying subjects who will likely develop cancer which ultimately could be used as an effective early detection method. Furthermore, by identifying genes associated with COPD, some of these may be shown to be effective drug- targets for chemoprevention strategies. Applicants found that a total of 1833 genes are differentially expressed (p<0.05) after applying a false-discovery rate correction. Applicants further found that gene expression intensities could be combined using well-known classifier algorithms [e.g., Linear Discriminant Analysis (LDA), or Support Vector Machine (SVM)] to generate "scores". The scores can then be used to distinguish COPD-positive and COPD-negative cases relative to a threshold.

Applicants found that gene signatures comprising different numbers of individual genes led to effective predictions of COPD. For a given combination of genes the sensitivity and specificity of the algorithm (or signature) was determined by comparison to previously diagnosed cases, with and without COPD. The sensitivity and specificity depends on the threshold value, and a Receiver Operator Characteristic (ROC) curve was constructed. An example is shown in Figure 1. In this case the overall Area Under the Curve (AUC) of the ROC curve is 0.81 and can be used as an indicator of overall accuracy of a gene-signature, where AUC=1 is 100% accuracy and AUC=0.5 is equivalent to a random predictor.

A list of the top 100 differentially expressed genes is shown in Table 2, along with the weight calculated for each gene. The greater the absolute value of the weight, the more significant the importance of the gene to differentiate subjects with and without COPD.

TABLE 2 - The top 100 differentially expressed genes for predicting COPD

GPR155 -5.15E-01

ITGA9 -5.14E-01

PTGFR -5.13E-01

ISLR -5.08E-01

SLC5A7 -5.07E-01

ZNF483 -5.03E-01

DPYSL3 5.02E-01

TNS3 -5.01E-01

FMNL2 -4.97E-01

GALE 4.95E-01

CNTN3 -4.95E-01

HSD17B13 -4.94E-01

PTPRM -4.93E-01

HLF -4.93E-01

PROS1 -4.90E-01

PLA2G4A 4.90E-01

KALI -4.89E-01

TCN1 4.88E-01

DPP4 -4.85E-01

GPR98 -4.84E-01

KCNA1 -4.83E-01

CABLES 1 -4.82E-01

PEG 10 -4.82E-01

PPP1R9A -4.81E-01

POLA2 -4.78E-01

C17orf37 4.78E-01

ABCC4 -4.78E-01

CA8 -4.76E-01

CYP2A13 -4.76E-01

SETBP1 -4.75E-01

ANKS1B -4.75E-01

CHP 4.75E-01

THSD4 -4.72E-01

MPDU1 4.72E-01

CD 109 4.71E-01

STK32A -4.70E-01

HHLA2 -4.70E-01

AMMECRl 4.69E-01

NPAS3 -4.68E-01

GXYLT2 -4.67E-01

KLF12 -4.67E-01

CA12 4.67E-01

C21orfl21 -4.67E-01

SH3BP4 4.66E-01

FABP6 -4.64E-01

GUCY1B3 4.64E-01

FUT3 4.62E-01

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention.

Accordingly, the foregoing description and drawings are by way of example only and the invention is described in detail by the claims that follow. Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.