US20190292600A1

US20190292600A1 - Nasal epithelium gene expression signature and classifier for the prediction of lung cancer

Info

Publication number: US20190292600A1
Application number: US16/300,947
Authority: US
Inventors: Avrum Spira; Marc Lenburg; Joseph Perez-Rogers; Christina Anderlind
Original assignee: Boston University
Current assignee: Boston University
Priority date: 2016-05-12
Filing date: 2017-05-12
Publication date: 2019-09-26
Also published as: WO2017197335A1; US20210381062A1; EP3455381A1; EP4180531A3; EP3455381A4; EP4180531A2

Abstract

Disclosed herein are assays and methods for diagnosis and prognosis of lung cancer using expression analysis of one or more genes from a biological sample comprising nasal epithelial cells. The assays and methods of the invention provide non-invasive means of accurately detecting the presence or absence of lung cancer relative to, for example, more invasive techniques, such as bronchoscopy. Similarly, in certain aspects, the assays and methods disclosed herein provide non-invasive means of accurately identifying the smoking history of a subject.

Description

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/335,391, filed May 12, 2016, which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Contract No. U01-CA152751 and U01-CA214182 awarded by the National Institutes of Health and under Contract No. W81XWH-11-2-0161 awarded by the U.S. Department of Defense. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Lung cancer is the deadliest form of cancer in the United States and the world. An estimated 221,000 new lung cancer diagnoses are expected in the United States in 2015, and approximately 158,000 men and women are expected to fall victim to the disease during the same time period. The high mortality rate is due, in part, to a failure in 70% of patients to detect lung cancer when it is localized and surgical resection remains feasible.
In 2011, the National Lung Screening Trial (NLST) demonstrated that annual screening of high-risk smokers by low-dose chest CT (LDCT) could lead to the detection of earlier stage lung cancers and reduce mortality by 20%. The expectation is that, similar to other cancers for which there are established screening programs (e.g., breast, prostate and colon cancers), regular lung cancer screening could lead to lung cancer becoming considerably less deadly. As a result, Medicare is now paying for lung cancer screening in defined high risk cohorts. In the NLST trial, there was, however, a considerable false-positive rate associated with CT screening (greater than 95%), with the overwhelming majority of nodules ultimately determined to be benign.
Together, these findings have led to the development of guidelines under which additional diagnostic procedures should be, performed in patients with screen-detected nodules, including those established by the Fleischner Society which recommends repeat imaging studies or invasive testing depending on the size of the lesion. Unfortunately, the diagnostic performance under these guidelines remains low and often results in a delay in the diagnosis of early stage lung cancer and unnecessary invasive procedures for those without disease.
With more than 9 million people in the United States meeting NLST screening eligibility criteria, there is a critical need for more accurate, non-invasive tools to prioritize patients for repeat imaging or invasive procedures following the detection of nodules by screening LDCT. Also needed are additional criteria for lung cancer screening eligibility. The current guidelines for determining screening eligibility are based on age and smoking history and present two fundamental challenges. First, even though these guidelines suggest the screening of almost 3% of the total United States population, they capture less than 30% of the cases of lung cancer that are diagnosed each year. Second, the prevalence of lung cancer among the screen-eligible cohort is only about 1%, indicating that the burden of screening could be greatly reduced if screening could be more accurately targeted. Taken together, these data suggest that there is a tremendous need and an opportunity to improve screening eligibility beyond age and smoking history.

SUMMARY OF THE INVENTION

Disclosed herein are assays and methods of diagnosing lung cancer and methods of identifying subjects at risk for developing lung cancer. The inventions disclosed herein provide non-invasive, or in certain embodiments minimally-invasive, methods for diagnosing lung cancer based in-whole or in-part on analysis of gene expression in nasal epithelial cells. Accordingly, provided herein are non-invasive and minimally invasive methods for the diagnosis, prognosis, monitoring and/or follow up of progression or success of treatment based upon the differential expression of certain genes in nasal epithelial cells (e.g., one or more of the 535 genes identified in Table 12 or Table 21).
In certain embodiments, disclosed herein are methods of diagnosing lung cancer in a subject, such methods comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes); and (b) comparing the expression of the one or more genes to a control sample of those genes taken from individuals without cancer; wherein the one or more genes are selected from the group consisting of genes in Tables 12, 13 or 21, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having lung cancer. In some aspects, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject not having lung cancer.
Also disclosed herein are methods of diagnosing lung cancer in a subject, such methods comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes); and (b) comparing the expression of the one or more genes to a control sample of those genes from individuals with cancer; wherein the one or more genes are selected from the group consisting of genes in Tables 12 or 13, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the subject not having lung cancer. In certain aspects, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having lung cancer.
In certain aspects, the inventions disclosed herein relate to methods of determining whether a subject has quit smoking comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes selected from the group consisting of genes in Tables 5 or 6 (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes); and (b) comparing the expression of the one or more genes to a control sample of those genes from non-smokers; wherein altered expression of the subject's genes relative to the control sample is indicative of the subject having quit smoking. In certain aspects, non-altered expression of the subject's one or more genes relative to the control sample is indicative of the subject not having quit smoking.
In still other embodiments, also disclosed herein are methods of determining whether a subject has quit smoking, such methods comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes selected from the group consisting of genes in Tables 5 or 6 (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes); and (b) comparing the expression of the one or more genes to a control sample of those genes obtained from smokers; wherein altered expression of the subject's genes relative to the control sample is indicative of the subject not having quit smoking. In some aspects, non-altered expression of the subject's one or more genes relative to the control sample is indicative of the subject having quit smoking.
In certain aspects, the present inventions also relate to methods of determining the likelihood that a subject has lung cancer, such methods comprising: (a) subjecting a biological sample comprising the subject's nasal epithelial cells to a gene expression analysis, wherein the gene expression analysis comprises comparing gene expression levels of one or more genes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes) selected from the group of genes identified in Tables 12 or 13 to the expression levels of a control sample of those genes from individuals without cancer; and (b) determining the likelihood that the subject has lung cancer by determining differential expression of the subject's one or more genes relative to the group of genes in Tables 12 or 13, wherein differential expression of the subject's genes relative to the control sample is indicative of the subject having a high likelihood of lung cancer. In some embodiments, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having a low likelihood of lung cancer.
In certain embodiments, the one or more genes comprise one or more of the leading edge genes identified in Table 21. For example, any of the methods disclosed herein may comprise, consist of or consist essentially of determining the differential expression of at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more of the leading edge genes identified in Table 21. In some aspects, the methods disclosed herein comprise determining the differential expression of all of the leading edge genes identified in Table 21.
In certain aspects, the inventions disclosed herein are directed to methods of determining the likelihood that a subject has lung cancer, such methods comprising: (a) subjecting a biological sample comprising the subject's nasal epithelial cells to a gene expression analysis, wherein the gene expression analysis comprises comparing gene expression levels of one or more genes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes) selected from the group of genes in Tables 12 or 13 to the expression levels of a control sample of those genes from individuals with cancer; and (b) determining the likelihood that the subject has lung cancer by determining differential expression of the subject's one or more genes relative to the group of genes in Tables 12 or 13, wherein differential expression of the subject's genes relative to the control sample is indicative of the subject having a low likelihood of lung cancer. In some embodiments, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having a high likelihood of lung cancer.
In any of the embodiments disclosed herein, at least about two genes are measured (e.g., at least two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred or more genes are measure). In some embodiments, at least about five genes are measured. In some embodiments, at least about ten genes are measured. In some embodiments, at least about twenty genes are measured. In still other embodiments, at least about thirty genes are measured. In yet other embodiments, at least about forty genes are measured. In still other embodiments, at least about fifty genes are measured.
In some embodiments, the 535 genes listed in Table 12 or Table 21 are grouped into one or more of the four clusters of related genes identified. For example, in some aspects, the genes measured comprise one or more of those genes identified in cluster 1 of Table 12. In some aspects, the genes measured comprise one or more of those genes identified in cluster 2 of Table 12. In some aspects, the genes measured comprise one or more of those genes identified in cluster 3 of Table 12. In some aspects, the genes measured comprise those genes identified in cluster 4 of Table 12. In yet another embodiment, the genes measured comprise at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, forty, fifty or more genes) from each of clusters 1, 2, 3 and 4 of Table 12.
In certain embodiments, the methods and assays disclosed herein are used in combination with one or more clinical risk factors (e.g., the subject's smoking status) for determining a subject's risk of having lung cancer or at risk of developing lung cancer. For example, such methods and assays may be combined with one or more clinical risk factors selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan, the location of the lesion or nodule (e.g., centrally located, peripherally located or both) and the amount of time since the subject quit smoking. Combining any of the methods and assays disclosed herein with, for example, a subject's positive smoking status may be more indicative of the subject having lung cancer and thereby enhance the predictive value and/or sensitivity of the methods and assays disclosed herein. Similarly, in some embodiments, the combination of the methods and assays disclosed herein and a subject's age (e.g., advanced age) may also be indicative of the subject having, or of being at increased risk of having lung cancer. In still other embodiments, the methods and assays disclosed herein comprise performing or reviewing the results of one or more imaging studies (e.g., chest X-ray, assessing the subject for the presence of a lung nodule or lesion greater than 3 cm on the subject's CT scan, assessing lesion or nodule location), which if positive, may be further indicative of the subject having lung cancer. In some embodiments, the methods and assays disclosed herein may further comprise a step of assessing the subject's time since quitting smoking, which if greater than 15 years may be indicative of the subject having lung cancer.
In certain aspects of any of the methods, compositions or assays disclosed herein, the one or more genes assessed comprise, consist of, or consist essentially of one or more genes from Table 14. In some embodiments of any of the methods, compositions or assays disclosed herein, the one or more genes comprise, consist of, or consist essentially of one or more genes from Table 15. In some embodiments of any of the methods compositions or assays disclosed herein, the one or more genes further comprise, consist of, or consist essentially of one or more genes from Table 13. In some embodiments, the one or more genes comprise, consist of, or consist essentially of all of the genes from Table 14. In some embodiments, the one or more genes comprise, consist of, or consist essentially of one or more genes from Table 13. In certain aspects, the one or more genes further comprise one or more genes from Table 5. In some other embodiments, the one or more genes further comprise one or more genes from Table 6.
In certain embodiments of any of the methods disclosed herein, the one or more genes (e.g., one or more genes from Table 12 or Table 21) are associated with DNA damage. In certain embodiments of any of the methods disclosed herein, the one or more genes (e.g., one or more genes from Table 12 or Table 21) are associated with regulation of apoptosis. In still other embodiments of any of the methods disclosed herein, the one or more genes (e.g., one or more genes from Table 12 or Table 21) are associated with immune system activation (e.g., one or more genes is associated with the interferon-gamma signaling pathway or associated with antigen presentation).
In some embodiments, expression of the one or more genes from the biological sample (e.g., a biological sample comprising nasal epithelial cells) is determined using a quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay or a oligonucleotide array assay.
In certain aspects of any of the methods disclosed herein, the method further comprises applying a gene filter to the expression to exclude specimens potentially contaminated with inflammatory cells.
In some embodiments, the methods and assays disclosed herein are useful for identifying subjects having, or of being at increased risk of having lung cancer. In certain aspects, the lung cancer is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, small cell cancer or non-small cell cancer.
As discussed above, in some aspects, the assays and methods disclosed herein rely in part on determining the differential expression of one or more genes in a subject's nasal epithelial cells (e.g., one or more of the genes set forth in Table 12 or Table 21). In some embodiments, the one or more genes comprise DNA. In some embodiments, the one or more genes comprise RNA. In some embodiments, the one or more genes comprise mRNA.
In some embodiments, the biological sample obtained from the subject comprises nasal epithelial cells. In some embodiments, the biological sample consists or consists essentially of nasal epithelial cells. In some embodiments, the biological sample does not comprise bronchial epithelial cells or bronchial epithelial tissue. In still other embodiments, the biological sample does not comprise cells or tissues from the bronchial airway.
In certain aspects of any of the inventions disclosed herein, if such method is indicative of the subject having lung cancer or of being at risk of developing lung cancer, the method further comprises treating the subject. Accordingly, in certain embodiments, any of the methods disclosed herein may further comprise a step of administering a cancer treatment to the subject (e.g., a treatment comprising one or more of chemotherapy, radiation therapy, immunotherapy, surgical intervention and combinations thereof). For example, in those embodiments where the methods and assays disclosed herein are indicative of a subject being at a higher risk of having or developing lung cancer, the subject may be subjected to a direct tissue sampling or biopsy of the nodule, under the presumption that the positive test indicates a higher likelihood of the nodule is a cancer. Conversely, in those instances where the methods and assays disclosed herein are indicative of a subject having a reduced risk of developing lung cancer, then the subject may be subjected to further imaging surveillance (e.g., a repeat computerized tomography scan to monitor whether the nodule grows or changes in appearance before doing a more invasive procedure), or a determination made to withhold a particular treatment (e.g., chemotherapy) on the basis of the subject's favorable or reduced risk of having or developing lung cancer.
Similarly, in certain aspects of any of the inventions disclosed herein, if such method is indicative of the subject having not quit smoking or of being a smoker, the method further comprises treating the subject. Accordingly, in certain embodiments, any of the methods disclosed herein may further comprise a step of administering a smoking-cessation treatment to the subject (e.g., a treatment comprising nicotine replacement therapy).
Also disclosed herein are minimally-invasive methods and assays useful for determining the likelihood that a subject does (or does not) have lung cancer, such methods and assays comprising a step of (a) detecting, by quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay or a oligonucleotide array assay, mRNA or cDNA expression levels in a sample comprising nasal epithelial cells from a subject; (b) determining mRNA or cDNA expression levels in the sample of nasal epithelial cells of two or more gene selected from the group consisting of the genes in Table 12, Table 13 or Table 21; and (c) based on the expression levels determined in step (b) (e.g., differentially expressed levels), determining a lung cancer risk-score that is indicative of the likelihood that the subject does not have lung cancer. In certain aspects, the subject has undergone an indeterminate or non-diagnostic bronchoscopy procedure. In certain embodiments, the genes comprise at least 1 gene from Table 13 (e.g., about one, two, three, four, five, six, seven, eight, nine or ten genes from Table 13). In some embodiments, the genes comprise at least 10 genes from Table 13 (e.g., about ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty genes from Table 13). In still other embodiments, the genes comprise at least 20 genes from Table 13 (e.g., about twenty one, twenty two, twenty three, twenty four, twenty five, twenty six, twenty seven, twenty eight, twenty nine or thirty genes from Table 13). In still other aspects, the genes comprise all of the genes from Table 13.
In certain embodiments, the methods and assays disclosed herein further comprise a step of determining one or more of the subject's clinical risk factors affecting the subject's risk for having lung cancer (e.g., one or more clinical risk factors selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan, lesion location and time since quitting smoking). In some embodiments, the subject's positive smoking status is indicative of the subject having lung cancer. In some aspects, the subject's advanced age is indicative of the subject having lung cancer. In some embodiments, the presence of a lung nodule greater than 3 cm on the subject's CT scan is indicative of the subject having lung cancer. In still other embodiments, the subject's time since quitting smoking greater than 15 years is indicative of the subject having lung cancer.
Also disclosed herein are compositions (e.g., diagnostic kits) and assays that comprise one or more nucleic acid probes, wherein each of the one or more nucleic acids probes specifically hybridizes with the expression products of five or more genes selected from the group of genes identified in any of Table 5, Table 6, Table 12, Table 13, Table 14, Table 15 or Table 21. In certain aspects, such one or more expression products comprise mRNA. In some aspects, such compositions measure expression of at least ten genes. In some aspects, such compositions measure expression of at least fifteen genes. In some aspects, such compositions measure expression of at least twenty genes. In some aspects, such compositions measure expression of at least thirty genes. In some embodiments, such compositions measure expression of at least forty genes. In still other embodiments, such compositions measure expression of at least fifty genes. In some embodiments, such compositions measure expression of at least one hundred genes.
In certain embodiments, the compositions (e.g., diagnostic kits) disclosed herein measure expression of those genes identified in cluster 1 of Table 12. In certain embodiments, the compositions disclosed herein measure expression of those genes identified in cluster 2 of Table 12. In yet other embodiments, the compositions disclosed herein measure expression of those genes identified in cluster 3 of Table 12. In still other embodiments, the compositions disclosed herein measure expression of those genes identified in cluster 4 of Table 12. In certain aspects, such compositions measure expression of one or more genes in Table 12 and comprise at least one gene from each of clusters 1-4. In certain aspects of any of the methods, assays or compositions disclosed herein, the one or more genes are associated with DNA damage. In certain aspects of any of the methods, assays or compositions disclosed herein, the one or more genes are associated with the regulation of apoptosis. In certain embodiments of any of the methods, assays or compositions disclosed herein, the one or more genes are immune system activation (e.g., associated with the interferon-gamma signaling pathway and/or antigen presentation).
The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts the characterization of 535 cancer-associated nasal epithelial genes in the training set. Five hundred thirty-five genes were differentially expressed by cancer status in the nasal training set (P<0.001) using a linear model that included cancer status, smoking status, pack-years, sex, age, and RIN as covariates. These genes were grouped into two co-expression clusters by unsupervised hierarchical clustering. Unsupervised hierarchical clustering of patients across these genes revealed two primary patient clusters.

FIGS. 2A-2B demonstrate the concordance between cancer-associated gene expression in bronchial and nasal epithelium. FIG. 2A shows that the 535 genes with cancer-associated expression in nasal epithelium were split into up- and downregulated gene sets, and the present inventors examined their distribution within all genes ranked from most down-regulated (left) to most upregulated (right) in the bronchial epithelium of patients with cancer using gene set enrichment analysis. The present inventors found that the genes with increased expression in nasal epithelium were enriched among the genes that are most induced in the bronchial epithelium of patients with cancer (top; P<0.001 by a two-sided permutation-based Kolmogorov-Smirnov-like test) while the reverse was true for genes with decreased expression in nasal epithelium (bottom; P<0.001 by a two-sided permutation based Kolmogorov-Smirnov-like test). Genes included in the core enrichment are shown in the green box. FIG. 2B depicts heatmaps and hierarchical clustering of the core enrichment genes in nasal (left) and bronchial (right) samples. All statistical tests were two-sided.

FIG. 3 shows clinicogenomic and clinical classifier performance in the validation set. Shown are the receiver operating characteristic (ROC) curves for the clinicogenomic (solid line) and clinical (dashed line) classifiers in the independent AEGIS-2 validation set. The area under the curve (AUC) was 0.81 (95% confidence interval [CI]=0.74 to 0.89) for the clinicogenomic classifier and 0.74 (95% CI=0.66 to 0.83) for the clinical classifier. The difference between ROC curves was statistically significantly different (P=0.01 by a two-sided Delong's test for correlated ROC curves).

FIG. 4 is a flowchart that illustrates data acquisition and processing workflow. Nasal epithelial samples from smokers with and without lung cancer were collected from 28 institutions across the U.S., Canada, and Europe as part of the AEGIS clinical trials. 557 samples were received by Boston University and run on Affymetrix Gene 1.0 ST microarrays. 31 samples were lost due to indeterminate cancer diagnosis at follow up. 18 samples were removed as part of the quality control process. The 526 remaining samples were RMA normalized and batch-corrected together, and then separated into a training set (AEGIS-1, n=375) and validation set (AEGIS-2, n=130).

FIG. 5 depicts the distribution of matched AEGIS-1 nasal and bronchial epithelial samples. Of the 375 patients in the nasal training set, 157 had a matched bronchial epithelium sample profiles as part of the study by Whitney et. al. The remaining 218 patients only had a nasal sample profiled as part of this study. The clinical model was derived using the union set of these samples (n=517).

FIG. 6 illustrates the correlation of bronchial genomic classifier in matched nasal and bronchial epithelium samples. Bronchial genomic classifier scores in matched nasal (y-axis) and bronchial (x-axis) samples (n=157). The scores from both tissues were statistically significantly correlated (R=70, p<0.001 by a two-sided Pearson's product-moment correlation test). The vertical and horizontal lines indicate the cut-point for binary classification reported by Whitney et al. Cancer samples are shown in green and benign samples are shown in grey.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are novel, non-invasive or minimally invasive assays and related methods that are useful for diagnosing lung cancer or determining a subject's previous smoking status, such assays and methods comprising a step of determining the expression of one or more genes in nasal epithelial cells of a subject. For example, in certain aspects the methods disclosed herein comprise a step of comparing the expression of one or more of the 535 genes set forth in Table 12 or Table 21 in a subject's nasal epithelial cells to expression of the same genes in a nasal epithelial cell from a control subject. In certain aspects, any of the methods disclosed herein further comprise applying a gene filter to the expression to exclude specimens potentially contaminated with inflammatory cells.
The assays and methods disclosed herein provide the first ever claim of a nasal epithelium gene expression classifier composed of the specific genes described herein and that can be used to predict the presence or absence of lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, small cell cancer or non-small cell cancer). Additionally, the assays and methods disclosed herein provide the first ever claim of a nasal epithelium gene expression classifier that can predict whether a subject is a current or former smoker. The assays and methods provided herein, whether used alone or in combination with other methods, provide useful information for health care providers to assist them in making early diagnostic and therapeutic decisions for a subject, thereby improving the likelihood that the subject's disease may be effectively treated. In some embodiments, methods and assays disclosed herein are employed in instances where other methods have failed to provide useful information regarding the lung cancer status of a subject.
Previous work from our group has demonstrated that gene expression in normal appearing bronchial and nasal epithelial cells is dramatically altered in current and former smokers (Zhang, et al.) and that several of these alterations persist for decades upon smoking cessation (Beane, et al.). The present inventors have extended these observations to show that gene expression in normal-appearing airway cells is also altered by smoking-related lung diseases such as COPD and lung cancer. For lung cancer, the present inventors measured gene expression in bronchial epithelial samples collected from a cohort of patients undergoing bronchoscopy for clinical suspicion of lung cancer and identified a panel of 80 genes that were indicative of the presence of lung cancer (Spira, et al., 2007) and which were independent of other clinical factors as a predictor of lung cancer (Beane, et al., 2008). More recently, a 232 gene signature was identified as differentially expressed in the bronchial epithelium of patients with lung cancer (Whitney, et al., 2015). This signature was ultimately used to develop a 23-gene bronchial genomic classifier (Whitney, et al., 2015; Silvestri, et al., 2015) that was prospectively validated in two independent cohorts consisting of over 600 patients.
The present inventions are based upon the surprising finding of a strong concordance between bronchial and nasal epithelium's response to cigarette smoke exposure, and our observation that lung disease alters gene expression in normal appearing nasal epithelium that is physically distant from the site of disease. The assays and methods disclosed herein are characterized by the accuracy with which they can discriminate lung cancer from non-lung cancer and their non-invasive or minimally-invasive nature. In some aspects, the assays and methods disclosed herein are based on detecting differential expression of one or more genes in nasal epithelial cells and such assays and methods are based on the discovery that such differential expression in nasal epithelial cells are useful for diagnosing cancer in the distant lung tissue. Accordingly, the inventions disclosed herein provide a substantially less invasive method for diagnosis, prognosis and follow-up of lung cancer using gene expression analysis of biological samples comprising nasal epithelial cells.
In contrast to conventional invasive methods, such as bronchoscopy, the assays and methods disclosed herein rely on expression of certain genes in a biological sample obtained from a subject. As the phrase is used herein, “biological sample” means any sample taken or derived from a subject comprising one or more nasal epithelial cells. 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.
Such biological samples comprising nasal epithelial cells may be obtained from a subject (e.g., a subject at risk for lung cancer) using a brush or a swab. The biological samples comprising nasal epithelial cells may be collected by any means known to one skilled in the art and, in certain embodiments, is obtained non-invasively. For example, in certain embodiments, a biological sample comprising nasal epithelial cells may be collected from a subject by nasal brushing. Similarly, nasal epithelial cells may be collected by brushing the inferior turbinate and/or the adjacent lateral nasal wall. For example, following local anesthesia with 2% lidocaine solution, a CYROBRUSH® (MedScand Medical, Malmd, Sweden) or a similar device, is inserted into the nare of the subject, for example the right nare, and under the inferior turbinate using a nasal speculum for visualization. The brush is turned (e.g., turned 1, 2, 3, 4, 5 times or more) to collect the nasal epithelial cells, which may then be subjected to analysis in accordance with the assays and methods disclosed herein.
In certain embodiments, the biological sample does not include or comprise bronchial airway epithelial cells. For example, in certain embodiments, the biological sample does not include epithelial cells from the mainstem bronchus. In certain aspects, the biological sample does not include cells or tissue collected from bronchoscopy. In some embodiments, the biological sample does not include cells or tissue isolated from a pulmonary lesion.
In certain embodiments, the subject has undergone an indeterminate or non-diagnostic bronchoscopy. In some embodiments, the method comprises determining that the subject does not have lung cancer based on the expression levels of one or more (such as, e.g., 2 or more) of the 535 genes set forth in Table 12 or Table 21 in a subject's nasal epithelial cells. In particular embodiments, the method comprises determining that the subject does not have lung cancer based on the expression levels in a nasal epithelial cell sample from the subject of one or more (such as, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 28, 29 or 30) genes listed in Table 13. In particular embodiments, the method comprises determining the subject does or does not have cancer by applying a classifier algorithm that is trained to differentiate cancer versus non-cancer based upon the expression of at least the 30 genes expressed in Table 13. In some such embodiments, the classifier is as shown in Table 17.
To isolate nucleic acids from the biological sample, the epithelial cells can be placed immediately into a solution that prevents nucleic acids from degradation. For example, if the nasal epithelial cells are collected using the CYTOBRUSH, and one wishes to isolate RNA, the brush is placed immediately into an RNA stabilizer solution, such as RNALATER®, AMBION®, Inc. One can also isolate DNA. After brushing, the device can be placed in a buffer, such as phosphate buffered saline (PBS) for DNA isolation.
The nucleic acids (e.g., mRNA) are then subjected to gene expression analysis. Preferably, the nucleic acids are isolated and purified. However, if techniques such as microfluidic devices are used, cells may be placed into such device as whole cells without substantial purification. In one embodiment, nasal epithelial cell gene expression is analyzed using gene/transcript groups and methods of using the expression profile of these gene/transcript groups in diagnosis and prognosis of lung diseases. In some embodiments, differential expression of the one or more genes determined with reference to the one or more of the 535 genes set forth in Table 12 or Table 21.
As used herein, the term “differential expression” refers to any qualitative or quantitative differences in expression of the gene or differences in the expressed gene product (e.g., mRNA) in the nasal epithelial cells of the subject. A differentially expressed gene may qualitatively have its expression altered, including an activation or inactivation, in, for example, the presence of absence of cancer and, by comparing such expression in nasal epithelial cell to the expression in a control sample in accordance with the methods and assays disclosed herein, the presence or absence of lung cancer may be determined.
In some embodiments, subjecting the nucleic acids to gene expression analysis may comprise directly measuring RNA (e.g., mRNA expression levels). In some embodiments, subjecting the nucleic acids to gene expression analysis may comprise detecting cDNAs produced from RNA expressed in the test sample, wherein, optionally, the cDNA is amplified from a plurality of cDNA transcripts prior to the detecting step. In some embodiments, subjecting the nucleic acids to gene expression analysis comprises labeling one or more of the nucleic acids.
In certain embodiments, the methods and assays disclosed herein are characterized as being much less invasive relative to, for example, bronchoscopy. The methods provided herein not only significantly increase the sensitivity or diagnostic accuracy of lung cancer or smoking status, but also make the analysis much less invasive and thus much easier for the subjects and clinician to perform. In some embodiments, the likelihood that the subject has lung cancer is also determined based on the presence or absence of one or more clinical risk factors or diagnostic indicia of lung cancer, such as the results of imaging studies. When the assays and methods of the present invention are combined with, for example, one or more relevant clinical risk factors (e.g., a subject's smoking history), the diagnosis of lung cancer may be dramatically enhanced, enabling the detection of lung cancer at an earlier stage, and by providing far fewer false negatives and/or false positives. As used herein, the term “clinical risk factors” refers broadly to any diagnostic indicia (e.g., subjective or objective diagnostic criteria) that would be relevant for determining a subject's risk of having or developing lung cancer. Exemplary clinical risk factors that may be used in combination with the methods or assays disclosed herein include, for example, imaging studies (e.g., chest X-ray, CT scan, etc.), the subject's smoking status or smoking history and/or the subject's age. In certain aspects, when such clinical risk factors are combined with the methods and assays disclosed herein, the predictive power of such methods and assays may be further enhanced.
In some embodiments, the biological sample comprising the subject's nasal epithelial cells are analyzed for the expression of certain genes or gene transcripts, either individually or in groups or subsets. In one embodiment, the inventions disclosed herein provide a group of genes (e.g., one or more of the genes listed in Table 12, Table 13 or Table 21) that may be analyzed to determine the presence or absence of lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, small cell cancer and/or non-small cell cancer) from a biological sample comprising the subject's nasal epithelial cells. In one embodiment, the inventions disclosed herein provide a group of genes (e.g., Tables 5 or 6) that may be analyzed to determine a subject's smoking status from a biological sample comprising the subject's nasal epithelial cells. For example, the biological sample may be analyzed to determine the expression of one or more genes listed in any of Table 5, Table 6, Table 12, Table 13, Table 14, Table 15 and/or Table 21, to thereby determine whether the subject has or is at risk of developing lung cancer. In certain embodiments, the nasal epithelial cells are analyzed using at least one and no more than 535 of the genes listed in Table 12 or Table 21. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or at least 10, at least 20, at least 30, at least 40 at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least or at maximum of 170, at least or at maximum of 180, at least or at maximum of 190, at least or at maximum of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 375, 380, 390, 400, 410, 420, 425, 450, 475, 500, 525 or at least 530 or at maximum of the 535 genes as listed on Table 12 or Table 21.
One example of the gene transcript groups useful in the diagnostic/prognostic assays and methods of the invention are set forth in Table 5, Table 6, Table 12, Table 13 or Table 21. The present inventors have determined that taking any group that has at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more of the Table 12 or Table 21 genes provides a much greater lung cancer diagnostic capability than chance alone. Similarly, the present inventors have determined that taking any group that has at least about 5, 10, 15, 20, 25, 30, 40, 50, 60 or more of the Tables 5 or 6 genes provides a much greater capability to determine a subject's smoking status than chance alone. Preferably one would analyze the nasal epithelial cells using more than about 20 of these genes, for example about 20-100 and any combination between, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and so on. In some instances, the present inventors have determined that one can enhance the sensitivity or diagnostic accuracy of the methods and assays disclosed herein by adding additional genes to any of these specific groups. For example, in certain aspects, the accuracy of such methods may approach about 70%, about 75%, about 80%, about 82.5%, about 85%, about 87.5%, about 88%, about 90%, about 92.5%, about 95%, about 97.5%, about 98%, about 99% or more by evaluating the differential expression of more genes from the set (e.g., the set of genes set forth in Tables 5, 6, 12, 13 or 21).
In some embodiments, the diagnosis of lung cancer is made by comparing the expression of the genes or groups of genes set forth in, for example Table 12 or Table 21, by the subject's nasal epithelial cells to a control subject or a control group (e.g., a positive control with a confirmed diagnosis of lung cancer). Similarly, in certain aspects, the determination of a subject's smoking status is made by comparing the expression of the genes or groups of genes from the subject's nasal epithelial cells to a control subject or a control group (e.g., a non-smoker negative control). In certain embodiments, an appropriate control is an expression level (or range of expression levels) of a particular gene that is indicative of a known lung cancer status. An appropriate reference can be determined experimentally by a practitioner of the methods disclosed herein or may be a pre-existing expression value or range of values. When an appropriate control is indicative of lung cancer, 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 lung cancer and the appropriate control may be indicative of lung cancer in the subject. When an appropriate control is indicative of lung cancer, a difference between an expression level determined from a subject in need of characterization or diagnosis of lung cancer and the appropriate reference may be indicative of the subject being free of lung cancer.
Alternatively, an appropriate control may be an expression level (or range of expression levels) of one or more genes that is indicative of a subject being free of lung cancer. For example, an appropriate control may be representative of the expression level of a particular set of genes in a reference (control) biological sample obtained from a subject who is known to be free of lung cancer. When an appropriate control is indicative of a subject being free of lung cancer, a difference between an expression level determined from a subject in need of diagnosis of lung cancer and the appropriate reference may be indicative of lung cancer in the subject. Alternatively, when an appropriate reference is indicative of the subject being free of lung cancer, 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 lung cancer and the appropriate reference level may be indicative of the subject being free of lung cancer.
The control groups can be or comprise one or more subjects with a positive lung cancer diagnosis, a negative lung cancer diagnosis, non-smokers, smokers and/or former smokers. Preferably, the genes or their expression products in the nasal epithelial cell sample of the subject are compared relative to a similar group, except that the members of the control groups may not have lung cancer. For example, such a comparison may be performed in the nasal epithelial cell sample from a smoker relative to a control group of smokers who do not have lung cancer. Such a comparison may also be performed, e.g., in the nasal epithelial cell sample from a non-smoker relative to a control group of non-smokers who do not have lung cancer. Similarly, such a comparison may be performed in the nasal epithelial cell sample from a former smoker or a suspected smoker relative to a control group of smokers who do not have lung cancer. The transcripts or expression products are then compared against the control to determine whether increased expression or decreased expression can be observed, which depends upon the particular gene or groups of genes being analyzed, as set forth, for example, in Table 12 or Table 21. In certain embodiments, at least 50% of the gene or groups of genes subjected to expression analysis must provide the described pattern. Greater reliability is obtained as the percent approaches 100%. Thus, in one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the one or more genes subjected to expression analysis demonstrate an altered expression pattern that is indicative of the presence or absence of lung cancer, as set forth in, for example, Table 12 or Table 21. Similarly, in one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the one or more genes subjected to expression analysis demonstrate an altered expression pattern that is indicative of the subject's smoking status, as set forth in, for example, Table 5 or Table 6.
Any combination of the genes and/or transcripts of Table 12 or Table 21 can be used in connection with the assays and methods disclosed herein. In one embodiment, any combination of at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80, 80-90, 90-100, 100-120, 120-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, and up to about 535 genes selected from the group consisting of genes or transcripts as shown in the Table 12 or Table 21.
The analysis of the gene expression of one or more genes may be performed using any gene expression methods known to one skilled in the art. Such methods include, but are not limited to expression analysis using nucleic acid chips (e.g. Affymetrix chips) and quantitative RT-PCR based methods using, for example real-time detection of the transcripts. Analysis of transcript levels according to the present invention can be made using total or messenger RNA or proteins encoded by the genes identified in the diagnostic gene groups of the present invention as a starting material. In certain embodiments the analysis is or comprises an immunohistochemical analysis with an antibody directed against proteins comprising at least about 10-20, 20-30, preferably at least 36, at least 36-50, 50, about 50-60, 60-70, 70-80, 80-90, 96, 100-180, 180-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-535 proteins encoded by the genes and/or transcripts as shown in Table 12 or Table 21.
In one embodiment, the analysis is performed analyzing the amount of proteins encoded by one or more of the genes listed in Table 12 or Table 21 and present in the sample. In one embodiment the analysis is performed using DNA by analyzing the gene expression regulatory regions of the airway transcriptome genes using nucleic acid polymorphisms, such as single nucleic acid polymorphisms or SNPs, wherein polymorphisms known to be associated with increased or decreased expression are used to indicate increased or decreased gene expression in the individual. In one embodiment, the present invention uses a minimally invasive sample procurement method for obtaining nasal epithelial cell RNA (e.g., mRNA) that can be analyzed by expression profiling, for example, by array-based gene expression profiling. These methods can be used to determine if nasal epithelial cell gene expression profiles are affected by cancer. The methods disclosed herein can also be used to identify patterns of gene expression that are diagnostic of lung disorders/diseases, for example, cancer, and to identify subjects at risk for developing lung cancer. All or a subset of the genes identified according to the methods described herein can be used to design an array, for example, a microarray, specifically intended for the diagnosis or prediction of lung disorders or susceptibility to lung disorders. The efficacy of such custom-designed arrays can be further tested, for example, in a large clinical trial of smokers.
In some embodiments, the gene expression levels are determined by RT-PCR, DNA microarray hybridization, RNASeq, or a combination thereof. In some embodiments, one or more of the gene expression products is labeled. For example, a mRNA (or a cDNA made from such an mRNA) from a nasal epithelial cell sample may be labeled.
The methods of analyzing expression and/or determining an expression profile of the one or more genes include, for example, Northern-blot hybridization, ribonuclease protection assay, and reverse transcriptase polymerase chain reaction (RT-PCR) based methods. In certain aspects, the different RT-PCR based techniques are a suitable quantification method for diagnostic purposes of the present invention, because they are very sensitive and thus require only a small sample size which is desirable for a diagnostic test. A number of quantitative RT-PCR based methods have been described and are useful in measuring the amount of transcripts according to the present invention. These methods include RNA quantification using PCR and complementary DNA (cDNA) arrays (Shalon, et al., Genome Research 6(7):639-45, 1996; Bernard, et al., Nucleic Acids Research 24(8): 1435-42, 1996), real competitive PCR using a MALDI-TOF Mass spectrometry based approach (Ding, et al., PNAS, 100: 3059-64, 2003), solid-phase mini-sequencing technique, which is based upon a primer extension reaction (U.S. Pat. No. 6,013,431, Suomalainen, et al., Mol. Biotechnol. June; 15(2): 123-31, 2000), ion-pair high-performance liquid chromatography (Doris, et al., J. Chromatogr. A May 8; 806(1):47-60, 1998), and 5′ nuclease assay or real-time RT-PCR (Holland, et al., Proc Natl Acad Sci USA 88: 7276-7280, 1991).
Additional approaches to assess gene expression of the one or more genes are known in the art and may include but are not limited to one or more of the following: additional cytological assays, assays for specific proteins or enzyme activities, assays for specific expression products including protein or RNA or specific RNA splice variants, in situ hybridization, whole or partial genome expression analysis, microarray hybridization assays, serial analysis of gene expression (SAGE), enzyme linked immunoabsorbance assays, mass-spectrometry, immunohistochemistry, blotting, sequencing, RNA sequencing, DNA sequencing (e.g., sequencing of cDNA obtained from RNA); Next-Gen sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing. For example, gene expression product levels may be determined according to the methods described in Kim, et. al. (Lancet Respir Med. 2015 June; 3(6):473-82, incorporated herein in its entirety, including all supplements). As used herein, the terms “assaying” or “detecting” or “determining” are used interchangeably in reference to determining gene expression product levels, and in each case, it is contemplated that the above-mentioned methods of determining gene expression product levels are suitable for detecting or assaying gene expression product levels. Gene expression product levels may be normalized to an internal standard such as total mRNA or the expression level of a particular gene including but not limited to glyceraldehyde 3 phosphate dehydrogenase, or tubulin.
In various embodiments, a sample comprises cells harvested from a tissue, e.g., in some embodiments the sample comprises cells harvested from a nasal epithelial cell sample. In certain embodiments, the cells may be harvested from a sample using standard techniques known in the art or disclosed herein. For example, in one embodiment, cells are harvested by centrifuging a cell sample and re-suspending the pelleted cells. The cells may be re-suspended in a buffered solution such as phosphate-buffered saline (PBS). After centrifuging the cell suspension to obtain a cell pellet, the cells may be lysed to extract nucleic acid, e.g., messenger RNA. All samples obtained from a subject, including those subjected to any sort of further processing, are considered to be obtained from the subject.
The sample, in one embodiment, is further processed before detection of the gene expression products is performed as described herein. For example, mRNA in a cell or tissue sample may be separated from other components of the sample. The sample may be concentrated and/or purified to isolate mRNA in its non-natural state, as the mRNA is not in its natural environment. For example, studies have indicated that the higher order structure of mRNA in vivo differs from the in vitro structure of the same sequence (see, e.g., Rouskin et al. (2014). Nature 505, pp. 701-705, incorporated herein in its entirety for all purposes).
mRNA from the sample in one embodiment, is hybridized to a synthetic DNA probe, which in some embodiments, includes a detection moiety (e.g., detectable label, capture sequence, barcode reporting sequence). Accordingly, in these embodiments, a non-natural mRNA-cDNA complex is ultimately made and used for detection of the gene expression product. In another embodiment, mRNA from the sample is directly labeled with a detectable label, e.g., a fluorophore. In a further embodiment, the non-natural labeled-mRNA molecule is hybridized to a cDNA probe and the complex is detected.
In one embodiment, once the mRNA is obtained from a sample, it is converted to complementary DNA (cDNA) in a hybridization reaction or is used in a hybridization reaction together with one or more cDNA probes. cDNA does not exist in vivo and therefore is a non-natural molecule. Furthermore, cDNA-mRNA hybrids are synthetic and do not exist in vivo. Besides cDNA not existing in vivo, cDNA is necessarily different than mRNA, as it includes deoxyribonucleic acid and not ribonucleic acid. The cDNA is then amplified, for example, by the polymerase chain reaction (PCR) or other amplification method known to those of ordinary skill in the art. For example, other amplification methods that may be employed include the ligase chain reaction (LCR) (Wu and Wallace, Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988), incorporated by reference in their entirety for all purposes, transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989), incorporated by reference in its entirety for all purposes), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990), incorporated by reference in its entirety for all purposes), incorporated by reference in its entirety for all purposes, and nucleic acid based sequence amplification (NASBA). Guidelines for selecting primers for PCR amplification are known to those of ordinary skill in the art. See, e.g., McPherson et al., PCR Basics: From Background to Bench, Springer-Verlag, 2000, incorporated by reference in their entirety for all purposes. The product of this amplification reaction, i.e., amplified cDNA is also necessarily a non-natural product. First, as mentioned above, cDNA is a non-natural molecule. Second, in the case of PCR, the amplification process serves to create hundreds of millions of cDNA copies for every individual cDNA molecule of starting material. The number of copies generated are far removed from the number of copies of mRNA that are present in vivo.
In one embodiment, cDNA is amplified with primers that introduce an additional DNA sequence (e.g., adapter, reporter, capture sequence or moiety, barcode) onto the fragments (e.g., with the use of adapter-specific primers), or mRNA or cDNA gene expression product sequences are hybridized directly to a cDNA probe comprising the additional sequence (e.g., adapter, reporter, capture sequence or moiety, barcode). Amplification and/or hybridization of mRNA to a cDNA probe therefore serves to create non-natural double stranded molecules from the non-natural single stranded cDNA, or the mRNA, by introducing additional sequences and forming non-natural hybrids. Further, as known to those of ordinary skill in the art, amplification procedures have error rates associated with them. Therefore, amplification introduces further modifications into the cDNA molecules. In one embodiment, during amplification with the adapter-specific primers, a detectable label, e.g., a fluorophore, is added to single strand cDNA molecules. Amplification therefore also serves to create DNA complexes that do not occur in nature, at least because (i) cDNA does not exist in vivo, (i) adapter sequences are added to the ends of cDNA molecules to make DNA sequences that do not exist in vivo, (ii) the error rate associated with amplification further creates DNA sequences that do not exist in vivo, (iii) the disparate structure of the cDNA molecules as compared to what exists in nature, and (iv) the chemical addition of a detectable label to the cDNA molecules.
In some embodiments, the expression of a gene expression product of interest is detected at the nucleic acid level via detection of non-natural cDNA molecules.
The gene expression products described herein include RNA comprising the entire or partial sequence of any of the nucleic acid sequences of interest, or their non-natural cDNA product, obtained synthetically in vitro in a reverse transcription reaction. The term “fragment” is intended to refer to a portion of the polynucleotide that generally comprise at least 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,200, or 1,500 contiguous nucleotides, or up to the number of nucleotides present in a full length gene expression product polynucleotide disclosed herein. A fragment of a gene expression product polynucleotide will generally encode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up to the total number of amino acids present in a full-length gene expression product protein of the invention.
In certain embodiments, a gene expression profile may be obtained by whole transcriptome shotgun sequencing (“WTSS” or “RNAseq”; see, e.g., Ryan et. al. BioTechniques 45: 81-94), which makes the use of high-throughput sequencing technologies to sequence cDNA in order to about information about a sample's RNA content. In general terms, cDNA is made from RNA, the cDNA is amplified, and the amplification products are sequenced.
After amplification, in some embodiments, the cDNA may be sequenced using any convenient method. For example, the fragments may be sequenced using Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (the SOLiD platform) or Life Technologies' Ion Torrent platform. Examples of such methods are described in the following references: Margulies et al (Nature 2005 437: 376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005 309: 1728); Imelfort et. al. (Brief Bioinform. 2009 10:609-18); Fox et. al. (Methods Mol Biol. 2009; 553:79-108); Appleby et. al. (Methods Mol Biol. 2009; 513: 19-39) and Morozova (Genomics. 2008 92:255-64), which are all incorporated by reference for the general descriptions of the methods and the particular steps of the methods, including all starting products, reagents, and final products for each of the steps. As would be apparent, forward and reverse sequencing primer sites that compatible with a selected next generation sequencing platform may be added to the ends of the fragments during the amplification step.
In other embodiments, the products may be sequenced using nanopore sequencing (e.g. as described in Soni et. al. Clin Chem 53: 1996-2001 2007, or as described by Oxford Nanopore Technologies). Nanopore sequencing is a single-molecule sequencing technology whereby a single molecule of DNA is sequenced directly as it passes through a nanopore. A nanopore is a small hole, of the order of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential (voltage) across it results in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows is sensitive to the size and shape of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule obstructs the nanopore to a different degree, changing the magnitude of the current through the nanopore in different degrees. Thus, this change in the current as the DNA molecule passes through the nanopore represents a reading of the DNA sequence. Nanopore sequencing technology as disclosed in U.S. Pat. Nos. 5,795,782, 6,015,714, 6,627,067, 7,238,485 and 7,258,838 and U.S. patent application publications US2006003171 and US20090029477.
In some embodiments, the gene expression product of the subject methods is a protein, and the amount of protein in a particular biological sample may be analyzed using a classifier derived from protein data obtained from cohorts of samples. The amount of protein may be determined by one or more of the following: enzyme-linked immunosorbent assay (ELISA), mass spectrometry, blotting, or immunohistochemistry.
In some embodiments, gene expression product markers and alternative splicing markers may be determined by microarray analysis using, for example, Affymetrix arrays, cDNA microarrays, oligonucleotide microarrays, spotted microarrays, or other microarray products from Biorad, Agilent, or Eppendorf. Microarrays provide particular advantages because they may contain a large number of genes or alternative splice variants that may be assayed in a single experiment. In some cases, the microarray device may contain the entire human genome or transcriptome or a substantial fraction thereof allowing a comprehensive evaluation of gene expression patterns, genomic sequence, or alternative splicing. Markers may be found using standard molecular biology and microarray analysis techniques as described in Sambrook Molecular Cloning a Laboratory Manual 2001 and Baldi, P., and Hatfield, W. G., DNA Microarrays and Gene Expression 2002.
Microarray analysis generally begins with extracting and purifying nucleic acid from a biological sample, (e.g. a biopsy or fine needle aspirate) using methods known to the art. For expression and alternative splicing analysis it may be advantageous to extract and/or purify RNA from DNA. It may further be advantageous to extract and/or purify niRNA from other forms of RNA such as tRNA and rRNA.
Purified nucleic acid may further be labeled with a fluorescent label, radionuclide, or chemical label such as biotin, digoxigenin, or digoxin for example by reverse transcription, polymerase chain reaction (PCR), ligation, chemical reaction or other techniques. The labeling may be direct or indirect which may further require a coupling stage. The coupling stage can occur before hybridization, for example, using aminoallyl-UTP and NHS amino-reactive dyes (like cyanine dyes) or after, for example, using biotin and labelled streptavidin. In one example, modified nucleotides (e.g. at a 1 aaUTP: 4 TTP ratio) are added enzymatically at a lower rate compared to normal nucleotides, typically resulting in 1 every 60 bases (measured with a spectrophotometer). The aaDNA may then be purified with, for example, a column or a diafiltration device. The aminoallyl group is an amine group on a long linker attached to the nucleobase, which reacts with a reactive label (e.g. a fluorescent dye).
The labeled samples may then be mixed with a hybridization solution which may contain sodium dodecyl sulfate (SDS), SSC, dextran sulfate, a blocking agent (such as COT1 DNA, salmon sperm DNA, calf thymus DNA, PolyA or PolyT), Denhardt's solution, formamine, or a combination thereof.
A hybridization probe is a fragment of DNA or RNA of variable length, which is used to detect in DNA or RNA samples the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions) into single DNA strands and then hybridized to the target DNA.
To detect hybridization of the probe to its target sequence, the probe is tagged (or labeled) with a molecular marker; commonly used markers are 32P or Digoxigenin, which is nonradioactive antibody-based marker. DNA sequences or RNA transcripts that have moderate to high sequence complementarity (e.g. at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more complementarity) to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Detection of sequences with moderate or high complementarity depends on how stringent the hybridization conditions were applied; high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized.
A mix comprising target nucleic acid to be hybridized to probes on an array may be denatured by heat or chemical means and added to a port in a microarray. The holes may then be sealed and the microarray hybridized, for example, in a hybridization oven, where the microarray is mixed by rotation, or in a mixer. After an overnight hybridization, non-specific binding may be washed off (e.g. with SDS and SSC). The microarray may then be dried and scanned in a machine comprising a laser that excites the dye and a detector that measures emission by the dye. The image may be overlaid with a template grid and the intensities of the features (e.g. a feature comprising several pixels) may be quantified.
Various kits may be used for the amplification of nucleic acid and probe generation of the subject methods. Examples of kit that may be used in the present invention include but are not limited to Nugen WT-Ovation FFPE kit, cDNA amplification kit with Nugen Exon Module and Frag/Label module. The NuGEN WT-Ovation™. FFPE System V2 is a whole transcriptome amplification system that enables conducting global gene expression analysis on the vast archives of small and degraded RNA derived from FFPE samples. The system is comprised of reagents and a protocol required for amplification of as little as 50 ng of total FFPE RNA. The protocol may be used for qPCR, sample archiving, fragmentation, and labeling. The amplified cDNA may be fragmented and labeled in less than two hours for GeneChip™. 3′ expression array analysis using NuGEN's FL-Ovation™. cDNA Biotin Module V2. For analysis using Affymetrix GeneChip™. Exon and Gene ST arrays, the amplified cDNA may be used with the WT-Ovation Exon Module, then fragmented and labeled using the FLOvation™. cDNA Biotin Module V2. For analysis on Agilent arrays, the amplified cDNA may be fragmented and labeled using NuGEN's FL-Ovation™. cDNA Fluorescent Module.
In some embodiments, Ambion WT-expression kit may be used. Ambion WT-expression kit allows amplification of total RNA directly without a separate ribosomal RNA (rRNA) depletion step. With the Ambion™ WT Expression Kit, samples as small as 50 ng of total RNA may be analyzed on Affymetrix™. GeneChip™ Human, Mouse, and Rat Exon and Gene 1.0 ST Arrays. In addition to the lower input RNA requirement and high concordance between the Affymetrix™ method and TaqMan™ real-time PCR data, the Ambion™. WT Expression Kit provides a significant increase in sensitivity. For example, a greater number of probe sets detected above background may be obtained at the exon level with the Ambion™. WT Expression Kit as a result of an increased signal-to-noise ratio. Ambion™-expression kit may be used in combination with additional Affymetrix labeling kit. In some embodiments, AmpTec Trinucleotide Nano mRNA Amplification kit (6299-A15) may be used in the subject methods. The ExpressArt™ TRinucleotide mRNA amplification Nano kit is suitable for a wide range, from 1 ng to 700 ng of input total RNA. According to the amount of input total RNA and the required yields of aRNA, it may be used for 1-round (input >300 ng total RNA) or 2-rounds (minimal input amount 1 ng total RNA), with aRNA yields in the range of >10 μg. AmpTec's proprietary TRinucleotide priming technology results in preferential amplification of mRNAs (independent of the universal eukaryotic 3′-poly(A)-sequence), combined with selection against rRNAs. More information on AmpTec Trinucleotide Nao mRNA Amplification kit may be obtained at www.amp-tec.com/products.htm. This kit may be used in combination with cDNA conversion kit and Affymetrix labeling kit.
The raw data may then be normalized, for example, by subtracting the background intensity and then dividing the intensities making either the total intensity of the features on each channel equal or the intensities of a reference gene and then the t-value for all the intensities may be calculated. More sophisticated methods, include z-ratio, loess and lowess regression and RMA (robust multichip analysis), such as for Affymetrix chips.
In some embodiments, the above described methods may be used for determining transcript expression levels for training (e.g., using a classifier training module) a classifier to differentiate whether a subject is a smoker or non-smoker. In some embodiments, the above described methods may be used for determining transcript expression levels for training (e.g., using a classifier training module) a classifier to differentiate whether a subject has cancer or no cancer, e.g., based upon such expression levels in a sample comprising cells harvested from a nasal epithelial cell sample.
The presently described gene expression profile can also be used to screen for subjects who are susceptible to or otherwise at risk for developing lung cancer. For example, a current smoker of advanced age (e.g., 70 years old) may be at an increased risk for developing lung cancer and may represent an ideal candidate for the assays and methods disclosed herein. Moreover, the early detection of lung cancer in such a subject may improve the subject's overall survival. Accordingly, in certain aspects, the assays and methods disclosed herein are performed or otherwise comprise an analysis of the subject's clinical risk factors for developing cancer. For example, one or more clinical risk factors selected from the group consisting of advanced age (e.g., age greater than about 40 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years or more), smoking status, the presence of a lung nodule greater than 3 cm on CT scan, the lesion or nodule location (e.g., centrally located, peripherally located or both) and the time since the subject quit smoking. In certain embodiments, the assays and methods disclosed herein further comprise a step of considering the presence of any such clinical risk factors to inform the determination of whether the subject has lung cancer or is at risk of developing lung cancer.
As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. In certain embodiments, the subject is a mammal (e.g., a primate or a human). In particular embodiments, the subject is a human. 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 cancer. The subject may have a cancer-free personal or family history. The subject may exhibit one or more symptoms of lung cancer or other lung disorder (e.g., emphysema, COPD). For example, the subject may have a new or persistent cough, worsening of an existing chronic cough, blood in the sputum, 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 an individual 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). The subject may be an individual who has undergone an indeterminate or non-diagnostic bronchoscopy. The subject may be an individual who has undergone an indeterminate or non-diagnostic bronchoscopy and who has been recommended to proceed with an invasive lung procedure (e.g., transthoracic needle aspiration, mediastinoscopy, lobectomy, or thoracotomy) based upon the indeterminate or non-diagnostic bronchoscopy. The terms, “patient” and “subject” are used interchangeably herein. In some embodiments, the subject is at risk for developing lung cancer. In some embodiments, the subject has lung cancer and the assays and methods disclosed herein may be used to monitor the progression of the subject's disease or to monitor the efficacy of one or more treatment regimens.
In certain aspects, the methods and assays disclosed herein are useful for determining a treatment course for a subject. For example, such methods and assays may involve determining the expression levels of one or more genes (e.g., one or more of the genes set forth in Table 12 or Table 21, or one or more or all of the genes set forth in Table 13) in a biological sample obtained from the subject, and determining a treatment course for the subject based on the expression profile of such one or more genes. In some embodiments, the treatment course is determined based on a lung cancer risk-score derived from the expression levels of the one or more genes analyzed. The subject may be identified as a candidate for a lung cancer therapy based on an expression profile that indicates the subject has a relatively high likelihood of having lung cancer. The subject may be identified as a candidate for an invasive lung procedure (e.g., transthoracic needle aspiration, mediastinoscopy, lobectomy, or thoracotomy) based on an expression profile that indicates the subject has a relatively high likelihood of having lung cancer (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%). In certain aspects, a relatively high likelihood of having lung cancer means greater than about a 65% chance of having lung cancer. In certain aspects, a relatively high likelihood of having lung cancer means greater than about a 70% chance of having lung cancer. In certain aspects, a relatively high likelihood of having lung cancer means greater than about a 75% chance of having lung cancer. In certain aspects, a relatively high likelihood of having lung cancer means greater than about an 80-85% chance of having lung cancer. The subject may be identified as not being a candidate for a lung cancer therapy or an invasive lung procedure based on an expression profile 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 lung cancer. In certain aspects, a relatively low likelihood of having lung cancer means less than about a 35% chance of having lung cancer. In certain aspects, a relatively low likelihood of having lung cancer means less than about a 30% chance of having lung cancer. In certain aspects, a relatively low likelihood of having lung cancer means less than about a 25% chance of having lung cancer. In certain aspects, a relatively low likelihood of having lung cancer means less than about a 35% chance of having lung cancer. In certain aspects, a relatively low likelihood of having lung cancer means less than about a 20-25% chance of having lung cancer. Accordingly, in certain aspects of the present inventions, if the methods disclosed herein are indicative of the subject having lung cancer or of being at risk of developing lung cancer, such methods may comprise a further step of treating the subject (e.g., administering to the subject a treatment comprising one or more of chemotherapy, radiation therapy, immunotherapy, surgical intervention and combinations thereof).
In certain aspects, if the methods and assays disclosed herein are indicative of a subject being at a higher risk of having or developing lung cancer, the subject may be subjected to more invasive monitoring, such as a direct tissue sampling or biopsy of the nodule, under the presumption that the positive test indicates a higher likelihood of the nodule is a cancer. Alternatively, on the basis of the methods and assays disclosed herein being indicative of a subject's higher risk of having or developing lung cancer, in certain embodiments an appropriate therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject. Conversely, in those instances where the methods and assays disclosed herein are indicative of a subject having a reduced risk of developing lung cancer, then in certain aspects the subject may be subjected to further confirmatory testing, such as further imaging surveillance (e.g., a repeat CT scan to monitor whether the nodule grows or changes in appearance before doing a more invasive procedure), or a determination made to withhold a particular treatment (e.g., chemotherapy or radiation therapy) on the basis of the subject's favorable or reduced risk of having or developing lung cancer. In some embodiments, the assays and methods disclosed herein may be used to confirm the results or findings from a more invasive procedure, such as direct tissue sampling or biopsy. For example, in certain aspects the assays and methods disclosed herein may be used to confirm or monitor the benign status of a previously biopsied nodule or lesion.
In some embodiments, the methods and assays disclosed herein are useful for determining a treatment course for a subject that has undergone an indeterminate or non-diagnostic bronchoscopy does not have lung cancer, wherein the method comprises determining the expression levels of one or more genes (e.g., one or more of the genes set forth in Table 12 or Table 21, or one or more or all of the genes set forth in Table 13) in a sample of nasal epithelial cells obtained from the subject, and determining whether the subject that has undergone an indeterminate or non-diagnostic bronchoscopy does or does not have lung cancer or is not at risk of developing lung cancer. In some such embodiments, the method comprises determining a lung cancer risk-score derived from the expression levels of the one or more genes analyzed. In particular embodiments, the subject that has undergone an indeterminate or non-diagnostic bronchoscopy would have typically been identified as being a candidate for an invasive lung procedure (e.g., transthoracic needle aspiration, mediastinoscopy, lobectomy, or thoracotomy) based upon such indeterminate of non-diagnostic bronchoscopy result, but the subject is instead identified as being a candidate for a non-invasive procedure (e.g., monitoring by CT scan) because the subjects expression levels of the one or more genes (e.g., one or more of the genes set forth in Table 12 or Table 21, or one or more or all of the genes set forth in Table 13) in the sample of nasal epithelial cells obtained from the subject indicates that the subject has a low risk of having lung cancer (e.g., in some embodiments the instant method indicates that the subject has a greater than 60% chance of not having cancer, or a greater than 70%, 80%, or greater than 90% chance of not having cancer). In some embodiments, the subject may be identified as a candidate for an invasive lung cancer therapy based on an expression profile that indicates the subject has a relatively high likelihood of having lung cancer (e.g., in some embodiments the instant method indicates that the subject has a greater than 60% chance of having cancer, or a greater than 70%, 80%, or greater than 90% chance of having cancer). Accordingly, in certain aspects of the present inventions, if the methods disclosed herein are indicative of the subject having lung cancer or of being at risk of developing lung cancer, such methods may comprise a further step of treating the subject (e.g., administering to the subject a treatment comprising one or more of chemotherapy, radiation therapy, immunotherapy, surgical intervention and combinations thereof).
In some cases, an expression profile 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 elect to monitor the subject and repeat the assays or methods at one or more later points in time, or undertake further diagnostics procedures to rule out lung cancer, or make a determination that cancer is present, soon after the subject's lung cancer risk determination was made. Also contemplated herein is the inclusion of one or more of the genes and/or transcripts presented in, for example, Table 5, Table 6, Table 12, Table 13, Table 14, Table 15 or Table 21, into a composition or a system for detecting lung cancer in a subject. For example, any one or more genes and or gene transcripts from Table 12, Table 13 or Table 21 may be added as a lung cancer marker for a gene expression analysis. In some aspects, the present inventions relate to compositions that may be used to determine the expression profile of one or more genes from a subject's biological sample comprising nasal epithelial cells. For example, compositions are provided that consist essentially of nucleic acid probes that specifically hybridize with one or more genes set forth in Table 12, Table 13 or Table 21. These compositions may also include probes that specifically hybridize with one or more control genes and may further comprise appropriate buffers, salts or detection reagents. In certain embodiments, such 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 compositions described herein may be assembled into diagnostic or research kits to facilitate their use in one or more diagnostic or research applications. In some embodiments, such kits and diagnostic compositions are provided that comprise one or more probes capable of specifically hybridizing to up to 5, up to 10, up to 25, up to 50, up to 100, up to 200, up to 300, up to 400, up to 500 or up to 535 genes set forth in Table 12, Table 13 or Table 21 or their expression products (e.g., mRNA). In some embodiments, each of the nucleic acid probes specifically hybridizes with one or more genes selected from those genes set forth in Table 12, Table 13 or Table 21, or with a nucleic acid having a sequence complementary to such genes. In some aspects, 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 probes specifically hybridizes with one or more genes selected from group of set forth in Table 12, Table 13 or Table 21, or with a nucleic acid having a sequence complementary to such genes.
A kit may include one or more containers housing one or more of the components provided in this disclosure 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 and/or disposition of these compositions. Kits may contain the components in appropriate concentrations or quantities for running various experiments.
The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or the entire group members are present in, employed in or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

EXAMPLES

Previous work from our lab has shown that bronchial and nasal epithelium exhibit a common physiological response to tobacco smoke exposure (Zhang, et al., Phys. Gen. 2011). Given this relationship and the demonstrated utility of bronchial gene expression as a diagnostic marker of lung cancer, the present inventors sought to test the hypothesis that the cancer-associated expression profiles observed in the bronchial airways might also be detectable in nasal epithelium. Detecting the cancer-associated airway field of injury via nasal epithelium would offer a faster, non-invasive and cheaper alternative to sampling bronchial epithelium, and thereby expand the clinical settings where airway gene expression would have utility in evaluating patients for lung cancer.
In the following studies, the present inventors identified genes with cancer-associated expression profiles in nasal epithelium using samples obtained from current and former smokers undergoing bronchoscopy for clinical suspicion of lung cancer as part of the Airway Epithelium Gene Expression in the Diagnosis of Lung Cancer (AEGIS) clinical trials. The inventors demonstrated that the cancer-associated field of injury observed in bronchial epithelium extended to the nose and that nasal epithelial gene expression adds information about lung cancer that is distinct from clinical risk factors. These findings suggest that nasal gene expression may be useful in determining the cancer status of indeterminate pulmonary nodules.

Example 1—Lung Cancer-Associated Gene Expression in Nasal Epithelium

To identify genes whose expression is associated with lung cancer status in nasal epithelium and to compare the relationship between nasal and bronchial cancer-associated gene expression, the present inventors used existing microarray data from 299 bronchial epithelium samples from patients in the AEGIS clinical trials (Whitney, et al., BMC Med Gen 2015) and generated novel microarray data from 554 nasal epithelium samples obtained from patients in the same trials. All samples were collected from consenting patients who were undergoing bronchoscopy for clinical suspicion on lung cancer. 424 nasal samples were collected from patients enrolled in the AEGIS-1 trial and 130 were from patients in the AEGIS-2 trial (FIG. 4). Thirty one patients from the AEGIS-1 cohort had an indeterminate cancer diagnosis or were lost to follow up and were removed from the study. The present inventors additionally removed 18 microarray samples from the AEGIS-1 dataset that did not meet minimum quality standards (Table 11). No samples were removed from the AEGIS-2 dataset. The remaining 375 samples from the AEGIS-1 cohort were used as a training set in which all data analyses and model building were performed, while the 130 samples from the AEGIS-2 cohort were used solely to validate the predictive models described herein (Table 1, below). The distribution of cancer stages was slightly skewed toward later-stage cancers in the validation set (Table 7). Lung cancer patients tended to have larger nodules than patients with benign diagnoses in both the training and validation sets (P<0.001 for both comparisons) (Table 8) while patient age was statistically significantly higher among cancer patients in the training set (P<0.001). The gene expression data from these samples has been deposited in the NCBI Gene Expression Omnibus under accession number GSE80796. The nasal samples were selected from a larger pool of banked tissue samples and were well balanced for clinical covariates between cancer and benign classes (see, Table 1, below). The cases and controls whose samples were used in the training set varied by both age (p=0.0002) and mass size (p=1.4e−12), while in the test set they varied only by mass size (p=3.8e−08).
Differential expression analysis via linear modeling revealed 535 genes that were significantly associated with cancer status in our training set (p<0.001), as illustrated in FIG. 1 (see, Table 21, below). Of these, 43 genes were upregulated in cancer patients compared to controls, while 492 were down-regulated, but there was heterogeneity in the expression of these genes within the cases and controls. Unsupervised hierarchical clustering separated the samples into two primary clusters, as depicted in FIG. 1. The distribution of cases and controls was significantly skewed between the two patient clusters (p=0.0002; FIG. 1 and FIGS. 2A-2B), with Patient Cluster 1 enriched for patients with lung cancer and Patient Cluster 2 enriched for controls (Table 12).
Several distinct patterns of gene co-expression were also observed within these 535 genes and consensus clustering identified four distinct co-expression clusters (Table 12). The smallest of the four clusters contained 43 genes that were up-regulated in samples from patients with cancer relative to controls. The other three clusters were down-regulated in patients with cancer relative to controls (FIG. 1). Genes that were downregulated in patients with lung cancer were enriched for genes associated with DNA damage, regulation of apoptosis, and processes involved in immune system activation including the interferon-gamma signaling pathway and antigen presentation (see, Table 2 below). Among genes that were upregulated in lung cancer patients, the present inventors found enrichment for genes involved in endocytosis and ion transport (see, Table 2 below). A complete list of the 535 genes and their respective cluster memberships is provided in Table 12, below.
To summarize the behavior of each cluster, the average expression of all genes in a cluster was computed for each sample. Each of the four cancer cluster means was strongly associated with cancer status (p<0.001), as shown in Table 2, below. The present inventors assessed the gene functions enriched in each of these four clusters using the Reactome and GO databases accessed through the web-based program EnrichR (Chen, et al., 2013 BMC Bioinfo). A complete list of statistically significantly enriched pathways and GO categories (FDR<0.05) is shown in Tables 13, 22 and 23, below. Clusters 1, 2, and 3 were enriched for genes involved in the regulation of apoptosis, immune system signaling, and xenobiotic detoxification, respectively. Cluster 4 was enriched for genes involved in ion transport.

Example 2—Similarities in Cancer-Associated Gene Expression Changes Between Nasal and Bronchial Epithelium

Given the strong concordance in smoking-related gene expression between nasal and bronchial epithelium, the present inventors next sought to determine if a shared pattern of cancer-related gene expression might exist between the nose and bronchus by leveraging microarray data from 299 bronchial epithelium samples obtained from AEGIS-1 patients (Whitney, et al., BMC Medical Genomics). One hundred and fifty-seven of the 299 bronchial samples came from the same patients as those in our nasal training set (Table 9 and FIG. 5). This analysis revealed significant enrichment (p<0.001) of both nasal gene sets in the bronchial ranked list, suggesting that the gene expression differences associated with the presence of lung cancer in nasal epithelium were also significantly concordantly altered in the bronchial epithelium (FIG. 2A). The expression profiles in both nasal and bronchial epithelium of the genes with the most concordant differential expression profiles between the two tissues and that contribute most strongly to this enrichment by GSEA (the “leading edge” genes) are shown in FIG. 2B and are listed in Table 21. Unsupervised hierarchical clustering of the leading edge genes organized the samples into two primary groups in each tissue. Importantly, the proportion of cancer patients in Patient Cluster 1 was significantly higher than the proportion found in Patient Cluster 2 in the bronchial (p=0.0358) samples, further demonstrating that the genes with cancer-associated expression in the nasal epithelium are part of a shared field of lung-cancer associated injury that encompasses both the nose and bronchus.
To further corroborate the hypothesis of a shared field of lung-cancer associated injury, the present inventors also examined the nasal expression patterns of genes previously found to be associated with lung cancer in bronchial epithelium (Whitney, et al., BMC Med Genomics 2015). Whitney, et al. previously reported a gene-expression signature of 232 genes grouped in 11 distinct co-expression clusters from bronchial epithelial samples that were strongly associated with the presence of lung cancer. Using the mean expression values of the genes in each of these clusters as a summary of the expression of each cluster in each patient, the present inventors found that eight of these clusters were significantly associated with the presence or absence of lung cancer (p<0.05) in the training set (Table 3, below). Among the clusters most associated with cancer were genes involved in cell cycle, response to retinoic acid, and the innate immune response (Table 3). Based on the concordant expression of cancer-associated genes in bronchial and nasal epithelium, the present inventors computed the bronchial lung cancer classifier risk score (Whitney, et al., BMC Med Gen 2015) for each of the samples in our nasal training set. The risk scores computed on matched bronchial and nasal samples were highly correlated (R=0.70, p<0.001, n=157) and the classifier had a sensitivity of 81% and AUC of 0.65 (p=8.1e−13, n=375) in the entire training set (FIG. 3). Taken together, these results suggest that some of the lung-cancer associated gene expression differences are similar in nasal and bronchial epithelium.

Example 3

To determine if nasal gene expression could serve as a predictor of lung cancer status, the present inventors selected the thirty most statistically significantly differentially expressed genes (P<0.001) from among the 535 genes with cancer-associated nasal gene expression for use in a weighted-voting biomarker (Table 13). The biomarker panel size of 30 genes was chosen as the smallest number of genes that achieved maximal performance in cross-validation. This biomarker had an AUC of 0.69 (n=375, 95% CI=0.63 to 0.75, P<0.001) in cross validation in the training set. Twenty-two of the 30 genes were also statistically significantly correlated between matched bronchial and nasal samples (mean R=0.29, range=0.16-0.49, P<0.05). In order to evaluate the potential for the nasal gene expression biomarker to add to clinical risk factors for lung cancer detection, the present inventors developed a clinical risk factor model and tested whether incorporating the gene-expression biomarker enhanced its performance. The computation of the clinical factor model biomarker score was derived from the following model,
x=(−4.65244938)+(−0.24676442*SMK)+(−1.16932025*TSQ1)+(0.12091159*TSQ2)+(0.07136355*AGE)+(1.22446427*BMS1)+(2.65403176*BMS2),
where, SMK=1 if former smoker and 0 if current smoker, TSQ1=1 if time since quit smoking is >=15 years, and 0 otherwise, T′SQ2=1 if time since quit smoking is unknown, and 0 otherwise, AGE=the patient's numeric age in years, BMS1=1 if patient's mass size is <3 cm, and 0 otherwise, and BMS2=1 if patient's mass size is >=3 cm, and 0 otherwise; then
$Clinical Factor Model Biomaker Score = \frac{e^{x}}{1 + e^{x}},$
where a patient is predicted cancer positive if the clinical factor model biomarker score is greater than 0.5823596, and cancer negative otherwise.
Gould previously identified smoking status, time since quit, age, and mass size as important clinical risk factors of lung cancer for patients with solitary pulmonary nodules (Gould, et al., Chest 2007). However, self-reported smoking status and time since quit which have been shown to be inconsistent with serum cotinine levels, especially in newly diagnosed lung cancer patients (Lewis, et al, Biomarkers 2003; Morales, et al., CCC 2013) and the inventors therefore used an approach similar to that described by Whitney, et al., to identify gene expression profiles that could serve as their surrogates. Two logistic regression models, including 5 and 2 genes, respectively, were derived in the training set to predict smoking status and time since quit (<15 y, >15 y) (Tables 14 and 15), where the equations associated with Tables 14 and 15 are respectively shown below,
$Clinical Risk Factors with Genomic Correlates Model Score = \frac{e^{x}}{1 + e^{x}},$
These classifiers had AUC values of 0.89 (p<2.2e−16, n=375) and 0.75 (p=0.0001, n=319) in the training set, respectively. Consistent with what has been reported for bronchial epithelial gene expression, the present inventors could not identify a gene expression predictor of patient age (Whitney, et al., BMC Med Gen 2015); nor were the present inventors able to identify a robust gene expression correlate of mass size. Collectively, the gene expression correlates for smoking status and time since quit as well as numerical age and categorized mass size (<3 cm, ≥3 cm, infiltrates) were used to model lung cancer using logistic regression in the training set (Table 16) and derived from the following model, where
x=−5.14689+(Genomic_Smoking_Status_Score*1.82244)+(Genomic_Time Since_Quit Score*2.31235)+(AGE*0.04947)+(BMS1*1.27246)+(BMS2*2,59898),
where, AGE=the patient's numeric age in years, BMS1=1 if patient's mass size is <3 cm, and 0 otherwise, BMS2=1 if patient's mass size is >=3 cm, and 0 otherwise, and
$x = - 24.1410 + (Expression_of_Probeset_8051583 * 0.2521) + (Expression_of_Probeset_7990391 * 0.0544) + (Expression_of_Probeset_7942693 * 2.5181) + (Expression_of_Probeset_8080578 * 1.7191) + (Expression_of_Probeset_8033257 * - 0.4727)$ $Genomic Smoking Status Score = \frac{e^{x}}{1 + e^{x}}; and$ $x = - 1.8161 + (Expression_of_Probeset_7990391 * 0.5726) + (Expression_of_Probeset_8051583 * - 0.4519)$ $Genomic Time Since Quit Score = \frac{e^{x}}{1 + e^{x}} .$
where a patient is predicted cancer positive if clinical risk factors with genomic correlates model score is greater than 0.4969356, and cancer negative otherwise.
These risk factors were further combined with the cancer-associated gene expression classifier into a single logistic regression clinicogenomic classifier, the parameters of which were also derived in the training set (Table 17) and from the following model, where,
x=−4.1504024+(Genomic_Smoking_Status_Score*0.7534516)+(Genomic_Time Since_Quit Score*0.3276714)+(GenomicSancer_Classifer_Score*0.6629011)+(AGE*0.0452670)+(BMS1*1.3423457)+(BMS2*2.6932782),
where, AGE=the patient's numeric age in years, BMS1=1 if patient's mass size is <3 cm, and 0 otherwise, BMS2=1 if patient's mass size is >=3 cm, and 0 otherwise, and
$Clinicogenomic with Genomic Correlates Model Score = \frac{e^{x}}{1 + e^{x}},$
where a patient is predicted cancer positive if clinicogenomic with genomic correlates model score is greater than 0.4590236, and cancer negative otherwise.
The performance of the clinical and clinicogenomic models was evaluated using an independent set of nasal samples (n=130) from the AEGIS-2 clinical trial that were not used in the development of either classifier. The clinicogenomic model yielded an AUC of 0.80 in the validation set which was significantly higher than the AUC of 0.76 achieved by the clinical risk factor model alone (p=0.05). Operating points for binary classification in both models were chosen to achieve 50% specificity in the training set. The addition of cancer-associated gene expression to the clinical risk-factor model resulted in a significant increase in sensitivity from 0.85 to 0.94 (p=0.04) and increase in negative predictive value from 0.73 to 0.87 (Table 18). Importantly, the clinicogenomic model showed improvements in sensitivity from 63% to 88% over the clinical model in subjects with lesion size <3 cm and showed stable or improved performance in patients with lesions >3 cm or ill-defined infiltrates (Table 18). Consistently higher sensitivity was also observed with the clinicogenomic model in patients with central and/or peripheral nodules compared to the clinical model (Table 19). Furthermore, the addition of cancer-associated gene expression to clinical risk factors improved prediction sensitivity across all stages and cell types of disease (Table 20). Collectively, these data suggest that nasal gene expression captures molecular information about the likelihood of lung cancer that is independent of clinical factors and therefore has the potential to improve lung cancer detection.

Example 4

In an alternative approach, the present inventors built clinical and clinicogenomic models that used reported clinical values instead of a mixture of reported clinical values and gene-expression predicted clinical values as in Example 3. In choosing which clinical risk factors to include, the present inventors again relied on a study in which Gould et al. identified smoking status, time since quit, age, and mass size as important clinical risk factors of lung cancer for patients with solitary pulmonary nodules (Gould, et al., Chest 2007). Patient age, smoking status (current, former), time since quit (<15 years, >15 years, unknown), and categorized mass size (<3 cm, >3 cm, infiltrates) were used to create a clinical risk factor model for lung cancer using logistic regression. The training set for this model consisted of the nasal training set used to derive the gene expression classifier as well as clinical data from an additional 142 patients from the AEGIS-1 cohort for a total training set of 517 patients for the clinical model (see, FIG. 5). A clinicogenomic logistic regression model that incorporated the clinical factors and the nasal gene expression classifier score was derived in the 375 training set samples with nasal gene expression. The genomic cancer classifier score used to calculate the clinicogenomic biomarker score was derived from the following model,
Gene_1_score=−0.076842874545387*(Expression_of_probeset_8091385−10.223361024585)
Gene_2_score=−0.066812409800121*(Expression_of_probeset_8115147−10.4979919874352)
Gene_3_score=−0.0508738437722716*(Expression_of_probeset_8034420−7.74862668913246)
Gene_4_score=−0.0853002904314322*(Expression_of_probeset_8075720−6.02260696919916)
Gene_5_score=−0.0663441276969046*(Expression_of_probeset_7940775−8.60283524794079)
Gene_6_score=−0.100361459561592*(Expression_of_probeset_8125463−5.76219176807997)
Gene_7_score=−0.0731786032726885*(Expression_of_probeset_7912638−5.80836005908298)
Gene_8_score=−0.0588577574308188*(Expression_of_probeset_7978123−7.81869896068138)
Gene_9_score=−0.0291537526685959*(Expression_of_probeset_7937217−7.99754044283416)
Gene_10_score=−0.059579001469581*(Expression_of_probeset_8002133−6.76231617487145)
Gene_11_score=−0.0539204890593068*(Expression_of_probeset_8084895−9.25452952745888)
Gene_12_score=−0.0435216311590311*(Expression_of_probeset_8180166−9.66750825451152)
Gene_13_score=−0.102616463622019*(Expression_of_probeset_8179331−5.87582547195644)
Gene_14_score=−0.256702735040285*(Expression_of_probeset_8146092−6.84033653454892)
Gene_15_score=−0.0471515312903042*(Expression_of_probeset_7898115−6.1806473478809)
Gene_16_score=−0.0978767707892084*(Expression_of_probeset_8117476−6.42634821287224)
Gene_17_score=−0.112823826752702*(Expression_of_probeset_8180078−7.19373066084955)
Gene_48_score=−0.0489348626366957*(Expression_of_probeset_8092978−10.4325518383754)
Gene_19_score=−0.042561683753686*(Expression_of_probeset_7925876−7.26663202627375)
Gene_20_score=−0.040517314218441*(Expression_of_probeset_7940160−8.41904220936401)
Gene_21_score=−0.0255314067182751*(Expression_of_probeset_8076998−9.90620981343659)
Gene_22_score=−0.0298478887838912*(Expression_of_probeset_8179041−11.3092804247355)
Gene_23_score=−0.152455958242676*(Expression_of_probeset_8145317−4.99539634280867)
Gene_24_score=−0.0733338563077433*(Expression_of_probeset_8180049−6.54533529834041)
Gene_25_score=−0.0563089183829938*(Expression_of_probeset_7993195−6.13360660846907)
Gene_26_score=−0.0595673359556534*(Expression_of_probeset_7929882−5.9425809217138)
Gene_27_score=−0.0292004329271551*(Expression_of_probeset_8179049−10.6201119280024)
Gene_28_score=−0.0421648259067651*(Expression_of_probeset_7947815−7.74324780382519)
Gene_29_score=−0.0815827122613575*(Expression_of_probeset_8096070−7.28569239691227)
Gene_30_score=−0.0326333009894926*(Expression_of_probeset_8063000−10.9610191238719),
where,
Genomic Cancer Classifier Score=Gene₁ _score+Gene₂ _score+Gene₃ _score+Gene₄ _score+Gene₅ _score+Gene₆ _score+Gene₇ _score+Gene₈ _score+Gene₉ _score+Gene₁₀ _score+Gene₁₁ _score+Gene₁₂ _score+Gene₁₃ _score+Gene₁₄ _score+Gene₁₅ _score+Gene₁₆ _score+Gene₁₇ _score+Gene₁₈ _score+Gene₁₉ _score+Gene₂₀ _score+Gene₂₁ _score+Gene₂₂ _score+Gene₂₃ _score+Gene₂₄ _score+Gene₂₅ _score+Gene₂₆ _score+Gene₂₇ _score+Gene₂₈ _score+Gene₂₉ _score+Gene₃₀ _score
and the clinicogenomic biomarker score was derived using the following equation,
x=(−3.56652108)+(−0.01621785*SMK)+(−0.24792934*TSQ1)+(0.52981359*TSQ2)+(0.04180910*AGE)+(1.29057600*BMS1)+(2.70293937 *BMS2)+(0.68513004*Genomic_cancer_classifier_score),
where SMK=1 if former smoker and 0 if current smoker, TSQ1=1 if time since quit smoking is >=15 years, and 0 otherwise, TSQ2=1 if time since quit smoking is unknown, and 0 otherwise, AGE=the patient's numeric age in years, BMS1=1 if patient's mass size is <3 cm, and 0 otherwise BMS2=1 if patient's mass size is >=3 cm, and 0 otherwise; then
$Clinicogenomic Model Biomaker Score = \frac{e^{x}}{1 + e^{x}},$
where a patient is predicted cancer positive if the clinicogenomic model biomarker score is greater than 0.4673243, and cancer negative otherwise.
The performance of the clinical and clinicogenomic models was evaluated using an independent set of nasal samples (n=130) from the AEGIS-2 clinical trial that were not used in the development of the classifier. The clinicogenomic model yielded an AUC of 0.81 (95% CI=0.74 to 0.89) in the validation set, which was statistically significantly higher than the AUC of 0.74 (95% CI=0.66 to 0.83) achieved by the clinical risk-factor model alone (P=0.01) (FIG. 3). Operating points for binary classification were chosen to maximize training set sensitivity with specificity of 50% or greater for both models. The addition of cancer-associated gene expression to the clinical risk factor model increased sensitivity from 0.79 (95% CI=0.67 to 0.88) to 0.91 (95% CI=0.81 to 0.97, P=0.03) and negative predictive value from 0.73 (95% CI=0.58 to 0.84) to 0.85 (95% C1=0.69 to 0.94, P=0.03) (Table 4). The negative likelihood ratio of the clinicogenomic classifier was consistent between training (0.18; 95% CI=0.12 to 0.28) and validation (0.18; 95% CI=0.08 to 0.39) sets. Additionally, in subjects with either lesion size less than 3 cm or peripheral lesions, the clinicogenomic model had a negative predictive value of 0.85 (95% CI-0.65 to 0.96) or 0.93 (95% CI-0.66 to 1.00), respectively (Table 10).

DISCUSSION

In the foregoing studies, the present inventors explored whether the airway field of injury in lung cancer extends to nasal epithelium and determined that there are gene expression alterations in the nasal epithelium of patients with lung cancer compared to those with benign diagnoses. It was observed that the lung cancer-associated gene expression patterns previously identified in the bronchial epithelium are highly concordant with those observed in nasal epithelium. Finally, the present inventors showed that the addition of nasal gene expression to clinical risk factors of disease improves diagnostic sensitivity and negative predictive value of a clinical factor model. These findings strengthen the “field of injury” hypothesis in which lung disease is able to influence the gene expression phenotype of normal-appearing cells throughout the airway; and perhaps more excitingly, suggest the potential for biomarkers based on nasal epithelial gene expression that could be used for lung cancer detection.
While previous studies have validated the existence of bronchial airway gene expression alterations in patients with lung cancer and demonstrated their clinical utility in lung cancer detection (Silvestri, et al. NEJM 2015), little is known about the physiological processes responsible for this “field of injury.” One hypothesis for the presence of lung cancer-associated alterations in nasal and bronchial gene expression is that the subset of smokers who develop lung cancer exhibit a distinct genomic response to tobacco smoke exposure throughout all airway epithelial cells, consistent with the “etiological field effect” described by Lochhead, et al. for colon and other cancer types (Lochhead, et al., Mod Pathol. 2015), This paradigm suggests that the airway gene-expression signature is a risk marker for lung cancer as opposed to a direct consequence of the presence of lung cancer based on local or systemic factors produced by the tumor or its microenvironment (i.e., the “conventional field effect” defined by Lochhead, et al., Mod Pathol. 2015). Consistent with the etiological field effect hypothesis, the present inventors observed a concordant downregulation of genes associated with immune system activation in patients with lung cancer in both bronchial and nasal epithelium, which might suggest that an impaired immune response sets the stage for tumorigenesis in the lung microenvironment. Alternatively, despite the distance to the tumor, these cancer-associated gene expression differences may be a direct result of factors secreted by the tumor or its microenvironment, or some other consequence of the presence of the tumor consistent with the “conventional field effect” described above.
Mechanistically, it is intriguing that a number of genes with important roles in cancer-related processes are among the differentially expressed genes. Of the genes that were downregulated in patients with lung cancer, CASP10 and CD177 were among the most correlated genes between bronchial and nasal epithelium and are associated with the induction of apoptosis and activation of the immune response, respectively. The present inventors also identified a number of genes involved in the p53 pathway that were downregulated in patients with lung cancer, including BAK1, ST14, CD82, and MUC4. BAK1 is associated with the induction of apoptosis (Rosell, et al., The Lancet 2013; Gu, et al., Tumor Biol. 2014) and has been previously shown to be downregulated in the tumors of patients with non-small cell lung cancer (NSCLC) (Singhal, et al., Lung Cancer. 2008; 60(3):313-324.). STI4 has been described as a tumor suppressor in breast cancer and its overexpression associated with the inhibition of tumor cell migration and cell invasion (Wang, et al., J Biol Chem. 2009). The downregulation of CD82, which is a metastasis suppressor in prostate cancer (Dong, et al. Science 1995), has been shown to be correlated with poor survival in patients with lung adenocarcinoma (Adachi, et al., Cancer Res. 1996). MUC4, whose downregulation has been associated with increased tumor stage and poorer overall survival, has also been shown to play an oncogenic role in multiple cancers and is a tumor suppressor in NSCLC, acting as a modifier of p53 expression (Majhi, et al., J Thorac Oncol Off Publ Int Assoc Study Lung Cancer. 2013).
From a clinical perspective, the present inventors found that the addition of lung cancer-associated gene expression to established clinical risk factors improved the sensitivity and negative predictive value for detecting lung cancer; these are the key performance metrics for driving potential clinical utility in this setting (e.g., allowing physicians to avoid unnecessary invasive procedures in those with benign disease). This provides the first proof of concept for the use of nasal gene expression for lung cancer detection. The present inventors elected to establish the presence of a nasal field of lung cancer-associated injury using samples from the AEGIS trial given the unique availability of matched bronchial samples, despite the fact that these patients were undergoing bronchoscopy for suspected lung cancer. The demonstration of the added value of nasal gene expression for lung cancer detection in this setting sets the stage for the development of nasal gene expression biomarkers for lung cancer in other clinical settings where bronchoscopy is not frequently used because of lesion or nodule size or location, risk of complications, or cost. In particular, it will now be of interest to develop nasal biomarkers for patients with small peripheral nodules found incidentally or via screening as our current bronchoscopy-based cohort is enriched for patients with centrally located lesions. In the clinical setting of patients with small peripheral nodules, it is envisioned that a nasal biomarker for lung cancer with a low negative likelihood ratio (on par with the NLR observed by the present inventors for the nasal biomarker in the AEGIS samples) could be used to identify nodule patients who are at low risk of malignancy and can be managed by CT surveillance.
Our demonstration of a nasal field of injury for lung cancer extends our previous work which demonstrated a smoking-induced field of injury that is highly concordant between bronchial and nasal epithelium (Zhang, et al., Phys. Gen. 2011). In this study, the present inventors present multiple lines of evidence that the lung cancer-associated field of injury detectable in bronchial airway epithelium (Whitney, et al., BMC Med Gen 2015) is similarly altered in nasal epithelium. The present inventors also demonstrated both that the genes whose expression is altered in patients with cancer are highly concordant in bronchial and nasal epithelium and that they are involved in similar biological processes including the innate immune response, response to retinoic acid, cell cycle, and xenobiotic detoxification. Furthermore, the present inventors also show that a lung cancer gene expression biomarker developed for use with bronchial gene expression data was able to distinguish patients with and without cancer when used with nasal instead of bronchial data.
Despite the similarity between bronchial and nasal cancer-associated gene expression, there were also differences identified. The present inventors found some lung-cancer associated genes and pathways that are either nasal- or bronchial-specific (e.g. the decreased expression of genes involved in apoptosis in nasal epithelium from patients with lung cancer). The present inventors also found that we were able to achieve better biomarker performance in independent nasal data when we developed and trained the biomarker using nasal data. The presence of some differences between bronchial and nasal epithelial cancer-associated gene expression was consistent with our previous findings with regard to smoking—where most genes are similarly altered in bronchial and nasal epithelium and a minority were airway-location specific (Zhang, et al., Phys. Gen. 2011). Given the concordance of gene expression in the context of both lung cancer and cigarette smoke exposure, one could envision expanding the airway field of injury concept for the monitoring and treatment of other diseases such as chronic obstructive pulmonary disease (COPD).
The importance and potential impact of the foregoing studies derive from several key strengths. First, the patients came from a large number of academic and community hospitals and reflect a variety of practice settings and different geographical locales; thus the diversity of alternative benign diagnoses is represented. Second, the training and validation sets came from two separate clinical trials, which minimized the potential for the model to depend on locally confounding variables. Third, the samples were prospectively collected and cancer status was unknown at the time of collection. Fourth, the present inventors have shown that nasal gene expression identifies a source of lung cancer risk that is independent of major clinical risk factors. Rather than serving as an alternative to bronchoscopy, the present inventors envision that a nasal biomarker for lung cancer could be used more broadly to distinguish the subset of patients who might benefit from bronchoscopy or other invasive procedures from those whose imaging abnormalities can be managed by repeat imaging.
While the sensitivity of our nasal clinicogenomic classifier was high (88%) in patients with nodules less than 3 cm in our validation set, the number of patients in that subgroup was small (n=54) and further studies are needed to both validate this performance as well as determine if similar levels of performance are attained in the broader clinical setting where this test would ultimately be used.
Second, while we found that nasal gene expression is an independent predictor of lung cancer compared to clinical factors alone, the performance of our nasal classifier was not dramatically different from a clinical factor biomarker. The present inventors hypothesized that this finding stems in large part from the cohort characteristics (the high pre-test probability of cancer making clinical factors such as nodule size very predictive of lung cancer) and that in a lower cancer prevalence setting, such as indeterminate pulmonary nodules, the relative contribution of the clinical factors might be substantially less.
The importance and impact of the foregoing studies are further emphasized by a number of key strengths. First, the samples used in the studies came from a variety of academic and community hospitals and reflect a variety of practice settings and different geographical locales. Second, the training and validation sets used came from two separate clinical trials which minimized the potential for spurious trends in the data to influence the model and result in overfitting. Third, since it is unlikely that genomic profiles would be used independently from clinical risk factors in the evaluation of indeterminate pulmonary nodules, we incorporated known clinical risk factors of lung cancer or their genomic correlates directly into our classifier. Fourth, the samples were prospectively collected and cancer status was unknown at the time of collection. Finally, we showed the potential utility of sampling nasal epithelium as a faster, cheaper, and non-invasive alternative to sampling bronchial epithelium which can be easily be obtained to evaluate patients with suspect lung cancer.
Together, the findings demonstrate the existence of a cancer-associated airway field of injury that can be non-invasively sampled using nasal epithelium and that nasal gene expression harbors unique information about the presence of cancer that is independent of standard clinical risk factors. These findings, in particular the high NPV of nasal clinicogenomic biomarker, suggest that nasal epithelial gene expression can potentially be used in lung cancer detection and may be especially useful in the management of indeterminate pulmonary nodules.

Materials and Methods

Study Design & Population

Patients were enrolled at 28 medical centers in the US, Canada and Europe as part of two prospective observational studies within the Airway Epithelium Gene Expression in the Diagnosis of Lung Cancer (AEGIS) clinical trials (registered as NCT01309087 and NCT00746759). Inclusion and exclusion criteria have been previously described (Silvestri, et al. NEJM 2015). All patients were current or former cigarette smokers (defined as having smoked at least 100 cigarettes in their lifetime) undergoing bronchoscopy as part of their diagnostic workup for clinical suspicion of lung cancer and all samples were collected prospectively prior to diagnosis. The diagnosis of cancer/no cancer in this cohort has been previously described (Silvestri, et al. NEJM 2015). From among the 1067 nasal samples collected in AEGIS-1 and AEGIS-2, we selected 554 samples for initial inclusion in this study based on RNA yield and sample quality.

Nasal Epithelial Cell Collection & RNA Processing

Nasal epithelial cells were collected by brushing the lateral aspect of the inferior turbinate with a single sterile cytology brush. Brushings were immediately placed into an RNA preservative (Qiagen RNAProtect, Cat. 76526). Nasal epithelial cells were processed to isolate RNA using Qiagen miRNeasy Mini Kits (Cat. 217004) as per the manufacturer's protocol. RNA concentration and purity were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and RNA integrity (RIN) was assessed using the 2100 Bioanalyzer (Agilent Technologies). All samples were subsequently stored at −80° C. until processing on microarrays.

Microarray Processing

All procedures were performed as described in the GeneChip® Whole Transcript Sense Target Labeling Assay Manual (Affymetrix, Santa Clara, Calif.) and Ambion® WT Expression Kit Protocol (Life Technologies). In vitro transcription and cDNA fragmentation quality controls were carried out by running an mRNA Nano assay in the Agilent 2100 Bioanalyzer. The labeled fragmented DNA was hybridized to Affymetrix Gene 1.0 ST microarrays. The hybridized samples were washed and stained using Affymetrix fluidics. Microarrays were immediately scanned using Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, Calif.). The technical quality of the data from each sample was assessed using multiple quality metrics as described herein. Any sample that failed to achieve minimally acceptable thresholds for >3 quality metrics were excluded from further analysis. CEL files from all patient samples passing quality control were normalized using the Robust Multichip Average (RMA) algorithm (Irizzari, et al., Biostatistics 2003) and the Chip Definition File for the Affymetrix Gene 1.0 ST array provided by Affymetrix. Nasal and bronchial samples were normalized separately. ComBat (Johnson, et al., Biostats 2007) was used within each dataset to correct for microarray-processing batch effects. No covariates were included in the ComBat model.

Characterization of Cancer Associated Genes in Nasal Epithelium

Genes associated with cancer status in nasal epithelium were identified using empirical Bayes linear models (Smyth, SAGMB 2004) that corrected for smoking status, pack years, gender, age, and RIN. The most differentially expressed genes (p<0.001, n=535) were clustered using consensus hierarchical clustering (Monti et al., Machine Learning 2003, Wilkerson, et al. Bioinformatics 2010) with Pearson distance and Ward linkage. The sample dendrogram was cut to yield two groups of samples. The difference in the proportion of cancer samples to benign samples in each group was tested using a Pearson's Chi-squared test for count data. The optimal number of gene clusters was determined using the delta-area under the Cumulative Distribution Function curve as described by Monti et al. The mean of each cluster was computed and its association with cancer status was assessed using a Welch t-test. The functional enrichment of the genes in each cluster was determined using the web-based tool EnrichR (Chen, et al. 2013 BMC Bioinfo). A manual review of the literature was used to summarize the significant enrichments within each cluster into an overall cluster theme.

Pre-Ranked Gene Set Enrichment Analysis & Analysis of Core Enrichment

Gene Set Enrichment Analysis (GSEA) (Subramanian, et al. PNAS 2005) was used to determine if the genes with cancer-associated expression in nasal epithelium were concordantly enriched among the genes with cancer-associated expression in the bronchial epithelium. Briefly, the most differentially expressed genes were segregated into up-regulated and down-regulated gene sets. In bronchial epithelium samples, each gene's association with binary cancer status (I/O) was assessed using a Welch t-test. Moderated (empirical Bayes) t-statistics were computed for each gene and genes were subsequently ranked by t-statistic in the bronchial data in descending order. The pre-ranked function within the GSEA software package was then used to determine the enrichment of the two nasal gene sets among the top and bottom ranked genes in bronchial samples. Normalized enrichment scores, p-values, and FDR values were calculated using the GSEA software tool (Subramanian, et al. PNAS 2005). Genes on the leading edge of each enrichment plot (core enrichment) were identified based on the GSEA enrichment report. These genes were clustered in nasal samples using unsupervised hierarchical clustering with Ward linkage. Similar to the approach delineated above, the sample dendrogram was cut to yield two groups of samples and Pearson's Chi-squared test for count data was used to test the difference in the proportion of cancer samples to benign samples in each group.
Projection of Bronchial Clusters into Nasal Training Set
Eleven gene clusters previously identified as being associated with cancer in the bronchial epithelium (Whitney, et al. BMC Med Gen 2015) were projected into our nasal training set by taking the mean of the cluster genes per sample. The number of genes per cluster ranged from 1 to 47. The correlation of cluster means between matched bronchial and nasal samples was computed using Pearson's method. The association of each cluster mean with the presence or absence of cancer was computed using a Welch t-test.

Evaluation of the Bronchial Genomic Classifier

The bronchial genomic lung cancer classifier was implemented as previously described (Whitney, et al. BMC Med Gen 2015). The present inventors computed the classifier score for each of the bronchial and nasal samples from the AEGIS-1 clinical trial. After applying a mean-shift to the nasal data as previously described (Whitney, et al. BMC Med Gen 2015) and detailed below, the classifier score was computed for each nasal sample in the AEGIS-1 trial (n=375). The correlation of the classifier score between matched bronchial and nasal samples from the AEGIS-1 trial (n=157) was computed using Pearson's product-moment coefficient.

Derivation of Cancer Gene Expression Classifier

The 535 genes whose expression was associated with cancer status made up the initial pool of candidate genes for the lung cancer classifier. Weighted voting was chosen as the classification algorithm because of its proven utility in similar classification problems (Spira, et al. Nat Med 2007). The optimal number of genes for the classifier was determined using 100 random 80/20 splits of the training set. The number of genes that maximized the average AUC across the 100 iterations was used. The genes included in the final model were selected for, and the classifier trained, using the entire training set. Details regarding the cross-validation and gene selection processes are further described below.

Derivation of Genomic Correlates

Gene expression surrogates for smoking status (current/former) and time since quit (<15 y, >15 y) were derived as follows. Specifically, empirical Bayes t-tests were used to identify genes that were significantly associated with each variable. The top 10 most up-regulated and top 10 most down-regulated genes by t-statistic were initially selected, followed by a down-selection of genes using forward selection and the lasso in cross-validation. Methodological details regarding this procedure are outlined herein. The set of genes that maximized the average cross-validation AUC while minimizing the total number of genes in the model were included in the genomic correlate. Finally, a logistic regression model was trained to predict the variable using the selected genes.

Derivation of Clinical Risk Factor and Clinicogenomic Classifier

A clinical risk factor classifier was derived using logistic regression in the training set. This model included the genomic smoking status and time since quit classifier scores as well as age and mass size (<3 cm, >3 cm, infiltrates). A clinicogenomic classifier was derived in the training set using cross-validation. A penalized logistic regression model with cancer status as the dependent variable was derived using the penalized R package. Unpenalized independent variables in the model included the smoking status and time since quit genomic correlate prediction scores, patient age, and mass size. The cancer gene expression classifier prediction score was included as the only penalized independent variable in the model.

Statistical Analysis

Statistical differences in clinical covariates between patients with and without lung cancer were calculated using Fisher's exact test (categorical variables) or Welch t-test (continuous variables). Differential expression analyses were performed using linear modeling (limma R package) or Welch t-tests unless otherwise specified. For the differential expression analysis, a two-sided P value of less than 0.001 was considered evidence of statistically significant differential expression. Correlation coefficients were calculated using Pearson's product-moment coefficient. Accuracy of each model was assessed using standard measures including ROC curve AUC, sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). Differences between receiver operating characteristic (ROC) curve AUC were assessed using DeLong's test (DeLong, et al. Biometrics 1988) for correlated ROC curves. Operating points for binary classification were chosen as the threshold that maximized sensitivity while maintaining 50% specificity in the training set. Differences in sensitivity and specificity between models were assessed using McNemar's chi-squared test for count data (Agresti, Cat. Data Analysis 1990). Statistical differences in NPV between models were assessed using the generalized score statistic (Leisenring W, et al., Biometrics 2000) for paired analyses or a proportions test for unpaired analyses. All confidence intervals (CIs) were reported as two-sided binomial 95% confidence intervals. All statistical tests were two-sided, and a P value of less than 0.05 was considered statistically significant.

Cohort Selection

All samples used in this study were obtained from patients with suspect lung cancer enrolled in the AEGIS-1 and AEGIS-2 clinical trials. By nature of the inclusion criteria, these clinical trials were enriched for patients that were ultimately diagnosed with lung cancer. As a result, the present investigators were limited by the number of samples we could select that had a benign diagnosis at 1 year follow up. The inventors selected all benign samples with sufficient RNA yield after isolation and then selected cancer samples to match the clinical covariates of the benign group. As a result, the cancer and benign classes are very well balanced for the recorded clinical covariates (Table 1).

Microarray Quality Control

All analytical methods were carried out using the R statistical computing environment. The quality of each microarray CEL file was assessed using the ArrayQualityMetrics R package (Kauffmann, et al. Bioinformatics 2009). Nine metrics were computed per CEL file (6 prior to RMA normalization, and 3 post-RMA normalization and batch correction). See Table 11 for a description of each quality metric and associated thresholds used to evaluate CEL files. Samples failing at least three quality metrics were removed from all subsequent analyses.

Low-Level Expression Filter

For differential expression analyses in nasal samples, only probesets that were expressed in at least 5% of samples were included to reduce noise and data dimensionality. Background-level expression was determined by examining the expression level of Y-chromosome genes DDX3Y, KDMSD, RPS4Y1, and USP9Y represented by probesets 8176375, 8176578, 8176624, 8177232 in female samples from the training set. Probesets whose expression level did not exceed 1.5 positive standard deviations of the mean expression of the four Y-linked genes in at least 5% of samples were not considered in the analyses.

Nasal Gene Expression Shift for the Evaluation of the Bronchial Genomic Classifier

To account for the difference in gene expression intensity between bronchial and nasal tissues, the present inventors performed a gene-wise mean-shift which was estimated using nasal samples that had a matched bronchial sample in the training set in which the bronchial classifier was developed (n=157) (Whitney, et al. BMC Med Gen 2015). Specifically, the mean expression of each gene in nasal samples (n=157) was subtracted from its corresponding mean expression in bronchial samples. The difference was then added to that gene's expression level in all nasal samples. The bronchial genomic classifier was then evaluated on the mean-shifted nasal data.

Cross-Validation and Optimization of the Lung Cancer Gene Expression Classifier

The training set was randomly divided with 80% of samples belonging to an internal training set and the remaining 20% of samples belonging to an internal test set. Within each split of the data, the association of each gene's expression with cancer status was assessed using Student's t-test. The genes were ranked by absolute t-statistic and a varying number of the top-ranked genes were selected for inclusion in the weighted voting classifier. Classifiers composed of 5 to 100 genes were considered. The performance of each internally trained classifier was quantified using the AUC in the internal test set. This cross-validation procedure was repeated for 100 iterations. The AUC values across the 100 splits of the data were used to rank the models. The classifier size that maximized average cross-validation AUC while minimizing standard deviation and minimizing the number of genes in the classifier was selected as optimal. The genes included in this model were selected for using the entire training set. The final weighted voting classifier was trained using the entire training set and locked prior to evaluation in the validation set.

Derivation of Smoking and Time Since Quit Classifiers

4779 genes were significantly associated with smoking status (p<0.001). Among the top 20 most differentially expressed, 5 were selected for inclusion in a logistic regression model to optimize prediction of smoking status based on cross-validation (Table 14). Specifically, the present inventors used the lasso as a feature selection algorithm to reduce the number of genes in our final model. Using the nasal training set and top 20 genes as a starting point, we fit logistic regression models with binary smoking status (current/former) as the dependent variable and the 20 genes as independent variables using the lasso. The present inventors varied the values of the shrinkage parameter lambda to calculate the misclassification error rate in 10-fold cross-validation using the cv.glmnet function in the glmnet R package (Friedman, et al. JSS 2008). With increasing values of lambda, more genes are allowed to remain in the model. The present inventors iterated over each value of lambda and recorded which genes were included in the models as lambda increased. Using these sets of genes, we fit ordinary logistic regression models in 10-fold cross-validation and computed the average test set AUC for each subset of genes. The subset that obtained the highest average AUC while minimizing the number of genes in the model was considered optimal and those genes were included in the final logistic regression model which was trained using the entire training set. This model was able to distinguish between current and former smokers with an AUC of 0.89 in the training set (p<2.2e−16, n=375).
An identical process was employed for the derivation of the time-since-quit classifier. Specifically, 235 genes were significantly associated with whether a patient had quit smoking less than or greater than 15 years prior to sample collection (p<0.001) in a subset of the training set with valid time since quit clinical annotation (n=319). Among the top 20 most differentially expressed genes, 2 were chosen for inclusion in the final logistic regression model with time since quit as the dependent variable and the two genes as independent variables. This model was trained to optimize the prediction of time since quit (<15 y, >15 y) based on the cross-validation method described above (Table 15). This model had an AUC of 0.75 in the training set (p=0.0001, n=319).

TABLE 1

Clinical and demographic characteristics of patients who
contributed nasal epithelial samples

	AEGIS-1	AEGIS-2
	training	validation
Characteristic	set (n = 375)	set (n = 130)	P

Cancer Status, No.*			.006
Lung Cancer	243	66
Benign Disease	132	64
Smoking Status, No.*			.75
Current	140	46
Former	235	84
Sex, No.*			.75
Male	237	80
Female	138	50
Cumulative smoke	39.0 (26.9, 371)	34.8 (30.7, 130)	.17
exposure (SD, No.),
pack-y†
Time since quit (SD, No.),	7.6 (12.9, 309)	9.4 (13.4, 120)	.21
y†
Age (SD), y†	59.5 (10.4)	61.7 (11.5)	.06
Lesion size, No.*,‡			.89
>3 cm	171	59
≤3 cm	142	54
Infiltrate	44	17
Unknown	18	0
Lesion location, No.*,§			.16
Central	134	55
Peripheral	114	31
Central and peripheral	100	44
Unknown	27	0
Lung cancer histological			.45
type, No.*,\|\|
Small cell	40	8
Non-small cell	200	58
Adenocarcinoma	90	29
Squamous	72	17
Large cell	9	4
Not specified	29	8
Unknown	3	0
Diagnosis of benign	105	34	.13
condition, No.*
Infection	36	7
Sarcoidosis	21	12
Other	48	15

*P value calculated using two-sided Fisher's Exact test.
†P value calculated using two-sided Student's t test.
‡P value calculated comparing >3 cm vs ≤3 cm vs infiltrates.
§P value calculated comparing central vs peripheral vs central and peripheral.
\|\|P value calculated comparing non-small cell vs small cell.

TABLE 2

Functional characterization of genes with cancer-associated
expression in nasal epithelium

		False discovery
	Genes	rate

	Downregulated genes (n = 492)
	DNA damage
	Signal transduction involved in mitotic	<0.001
	DNA integrity checkpoint (GO: 1902400)
	Ubiquitin-dependent degradation of Cyclin D1	<0.001
	(reactome)
	Regulation of apoptosis (reactome)	<0.001
	G1/S DNA damage checkpoints (reactome)	<0.001
	Immune system activation
	Antigen presentation and processing of	<0.001
	exogenous antigen (GO: 0019884)
	Interferon-gamma signaling (reactome)	<0.001
	Upregulated genes (n = 43)
	Ion transport
	Response to magnesium ion (GO: 0032026)	0.01
	Regulation of endocytosis (GO: 0030100)	0.01
	Positive regulation of release of calcium ion	0.02
	into cytosol (GO: 0010524)

TABLE 3

Projection of previously reported bronchial cancer gene clusters
from Whitney, et al., into nasal training set

		No. of	Direction in
Cluster	Function	probesets	cancer	P*

1†	Innate immune	25	Down	<.001
2†	Mitotic cell cycle	47	Down	.05
3	Inflamation	45	Down	.83
4†	Resp. retinoic acid/	34	Up	.004
	cell cycle
5	NA	10	Up	.36
6	NA	21	Down	.02
7†	Submucosal gland markers	20	Up	.01
8	n/a	15	Up	.003
9†	Xenobiotic detoxification	7	Down	.15
10†	Cartilaginous markers	4	Down	.05
11	NA	1	Down	.03

*P value of two-sided t test measuring the difference in mean average expression of all genes in a cluster between cancer and benign nasal sample in the AEGIS-1 cohort,
†In bronchial genomic classifier described by Whitney et al. 2015

TABLE 4

Classifier performance in the validation set (n = 130).

Biomarker
performance metric	Clinical model	Clinicogenomic model	P

Area under the curve	0.74 (0.66 to 0.83)	0.81 (0.74 to 0.89)	.01
(95% CI)*
Sensitivity (95% CI)†	0.79 (0.67 to 0.88)	0.91 (0.81 to 0.97)	.03
Specificity (95% CI)†	0.58 (0.45 to 0.70)	0.52 (0.39 to 0.64)	.42
Negative predictive	0.73 (0.58 to 0.84)	0.85 (0.69 to 0.94)	.03
value (95% CI)‡
Positive predictive	0.66 (0.54 to 0.76)	0.66 (0.55 to 0.76)	.97
value (95% CI)‡
Accuracy (95% CI)§	0.68 (0.60 to 0.76)	0.72 (0.63 to 0.79)	.68

*P value comparing models calculated using Delong's two-sided test. CI = confidence interval.
†P value comparing models calculated using McNemar's two-sided chi-square test.
‡P value comparing models calculated using two-sided generalized score statistic.
§P value comparing models calculated using two-sided Fisher Exact test.

TABLE 5

Top smoking-associated genes in nasal epithelium

Probeset	Gene Name	P-Value	T-statistic

8051583	CYP1B1	9.44E−33	13.14626076
7990391	CYP1A1	1.01E−31	12.88384277
7942693	B3GNT6	3.24E−31	12.75410581
8041540	NA	6.32E−24	10.81589499
8092765	MB21D2	2.13E−23	10.66933402
8102800	SLC7A11	9.53E−22	10.20398693
8080578	CACNA2D3	6.89E−20	9.66548377
8041536	CYP1B1-AS1	7.01E−20	9.66326743
8049349	NA	2.68E−19	9.491193205
8046283	NA	5.19E−19	9.405496334
7913385	RAP1GAP	3.38E−18	9.160235486
8088106	TKT	7.69E−18	9.051261889
8070579	TFF1	8.34E−18	9.040457566
8134452	BHLHA15	1.32E−17	8.978834747
8149811	NKX3-1	1.74E−17	8.942194811
7958019	DRAM1	2.77E−17	−8.882399906
8011009	NA	4.40E−17	8.817561654
8103244	RNF175	4.52E−17	8.813842102
8053200	DQX1	4.85E−17	8.804194587
7969640	CLDN10	8.05E−17	8.735574427
8033257	C3	1.00E−16	−8.706025605
8072626	TIMP3	1.31E−16	8.669404178
8165406	NPDC1	1.77E−16	8.628299892
7984862	CYP1A2	2.47E−16	8.582059649
7992463	SLC9A3R2	2.67E−16	8.571682017
7991516	ADAMTS17	3.49E−16	8.534733027
8078405	CMTM7	3.62E−16	−8.529849006
8084630	LOC344887	4.71E−16	8.493365253
7901272	CYP4X1	5.53E−16	−8.47115011
8013384	NA	6.23E−16	8.454612492
7909946	FAM177B	8.14E−16	8.417323126
8143749	ZNF467	9.48E−16	8.396125375
7937465	TALDO1	1.35E−15	8.346624568
8151074	PDE7A	2.16E−15	−8.280542988
8103254	SFRP2	2.84E−15	8.24214866
8027381	NA	3.06E−15	8.231364256
8154295	IL33	3.33E−15	−8.219438241
7967544	SCARB1	4.86E−15	8.165894689
8066117	SAMHD1	6.77E−15	−8.1188439
7986838	OCA2	7.98E−15	8.095297454
7990138	GRAMD2	9.29E−15	−8.073612212
7970194	MCF2L	1.49E−14	8.005925946
8115261	CCDC69	1.56E−14	−7.999239917
7979658	GPX2	2.30E−14	7.943143325
8069764	NA	2.52E−14	7.929904611
8171435	PIR	3.15E−14	7.897651983
7937749	TNNT3	3.20E−14	7.89526945
8008289	TMEM92	3.57E−14	7.879450691
8075635	TIMP3	4.36E−14	7.850198962
7945169	TMEM45B	6.06E−14	7.802052905
7920025	NA	6.17E−14	7.799799308
7931417	JAKMIP3	6.58E−14	7.789872847
8135378	PRKAR2B	9.57E−14	−7.734766749
8067185	BMP7	1.10E−13	7.714188929
7997738	NA	2.15E−13	7.614758084
8059832	NA	2.66E−13	7.58290221
8028973	CYP2A13	3.45E−13	−7.544020294
8075375	SEC14L3	3.83E−13	−7.528616582
7920178	CRNN	5.55E−13	7.47248828
8068413	CBR3	6.16E−13	7.456682671
8061780	BPIFB2	6.35E−13	7.452269947
7940654	SCGB1A1	8.78E−13	−7.403044462
8011354	TRPV3	9.11E−13	7.397434445
8165538	ENTPD8	1.10E−12	7.368905918
8066493	SLPI	1.17E−12	−7.358999404
8152041	RNF19A	1.28E−12	−7.345510956
7996423	FBXL8	1.42E−12	7.329308846
8001082	NA	1.47E−12	7.324396485
8070567	TFF3	1.53E−12	7.318138631
7963313	GALNT6	1.90E−12	7.285014072
8048595	GMPPA	1.95E−12	7.281023948
7941214	POLA2	2.21E−12	−7.26184698
8123951	ADTRP	4.60E−12	−7.148014135
8061847	BPIFA2	4.94E−12	7.136919575
8125843	SPDEF	4.99E−12	7.13526023
8020653	CABYR	5.93E−12	7.108207007
8173106	ITIH6	6.40E−12	7.096462699
7920146	RPTN	6.41E−12	7.096242593
8022434	NA	7.45E−12	7.072503652
8143441	KIAA1147	8.53E−12	7.051156474
7986446	ALDH1A3	8.75E−12	7.047302473
7922200	SELP	1.06E−11	−7.017305467
7920185	LCE3D	1.08E−11	7.014432665
8095626	NA	1.27E−11	6.988403197
7990379	EDC3	1.53E−11	6.958349485
8069553	NRIP1	1.72E−11	−6.940070269
7934896	NA	1.73E−11	6.939591135
8070584	NA	1.91E−11	−6.923569828
7942007	LRP5	2.05E−11	6.911816552
8128818	WASF1	2.05E−11	6.911761175
8064375	SRXN1	2.21E−11	6.900342564
8096617	BANK1	2.39E−11	−6.887874563
8019316	PYCR1	2.43E−11	6.885218758
7944667	SORL1	2.71E−11	6.867419863
8098204	CPE	3.11E−11	6.845187685
7937696	KRTAP5-AS1	3.14E−11	6.843763116
8006836	LRRC37A11P	3.23E−11	−6.839185743
8037071	RABAC1	3.32E−11	6.834937234
8049799	ANO7	3.45E−11	6.828397833
8040103	ID2	3.46E−11	−6.828251035
8029754	FOXA3	3.55E−11	6.824011046
7996448	NOL3	3.80E−11	6.812881537
8078330	RBMS3	3.99E−11	−6.804918446
8018264	HID1	4.58E−11	6.782713122
7933423	NA	4.72E−11	6.778039944
8038747	KLK12	4.84E−11	6.773913581
7945680	NA	5.41E−11	6.755605977
7912552	NA	5.97E−11	6.739644374
7923792	SLC45A3	6.20E−11	6.733665835
8109049	SPINK7	6.26E−11	6.731918916
8018982	CANT1	6.28E−11	6.731555767
8023855	CYB5A	6.29E−11	−6.731178772
8161044	TPM2	6.86E−11	−6.71710533
8173933	PCDH19	7.02E−11	−6.713205189
7996430	HSF4	7.18E−11	6.709527598
8139909	NA	7.28E−11	6.707286953
7991283	RHCG	7.63E−11	6.699651343
7965873	IGF1	7.65E−11	6.699237529
7926900	MAP3K8	7.80E−11	6.696117595
8003889	NA	7.85E−11	6.695014937
7960771	CD163L1	9.40E−11	6.665441225
8004271	ACADVL	1.01E−10	6.65320783
7962895	FKBP11	1.08E−10	6.642382655
8132539	DBNL	1.11E−10	6.638028488
8115584	CCNJL	1.21E−10	6.623778994
8090420	TPRA1	1.22E−10	6.622795154
8124305	NA	1.23E−10	6.621294194
8163063	CTNNAL1	1.29E−10	−6.613782611
8007112	KRTAP4-7	1.41E−10	6.598801375
7952426	VSIG2	1.43E−10	6.596120908
7957167	TMEM19	1.49E−10	−6.58984449
8046790	NA	1.49E−10	6.589278946
7947110	E2F8	1.57E−10	−6.580665481
8143247	KIAA1549	1.60E−10	6.577764751
7943715	ZC3H12C	1.60E−10	−6.577429765
8116653	NA	1.69E−10	6.568249673
8180303	SAA2	1.90E−10	−6.549464434
8105523	KIF2A	2.08E−10	−6.533921134
8033043	FUT6	2.10E−10	6.532571092
8140668	SEMA3A	2.20E−10	−6.524747412
7983290	NA	2.22E−10	6.523319874
8097030	NA	2.30E−10	6.517656031
7920155	NA	2.64E−10	6.494186742
8165183	SEC16A	2.66E−10	6.492943101
8170662	NA	2.82E−10	6.483430835
7961514	MGP	2.91E−10	−6.478221997
7949383	SYVN1	2.96E−10	6.474998698
7991453	FAM174B	2.98E−10	6.474227362
8059244	CHPF	3.00E−10	6.472743896
8152148	UBR5	3.02E−10	−6.47174233
8009334	CACNG4	3.03E−10	6.471254201
8014768	NA	3.38E−10	−6.452806445
8168081	NA	3.43E−10	−6.450273709
8104180	AHRR	3.57E−10	6.443432623
8038735	KLK11	3.73E−10	6.436186186
8083569	TIPARP	3.81E−10	6.432662897
7995976	CPNE2	3.98E−10	−6.425069554
8099476	PROM1	4.26E−10	−6.413943176
7955119	C12orf54	4.31E−10	6.411926841
8129937	CITED2	4.34E−10	6.4107318
8160521	MOB3B	4.39E−10	−6.408533966
8052269	CCDC88A	4.44E−10	−6.406838489
8068401	CBR1	4.53E−10	6.403458275
8088560	ADAMTS9	4.69E−10	6.39751647
8173941	TSPAN6	4.77E−10	−6.394768754
8116651	NA	5.06E−10	6.384771502
8100701	TMPRSS11B	5.27E−10	6.37768863
7905486	CRCT1	5.29E−10	6.377217041
7913667	GALE	5.44E−10	6.372346454
7895178	NA	5.48E−10	6.37122193
7935180	PDLIM1	6.10E−10	−6.352793486
8079060	VIPR1	7.88E−10	6.309154241
8135933	FAM71F2	8.52E−10	6.295722961
7961891	BHLHE41	8.58E−10	−6.294551046
8096744	CYP2U1	8.78E−10	−6.290661637
8071107	SLC25A18	9.07E−10	6.285035409
8155824	TMC1	9.47E−10	6.27767347
8135601	MET	9.66E−10	−6.274282012
7991186	NTRK3	1.02E−09	6.264066436
8117207	ALDH5A1	1.12E−09	−6.248138359
8138381	AGR2	1.16E−09	6.24229081
7933933	NA	1.17E−09	6.240903303
8073992	PANX2	1.17E−09	6.240805704
8044548	IL36A	1.24E−09	6.230878097
7894476	NA	1.24E−09	6.230710063
8101637	HSD17B13	1.34E−09	−6.21801835
8059111	NA	1.34E−09	6.217924345
8034521	HOOK2	1.36E−09	6.215767634
8084891	FAM43A	1.37E−09	6.213873367
8126905	CRISP3	1.39E−09	6.211941375
8081880	ADPRH	1.40E−09	−6.210420773
8153002	NDRG1	1.51E−09	6.197083836
8002303	NQO1	1.51E−09	6.196974743
7932765	MPP7	1.53E−09	−6.194925885
7992071	MSLN	1.56E−09	6.191635457
7958200	EID3	1.61E−09	6.186294165
8102468	PRSS12	1.82E−09	−6.164183374
8044700	DPP10	1.87E−09	6.159971437
7901287	CYP4Z1	1.93E−09	−6.154421494
8039090	NA	1.96E−09	6.151730836

TABLE 6

Top 200 time since quit-associated genes in nasal epithelium

Probeset ID	Gene Name	P-Value	T-statistic

8051583	CYP1B1	1.15E−08	−5.864698521
7990138	GRAMD2	2.32E−07	5.28908934
7942693	B3GNT6	3.53E−07	−5.205136289
8070579	TFF1	4.34E−07	−5.163177999
7986838	OCA2	6.23E−07	−5.089401186
8027381	NA	7.25E−07	−5.058295771
8041540	NA	1.44E−06	−4.915174976
8001082	NA	1.44E−06	−4.914726373
8078405	CMTM7	1.50E−06	4.906436093
7990391	CYP1A1	1.57E−06	−4.89642192
8101828	TSPAN5	2.48E−06	−4.799287296
7901272	CYP4X1	2.61E−06	4.787944198
8041536	CYP1B1-AS1	2.75E−06	−4.776872722
8153002	NDRG1	4.35E−06	−4.676755458
8014768	NA	6.35E−06	4.593299291
8023855	CYB5A	7.21E−06	4.564723732
7940654	SCGB1A1	8.74E−06	4.521528076
7909946	FAM177B	1.03E−05	−4.484092157
8092765	MB21D2	1.15E−05	−4.45873449
8000117	CRYM	1.32E−05	4.428289791
8103244	RNF175	1.36E−05	−4.420878287
8007112	KRTAP4-7	1.41E−05	−4.41285066
7941214	POLA2	1.57E−05	4.38716782
7920025	NA	1.65E−05	−4.376220861
8009334	CACNG4	1.97E−05	−4.335205613
7961514	MGP	2.02E−05	4.329246764
8011009	NA	2.20E−05	−4.309581331
7958019	DRAM1	2.36E−05	4.292941953
8033257	C3	2.46E−05	4.283082198
7918517	WDR77	2.48E−05	4.281132252
8046283	NA	2.65E−05	−4.265297993
8053200	DQX1	2.74E−05	−4.257941513
8095626	NA	2.83E−05	−4.250377327
8180303	SAA2	2.92E−05	4.242811469
8028973	CYP2A13	2.99E−05	4.236737342
8143749	ZNF467	3.09E−05	−4.228935172
8020878	NA	3.34E−05	−4.210647807
7997500	NA	3.40E−05	4.206034939
8128818	WASF1	3.43E−05	−4.204333192
8115261	CCDC69	3.45E−05	4.202475526
8081214	GPR15	3.51E−05	−4.19870281
8078136	NA	4.35E−05	−4.14711833
8049349	NA	4.41E−05	−4.143953075
8071541	NA	4.49E−05	−4.139669844
8037467	ZNF229	5.00E−05	−4.113296508
8070584	NA	5.43E−05	4.092987565
8162059	SLC28A3	5.49E−05	4.090494754
8011354	TRPV3	5.71E−05	−4.080883688
8121768	PKIB	5.78E−05	4.077769044
7896169	NA	5.80E−05	4.077200723
7946983	SAA2	6.25E−05	4.058633159
7984862	CYP1A2	6.29E−05	−4.057111887
8119599	PTCRA	6.29E−05	−4.057001867
8097126	PP12613	6.35E−05	−4.05483528
8104930	SLC1A3	6.48E−05	4.049947014
8069764	NA	6.61E−05	−4.045055536
8102800	SLC7A11	6.68E−05	−4.042121587
8072626	TIMP3	6.73E−05	−4.040625022
7997738	NA	6.76E−05	−4.039368991
8152092	NA	7.09E−05	−4.02747718
8060325	DEFB132	7.35E−05	−4.018764666
8114898	NA	7.36E−05	4.018238782
7907849	LHX4	7.42E−05	−4.016271342
8146921	RDH10	7.46E−05	−4.015106746
7961891	BHLHE41	7.55E−05	4.011832721
7995976	CPNE2	7.62E−05	4.009555365
8037071	RABAC1	7.78E−05	−4.004631261
8081375	NXPE3	7.80E−05	4.004011589
8075635	TIMP3	7.82E−05	−4.003248222
8162216	SHC3	7.88E−05	4.001385938
7909545	TRAF5	7.92E−05	3.999998473
8151074	PDE7A	8.74E−05	3.975623905
7922174	F5	8.81E−05	3.973635577
8088369	NA	9.38E−05	−3.957921618
7928770	CDHR1	9.96E−05	−3.942750268
7929373	LGI1	0.000103668	3.932619259
7952436	ESAM	0.000106499	−3.925816763
8067858	NA	0.000107269	−3.923996515
7897210	DFFB	0.000112878	−3.911094694
8126905	CRISP3	0.000113792	−3.909052026
7904414	NA	0.00012457	−3.886049558
7934896	NA	0.000131895	−3.871471615
7965403	LUM	0.000132326	3.870638334
7940914	FKBP2	0.000132651	−3.870011418
8165932	NA	0.000141002	−3.854379166
8117547	PRSS16	0.00014542	−3.846460061
8067185	BMP7	0.000147035	−3.843622904
7895628	NA	0.000148842	−3.840481877
8046790	NA	0.000149095	−3.840044195
8062444	BPI	0.000156662	3.827293289
7944331	NA	0.000161854	−3.818877135
8066493	SLPI	0.000161907	3.81879309
7946977	NA	0.000163001	3.817051356
8130645	PARK2	0.000166155	3.81209577
7983252	NA	0.00016955	−3.806859517
8070567	TFF3	0.000172564	−3.802293333
7894196	NA	0.000176703	−3.796142803
8075375	SEC14L3	0.000180154	3.791119372
8036969	CYP2A6	0.000195574	3.769726836
7970194	MCF2L	0.000202681	−3.760397969
8174831	CT47B1	0.000211917	−3.748724945
8124437	NA	0.000232177	−3.724718008
8058765	FN1	0.00023511	3.721406766
7946903	USH1C	0.000237444	3.718800401
7920178	CRNN	0.000241145	−3.714715447
8119107	NA	0.000243721	−3.711907406
8036989	NA	0.000249824	3.705365209
8165538	ENTPD8	0.000258238	−3.69658766
8109049	SPINK7	0.000261177	−3.693584448
7960099	P2RX2	0.000261481	−3.69327571
8072328	SEC14L2	0.000264023	3.690706957
8161368	LOC100132167	0.000269317	3.685431484
7985871	MIR9-3	0.000270171	−3.684590383
8004394	SPEM1	0.000278264	−3.676733864
7982070	SNORD115-32	0.000283003	3.672233183
8082574	TRH	0.000286174	−3.669261344
7991186	NTRK3	0.00029042	−3.665329482
7900540	RIMKLA	0.000292574	−3.663355926
8080578	CACNA2D3	0.000294622	−3.661491289
8172573	SYP	0.000299006	−3.657541531
7922200	SELP	0.000301062	3.655708817
8088468	NPCDR1	0.000304757	3.652442615
7988327	PATL2	0.000307476	3.65006304
8075820	CACNG2	0.000323566	−3.636374828
7934215	SPOCK2	0.000324783	3.635365593
8017476	CSH2	0.000326981	−3.633551814
8173414	SLC7A3	0.000330053	−3.631036902
8103736	SCRG1	0.000331212	−3.630093458
8175393	ARHGEF6	0.000331608	3.62977143
7969640	CLDN10	0.000336817	−3.625574076
7892769	NA	0.000337111	3.625339236
7920185	LCE3D	0.000348019	−3.616750963
7931417	JAKMIP3	0.000354695	−3.611618979
8139909	NA	0.000359521	−3.607965147
7894088	NA	0.000364096	−3.604543506
7914921	NA	0.000366732	−3.602590392
7945680	NA	0.000372486	−3.598373012
8078619	ITGA9	0.000372991	3.598005497
8043682	LOC653924	0.000374957	−3.596580619
8117207	ALDH5A1	0.00038227	3.591339234
8096617	BANK1	0.000386498	3.588352716
7937696	KRTAP5-AS1	0.000393861	−3.583223045
8098204	CPE	0.000395509	−3.582086909
8076894	MLC1	0.0003991	−3.579627779
8095870	CCNG2	0.000405683	−3.575172113
8166447	PTCHD1	0.000417361	−3.567433053
8118995	LHFPL5	0.000427736	−3.560725903
8097030	NA	0.000437767	−3.554384169
8072344	NA	0.000441594	3.552000856
8155516	LOC100132167	0.000450392	3.546594339
8123951	ADTRP	0.000451665	3.545820491
8128247	BACH2	0.00046363	−3.538643231
7966878	NA	0.000463765	−3.538562745
7906988	LOC440700	0.000475937	−3.531439634
8170662	NA	0.000479846	−3.529188018
7938758	SAA1	0.000481853	3.528038906
7895743	NA	0.00048205	−3.527926213
7901287	CYP4Z1	0.000488219	3.524422872
8010057	NA	0.000498899	−3.518453969
7927560	FAM21A	0.000499926	3.517886391
8120715	NA	0.000502377	3.516536417
8167601	USP27X	0.000527994	3.502782171
8115327	SPARC	0.000528943	3.502284668
8139828	LOC441239	0.000533099	−3.500116162
7926170	DHTKD1	0.000535909	−3.49865877
8062557	PPP1R16B	0.000541925	3.495562545
8078330	RBMS3	0.000543871	3.494567757
7983447	SLC28A2	0.000543925	3.494540275
7983290	NA	0.000546906	−3.493023391
8116439	SCGB3A1	0.000550383	3.491263407
7966779	NOS1	0.000553633	−3.48962808
8019988	NA	0.000570334	3.481362864
8055314	LYPD1	0.000575959	−3.478630058
8088371	DNASE1L3	0.00057999	−3.476686921
8044700	DPP10	0.000580791	−3.476302542
8075600	BPIFC	0.000581413	−3.476004282
8106556	CMYA5	0.000582713	3.475381458
8019316	PYCR1	0.000589793	−3.472014063
8029754	FOXA3	0.000594623	−3.469738353
8154295	IL33	0.000595872	3.469157282
8171917	FTHL17	0.000596904	−3.468669173
8095422	STATH	0.000598118	−3.468101959
7982052	NA	0.000600475	3.467003893
7979658	GPX2	0.000606126	−3.464387193
8017867	FAM20A	0.000613706	3.460912633
8138381	AGR2	0.000614997	−3.460324637
8081880	ADPRH	0.000622628	3.456873835
8155824	TMC1	0.000630659	−3.453284349
8172658	NA	0.000631342	−3.452981264
8062971	NA	0.000636581	3.45066517
8052269	CCDC88A	0.000642416	3.44810603
7953943	GABARAPL1	0.000652833	−3.443591394
8104180	AHRR	0.000655203	−3.442574075
7934145	LRRC20	0.000659983	−3.440531661
8032249	ADAMTSL5	0.000664642	−3.438554616
7938683	OR7E14P	0.00067819	3.432877519
7981787	NA	0.000687427	−3.429067246
8044813	TMEM37	0.000703886	3.422395032
8022434	NA	0.000732313	−3.411207279
8121009	C6orf163	0.000751363	3.403934422

TABLE 7

Stage data on patients diagnosed with primary lung cancer.

	AEGIS-1	AEGIS-2
	Training Set	Validation Set
Lung Cancer Stage	(n = 243)	(n = 66)

Non-small cell lung cancer*, No.	200	58
1a, 1b	44	6
2a, 2b	13	4
3a, 3b	44	19
4	66	25
Uncertain	33	4
Small cell lung cancer†, No.	40	8
Extensive	18	8
Limited	16	0
Uncertain	6	0
Unknown, No.	3	0

*p =0.04 by two-sided Fisher's Exact test calculated for AEGIS-1 non-small cell lung cancer stage vs AEGIS-2 non-small cell lung cancer stage
†p = 0.02 by two-sided Fisher's Exact test calculated for AEGIS-1 small cell lung cancer stage vs AEGIS-2 small cell lung cancer stage

TABLE 8

Training and validation set demographics distributed based on cancer status.

AEGIS-1 Training Set

AEGIS-2 Validation Set

Characteristic	Cancer	Benign	P*	Cancer	Benign	P*

Total No.	243	132		66	64
Smoking Status, No.*			0.91			1.00
Current	90	50		23	23
Former	153	82		43	41
Gender, No.*			0.57			0.72
Male	151	86		42	38
Female	92	46		24	26
Mass Size, No.*			<0.001			<0.001
≥3 cm	140	31		46	13
<3 cm	80	62		16	38
Infiltrates	12	32		4	13
Unknown	11	7		0	0
Age, y (SD)†	61.1	56.6	<0.001	62.2	61.1	0.58
	(9.5)	(11.4)		(9.6)	(13.3)
Pack-years (SD)†	40.7	35.7	0.11	37.9	31.6	0.24
	(24.6)	(30.5)		(28.1)	(33.1)
Time Since Quit, yr.	6.7	8.9	0.17	6.4	12.2	0.02
(SD)†	(11.5)	(14.8)		(10.6)	(15.3)
RNA Integrity Number	4.4 (1.8)	4.3 (1.9)	0.92	4.7 (1.8)	4.3 (1.9)	0.24
(SD)†

*p-value calculated using a two-sided Fisher's Exact test to compare cancer vs. benign.
†p-value calculated using a two-sided Student t-test to compare cancer vs. benign.

TABLE 9

Clinical and demographic characteristics for patients with matched
nasal and bronchial epithelial samples included in this study.

	Matched
	Nasal	Non-matched Nasal
	Samples	Samples
Characteristic*	(n = 157)	(n = 218)

Cancer Status, No.
Lung Cancer	97	146
Benign Disease	60	72
Smoking Status, No.
Current	53	87
Former	104	131
Gender, No.
Male	104	85
Female	53	133
Cumulative Smoke Exposure,	37.8 (24.7)	39.9 (28.4)
pack-yr (SD)
Time Since Quit, y (SD)	8.1 (12.6)	7.0 (13.2)
Age, y (SD)	59.6 (11.2)	59.5 (9.8)
Lesion Size, No.
>3 cm	66	105
<3 cm	68	74
Infiltrate	17	27
Unknown	6	12
Lesion Location, No.
Central	55	79
Peripheral	51	63
Central and Peripheral	42	58
Unknown	9	18
Lung Cancer Histological Type, No.
Small-cell	14	26
Non-small-cell	83	117
Adenocarcinoma	41	49
Squamous	30	42
Large-cell	3	5
Not specified	9	20
Unknown	0	4
Diagnosis of Benign Condition, No.
Infection	15	21
Sarcoidosis	11	10
Other	18	30

*No statistically significant differences were observed between matched and unmatched nasal samples.

TABLE 10

Comparison of clinical risk-factor model and clinicogenomic classifiers in patient
subgroups stratified by lesion size and location*

			% Negative Predictive Value (95%
	Patients	% Sensitivity (95% CI)	CI)

	All	with	Clinical Risk-	Clinicogenomic	Clinical Risk-	Clinicogenomic
Group	Patients	Cancer	Factor Model	Model	Factor Model	Model

All patients	130	66	78.8 (67.0-87.9)	90.9 (81.3-96.6)	72.5 (58.3-84.1)	84.6 (69.5-94.1)
Lesion Size
<3 cm	54	16	50.0 (24.7-75.3)	75.0 (47.6-92.7)	77.1 (59.9-89.6)	84.6 (65.1-95.6)
≥3 cm	59	46	95.6 (85.2-99.5)	100.0 (88.7-100.0)	0.0 (0.0-90.6)	100.0 (1.3-100.0)
Infiltrates	17	4	0.0 (0.0-71.6)	50 (7-93)	71.4 (41.9-91.6)	83.3 (51.6-97.9)
Lesion
Location
Central	55	28	78.6 (59.0-91.7)	92.9 (76.5-99.1)	76.9 (56.4-91.0)	88.2 (63.6-98.5)
Peripheral	31	6	66.7 (22.2-95.7)	83.3 (35.9-99.6)	86.7 (59.5-98.3)	92.9 (66.1-99.8)
Both	44	32	81.2 (63.6-92.8)	90.6 (75.0-98.0)	40.0 (12.2-73.8)	62.5 (24.5-91.5)

*CI = confidence interval.

TABLE 11

Microarray quality control metrics and thresholds

Metric	Pre/Post RMA	Threshold

L1 Distance Between Arrays	Pre	353
Array Intensity Distribution	Pre	0.175
Relative Log Expression	Pre	0.162
Normalized Unscaled Standard Error	Pre	1.07
MA Plot Hoeffding's Statistic	Pre	0.15
Spatial Distribution of Feature Intensities	Pre	0.108
L1 Distance Between Arrays	Post	243
Array Intensity Distribution	Post	0.0272
MA Plot Hoeffding's Statistic	Post	0.15

TABLE 12

535 cancer-associated differentially expressed
genes in nasal epithelium

Probeset	Gene Symbol	Gene Cluster

7892618	NA	1
7892678	NA	1
7892766	NA	1
7892947	NA	1
7893061	NA	1
7893173	NA	1
7893248	NA	1
7893296	NA	1
7893333	NA	1
7893647	NA	1
7893862	NA	1
7894331	NA	1
7894501	NA	1
7894737	NA	1
7894926	NA	1
7895180	NA	1
7895602	NA	1
7895618	NA	1
7896201	NA	1
7896651	NA	1
7901110	AKR1A1	1
7904830	RNF115	1
7905938	SLC50A1	1
7906079	RAB25	1
7908147	TSEN15	1
7910416	URB2	1
7912412	MTOR	1
7914563	YARS	1
7914834	PSMB2	1
7915504	ELOVL1	1
7915578	TMEM53	1
7917359	ZNHIT6	1
7920971	C1orf85	1
7923483	RABIF	1
7923929	PIGR	1
7928630	EIF5AL1	1
7930031	GBF1	1
7930498	ACSL5	1
7930533	LOC143188	1
7930577	CASP7	1
7931778	PITRM1	1
7933760	CCDC6	1
7934133	PPA1	1
7934653	POLR3A	1
7934753	NA	1
7936284	XPNPEP1	1
7937217	ECHS1	1
7938834	NAV2	1
7940775	RARRES3	1
7944803	VWA5A	1
7950248	FCHSD2	1
7950906	CTSC	1
7951565	ARHGAP20	1
7952557	SRPR	1
7953395	COPS7A	1
7953981	ETV6	1
7958828	TRAFD1	1
7959153	COX6A1	1
7962869	DDX23	1
7963187	LIMA1	1
7967175	KDM2B	1
7969794	LOC100132099	1
7973314	OXA1L	1
7973564	PSME1	1
7979743	RDH11	1
7979757	ZFYVE26	1
7980146	NPC2	1
7981824	NA	1
7985959	GDPGP1	1
7987536	RMDN3	1
7988124	PPIP5K1	1
7988132	STRC	1
7989619	PPIB	1
7991323	PEX11A	1
7993223	CLEC16A	1
7996725	DUS2	1
7996908	SNTB2	1
7999791	NA	1
8002919	KARS	1
8005994	ERAL1	1
8006392	PSMD11	1
8006531	SLFN5	1
8006812	PSMB3	1
8007302	TUBG1	1
8007312	TUBG2	1
8007715	NMT1	1
8008139	UBE2Z	1
8009164	DCAF7	1
8010924	VPS53	1
8011599	ANKFY1	1
8012856	ELAC2	1
8013588	POLDIP2	1
8013641	PIGS	1
8014115	MYOD1	1
8014903	NA	1
8015545	RAB5C	1
8016099	EFTUD2	1
8021727	CNDP2	1
8026106	CALR	1
8027876	TMEM147	1
8028705	TIMM50	1
8028756	PSMC4	1
8031827	ZNFS87	1
8033912	DNMT1	1
8036010	PEPD	1
8042576	NAGK	1
8043100	TMSB10	1
8043197	VAMP8	1
8043937	CNOT11	1
8047403	CASP10	1
8048926	SP140L	1
8058914	AAMP	1
8059350	AP1S3	1
8059361	WDFY1	1
8062349	RPN2	1
8062981	PIGT	1
8063211	NCOA3	1
8063369	RNF114	1
8064522	IDH3B	1
8065832	TRPC4AP	1
8066939	B4GALT5	1
8075585	RTCB	1
8080938	MITF	1
8086028	GLB1	1
8088247	ARHGEF3	1
8088634	NA	1
8089544	CCDC80	1
8089568	CD200R1	1
8091385	CP	1
8091991	NA	1
8092169	TNFSF10	1
8092230	ZMAT3	1
8092541	LIPH	1
8093398	PCGF3	1
8093685	HIT	1
8095139	SRD5A3	1
8098547	NA	1
8102311	CASP6	1
8103911	IRF2	1
8105077	CARD6	1
8108558	SLC35A4	1
8108593	WDR55	1
8114145	VDAC1	1
8116096	DDX41	1
8117243	LRRC16A	1
8117321	TRIM38	1
8122013	L3MBTL3	1
8122803	NA	1
8123062	TMEM181	1
8123800	NA	1
8123951	ADTRP	1
8126588	XPO5	1
8126729	NA	1
8129254	MAN1A1	1
8131631	HDAC9	1
8133690	MDH2	1
8134091	CLDN12	1
8135422	BCAP29	1
8136095	AHCYL2	1
8136580	RAB19	1
8139392	DDX56	1
8147112	NA	1
8148059	DEPTOR	1
8153474	TSTA3	1
8154733	ACO1	1
8156770	GALNT12	1
8159249	MRPS2	1
8160914	VCP	1
8163452	FKBP15	1
8165866	STS	1
8168762	CSTF2	1
8169249	MID2	1
8170882	ATP6AP1	1
8173979	NOX1	1
8173999	XKRK	1
8175844	IDH3G	1
8179298	CSNK2B	1
8180343	RAC1	1
7897728	FBXO6	2
7898799	C1QC	2
7898805	C1QB	2
7906355	CD1E	2
7917561	GBP4	2
7919971	RFX5	2
7931951	SFMBT2	2
7934215	SPOCK2	2
7938035	TRIM22	2
7942569	SLCO2B1	2
7945962	TRIM21	2
7948274	UBE2L6	2
7949340	BATF2	2
7953428	CD4	2
7953993	BCL2L14	2
7960947	A2M	2
7964119	STAT2	2
7978123	PSME2	2
7980958	LGMN	2
7981290	WARS	2
7993195	NA	2
7995926	NLRC5	2
8006214	ADAP2	2
8010426	RNF213	2
8010454	RNF213	2
8026971	IFI30	2
8029536	AP0C1	2
8034304	ACP5	2
8057744	STAT1	2
8066214	TGM2	2
8066905	ZNFX1	2
8072710	APOL6	2
8072735	APOL1	2
8075720	APOL2	2
8082075	DTX3L	2
8086125	TRANK1	2
8090018	PARP9	2
8115147	CD74	2
8117435	BTN3A2	2
8117458	BTN3A1	2
8117476	BTN3A3	2
8117760	HLA-F	2
8117777	NA	2
8118556	NA	2
8118594	HLA-DPB1	2
8125463	NA	2
8125483	TAP2	2
8125993	ETV7	2
8140971	SAMD9L	2
8143327	PARP12	2
8145317	ADAMDEC1	2
8146092	IDO1	2
8161954	FRMD3	2
8177732	HLA-A	2
8178193	HLA-DRA	2
8178205	HLA-DQA2	2
8179019	HLA-F	2
8179041	NA	2
8179049	HLA-J	2
8179481	HLA-DRA	2
8179489	NA	2
8179495	PSMB9	2
8179519	HLA-DPB1	2
8179731	NA	2
8180003	NA	2
8180022	NA	2
8180029	HLA-DQB2	2
8180034	TAP2	2
8180049	PSMB8	2
8180061	TAP1	2
8180078	HLA-DMB	2
8180086	HLA-DMA	2
8180093	HLA-DQA	2
8180100	HLA-DPA1	2
7894264	NA	3
7895149	NA	3
7896038	NA	3
7896908	PUSL1	3
7897263	RNF207	3
7898115	TMEM51	3
7898161	EFHD2	3
7903827	STRIP1	3
7904050	MOV10	3
7905881	ADAM15	3
7908694	NAV1	3
7908793	NA	3
7909127	MFSD4	3
7909188	IKBKE	3
7912239	GPR157	3
7912374	SRM	3
7912496	MTHFR	3
7912537	NA	3
7912638	TMEM51-AS1	3
7913256	DDOST	3
7915184	NA	3
7915543	SLC6A9	3
7915659	HECTD3	3
7918394	EPS8L3	3
7919872	FAM63A	3
7920271	S100A4	3
7920291	NA	3
7920642	MUC1	3
7923662	PIK3C2B	3
7924150	TMEM206	3
7924823	JMJD4	3
7925876	NA	3
7929882	SEMA4G	3
7930537	TCF7L2	3
7931899	NA	3
7934196	PSAP	3
7934477	CAMK2G	3
7935058	MYOF	3
7935188	NA	3
7937518	TSPAN4	3
7937713	SYT8	3
7938519	MICALCL	3
7939546	CD82	3
7939665	MDK	3
7939767	MADD	3
7940160	DTX4	3
7940530	MYRF	3
7940798	MARK2	3
7941621	DPP3	3
7942697	NA	3
7944164	TMPRSS4	3
7945204	ST14	3
7945666	CTSD	3
7946781	PLEKHA7	3
7947815	ACP2	3
7948444	TCN1	3
7948588	SYT7	3
7949765	PPP1CA	3
7951309	MMP13	3
7951896	PCSK7	3
7952132	SLC37A4	3
7952290	TRIM29	3
7953341	TAPBPL	3
7953483	USP5	3
7955613	KRT7	3
7958989	PLBD2	3
7962842	NA	3
7964203	BA22A	3
7969414	KLF5	3
7976000	ADCK1	3
7976567	BDKRB1	3
7977046	TNFAIP2	3
7977249	INF2	3
7977511	TEP1	3
7978260	DHRS1	3
7983405	DUOXA2	3
7963478	C15orf48	3
7983512	SQRDL	3
7984779	PML	3
7985240	TMED3	3
7985620	ALPK3	3
7987230	LPCAT4	3
7988350	DUOX2	3
7990417	SCAMP2	3
7994737	NA	3
7997152	CHST4	3
7997158	NA	3
7997401	BCO1	3
7998222	MRPL28	3
7999909	GPRC5B	3
8000375	ARHGAP1	3
8000543	NA	3
8000811	MAPK3	3
8001030	PYCARD	3
8001552	CIAPIN1	3
8002133	PSMB10	3
8002421	VAC14	3
8005475	TRIM16L	3
8005661	NA	3
8006984	PSMD3	3
8007188	CNP	3
8007505	DHX8	3
8007620	GRN	3
8008664	AKAP1	3
8009666	RAB37	3
8009693	TMEM104	3
8010354	GAA	3
8011293	CLUH	3
8011516	ATP2A3	3
8011671	GGT6	3
8011713	CXCL16	3
8012126	CLDN7	3
8014768	NA	3
8017867	FAM20A	3
8018324	GGA3	3
8019211	NPLOC4	3
8019622	TMEM106A	3
8021301	RAB27B	3
8023043	PSTPIP2	3
8024687	TJP3	3
8028524	ACTN4	3
8029086	CEACAM5	3
8029098	CEACAM6	3
8029560	CLPTM1	3
8032789	STAP2	3
8034420	MAN2B1	3
8034589	FARSA	3
8037205	NA	3
8037222	CEACAM8	3
8037794	PRKD2	3
8038261	GYS1	3
8039389	PTPRH	3
8040365	TRIB2	3
8040698	SLC35F6	3
8040753	TMEM214	3
8043657	CNNM4	3
8045539	NA	3
8047738	NA	3
8048717	SGPP2	3
8050160	MBOAT2	3
8051298	GALNT14	3
8051322	XDH	3
8053214	AUP1	3
8053406	RETSAT	3
8054054	NA	3
8058390	RAPH1	3
8058973	ZNF142	3
8059222	DNPEP	3
8060353	RBCK1	3
8062041	ACSS2	3
8062251	NA	3
8062927	PI3	3
8063000	NA	3
8063078	CTSA	3
8063351	SLC9A8	3
8063893	ADRM1	3
8064613	SLC4A11	3
8065612	NOL4L	3
8065920	NA	3
8065948	FER1L4	3
8066513	SDC4	3
8068254	IL10RB	3
8068810	SLC37A1	3
8069399	NA	3
8070538	C2CD2	3
8072108	ASPHD2	3
8072926	H1F0	3
8073605	BIK	3
8076569	TTLL12	3
8076998	PLXNB2	3
8077082	LMF2	3
8080100	RAD54L2	3
8082797	TF	3
8084717	ST6GAL1	3
8084895	MUC20	3
8084929	SLCS1A	3
8085300	SEC13	3
8087485	NA	3
8088425	FAM3D	3
8090823	SLCO2A1	3
8092978	MUC4	3
8093230	NA	3
8096070	BMP3	3
8103025	ZNF827	3
8104079	FAT1	3
8106170	TMEM171	3
8114050	8-Sep	3
8115623	ATP10B	3
8118833	UHRF1BP1	3
8119926	TMEM63B	3
8122843	ESR1	3
8123606	SERPINB9P1	3
8125766	BAK1	3
8129677	SGK1	3
8133721	HSPB1	3
8136849	GSTK1	3
8136863	TMEM139	3
8137798	PSMG3	3
8139859	GUSB	3
8143575	EPHA1	3
8144880	SH2D4A	3
8145027	FAM160B2	3
8145669	NA	3
8146921	RDH10	3
8148548	PSCA	3
8148572	LY6E	3
8149330	CTSB	3
8150036	KIF13B	3
8150112	GSR	3
8152828	GSDMC	3
8153334	PSC4	3
8153342	LYPD2	3
8155707	NA	3
8156058	NA	3
8157362	ZNF618	3
8157381	ZNF618	3
8158167	LCN2	3
8158242	URM1	3
8158671	NA	3
8158684	NA	3
8158961	GTF3C5	3
8160670	AQP3	3
8161174	GNE	3
8162502	FBP1	3
8162729	TRIM14	3
8162744	CORO2A	3
8163505	HDHD3	3
8164535	CRAT	3
8164580	PTGES	3
8172280	SLC9A7	3
8175924	NAA10	3
8178115	CFB	3
8178561	ABHD16A	3
8179028	LOC554223	3
8179112	ABCF1	3
8179331	C2	3
8179351	CFB	3
8179364	SKIV2L	3
8179638	TRIM26	3
8180166	TAPBP	3
7892796	NA	4
7893130	NA	4
7894970	NA	4
7895574	NA	4
7896160	NA	4
7899502	RNU11	4
7902043	DNAJC6	4
7916506	C1orf168	4
7930612	NA	4
7932498	SKIDA1	4
7944765	NA	4
7953383	SCARNA10	4
7961710	ABCC9	4
7964631	FAM19A2	4
7971165	NA	4
7978407	PRKD1	4
7985317	CEMIP	4
7999291	C16orf89	4
8006504	FNDC8	4
8009380	SNORA38B	4
8013521	NA	4
8013523	NA	4
8043782	CNGA3	4
8045287	NA	4
8049530	LRRFIP1	4
8076223	NA	4
8089145	ABI3BP	4
8098604	ANKRD37	4
8101762	SNCA	4
8104141	PLEKHG4B	4
8107204	NA	4
8108180	NA	4
8127658	NA	4
8132248	NA	4
8147990	NA	4
8156358	NA	4
8165694	NA	4
8165696	NA	4
8165698	NA	4
8165700	NA	4
8165707	NA	4
8168868	ARMCX1	4
8175531	CDR1	4

TABLE 13

Genes and parameters of the genomic lung cancer weighted voting classifier

Probeset	Gene Symbol	Weight	Probeset	Gene Symbol	Weight

8091385	CP	−0.076842875	8117476	BTN3A3	−0.097876771
8115147	CD74	−0.06681241	8180078	HLA-DMB	−0.112823827
8034420	MAN2B1	−0.050873844	7925876	NA	−0.042561684
8075720	APOL2	−0.08530029	8092978	MUC4	−0.048934863
7940775	RARRES3	−0.066344128	7940160	DTX4	−0.040517314
8125463	NA	−0.10036146	8076998	PLXNB2	−0.025531407
7912638	TMEM51-AS1	−0.073178603	8179041	NA	−0.029847889
7978123	PSME2	−0.058857757	8145317	ADAMDEC1	−0.152455958
7937217	ECHS1	−0.029153753	8180049	PSMB8	−0.073333856
8002133	PSMB10	−0.059579001	7993195	NA	−0.056308918
8084895	MUC20	−0.053920489	7929882	SEMA4G	−0.059567336
8180166	TAPBP	−0.043521631	8179049	HLA-J	−0.029200433
8179331	C2	−0.102616464	7947815	ACP2	−0.042164826
8146092	IDO1	−0.256702735	8096070	BMP3	−0.081582712
7898115	TMEM51	−0.047151531	8063000	NA	−0.032633301

TABLE 14

Genes and parameters of the genomic smoking status
logistic regression model

	Probeset	Gene Symbol	Coefficient

Intercept	NA	−24.1410
8051583	CYP1B1	0.2521
7990391	CYP1A1	0.0544
7942693	B3GNT6	2.5181
8080578	CACNA2D3	1.7191
8033257	C3	−0.4727

TABLE 15

Genes and parameters of the genomic time since
quit logistic regression model

	Probeset	Gene Symbol	Coefficient

Intercept	NA	−1.8161
7990391	GRAMD2	0.5726
8051583	CYP1B1	−0.4519

TABLE 16

Genes and parameters of the clinical risk-factor lung cancer classifier

	Variable	Coefficient

	Intercept	−5.14689
	Genomic Smoking Classifier Score	1.82244
	Genomic Time Since Quit Classifier Score	2.31235
	Age	0.04947
	Mass Size (Infiltrate vs <3 cm)	1.27246
	Mass Size (Infiltrate vs >3 cm)	2.59898

TABLE 17

Genes and parameters of the clinicogenomic lung cancer classifier

	Variable	Coefficient

	Intercept	−4.1504024
	Genomic Smoking Classifier Score	0.7534516
	Genomic Time Since Quit Classifier Score	0.3276714
	Genomic Cancer Classifier Score	0.6629011
	Age	0.0452670
	Mass Size (Infiltrate vs <3 cm)	1.3423457
	Mass Size (Infiltrate vs >3 cm)	2.6932782

TABLE 18

Performance metrics of clinical risk-factor and clinicogenomic
classifiers in the independent AEGIS-2 validation set

Clinical Risk-
Factor	Clinico-
Model*	genomic	p-value

AUC	0.76	0.80	0.0495
Sens	0.85	0.94	0.0412
Spec	0.42	0.44	1.0000
NPV	0.73	0.87	—
PPV	0.60	0.63	—
ACC	0.64	0.69	—

TABLE 19

Comparison of clinical risk-factor model and clinicogenomic classifiers in lesion
size and location patient subgroups

Sensitivity (%)

			Clinical
		Patients with	Risk-Factor	Clinicogenomic
Group	All Patients	Cancer	Model	Model	p-value

All patients	130	66	85	94	0.0412
Lesion Size
<3 cm	54	16	63	88	0.1336
>=3 cm	59	46	100	100	1.0000
Infiltrates	17	4	0	50	n/a
Lesion Location
Central	55	28	89	96	0.4795
Peripheral	31	6	67	83	1.0000
Both	44	32	84	94	0.2482

TABLE 20

Comparison of clinical risk-factor and clinicogenomic classifiers
in disease stage and disease cell type subgroups

Sensitivity (%)

		Clinical
	Patients with	Risk-Factor	Clinicogenomic
Group	Cancer	Model	Model	p-value

Stage
1a, 1b	6	50	67	1.0000
2a, 2b	4	100	100	1.0000
3a, 3b	17	94	94	1.0000
4	22	86	95	0.4795
Extensive	7	71	100	n/a
Other	10	90	100	n/a
Cell Type
Adenocarinoma	26	85	92	0.4795
Squamous	17	82	94	0.4795
Small Cell	7	71	100	n/a
Unknown or NA	16	94	94	1.0000

TABLE 21

Probeset	Gene Symbol	Direction in Cancer

7892618	NA	down
7892678	NA	down
7892766	NA	down
7892947	NA	down
7893061*	NA	down
7893173	NA	down
7893248	NA	down
7893296*	NA	down
7893333	NA	down
7893647	NA	down
7893862	NA	down
7894331	NA	down
7894501	NA	down
7894737	NA	down
7894926	NA	down
7895180	NA	down
7895602*	NA	down
7895618	NA	down
7896201	NA	down
7896651	NA	down
7901110*	AKR1A1	down
7904830	RNF115	down
7905938	SLC50A1	down
7906079*	RAB25	down
7908147	TSEN15	down
7910416	URB2	down
7912412	MTOR	down
7914563	YARS	down
7914834*	PSMB2	down
7915504	ELOVL1	down
7915578*	TMEM53	down
7917359	ZNHIT6	down
7920971*	C1orf85	down
7923483*	RABIF	down
7923929*	PIGR	down
7928630	EIF5AL1	down
7930031	GBF1	down
7930498*	ACSL5	down
7930533	LOC143188	Down
7930577	CASP7	down
7931778	PITRM1	down
7933760*	CCDC6	down
7934133	PPA1	down
7934653	POLR3A	down
7934753	NA	down
7936284*	XPNPEP1	down
7937217*	ECHS1	down
7938834	NAV2	down
7940775	RARRES3	down
7944803*	VWA5A	down
7950248*	FCHSD2	down
7950906*	CTSC	down
7951565*	ARHGAP20	down
7952557	SRPR	down
7953395	COPS7A	down
7953981*	ETV6	down
7958828	TRAFD1	down
7959153	COX6A1	down
7962869	DDX23	down
7963187	LIMA1	down
7967175	KDM2B	down
7969794	LOC100132099	down
7973314*	OXA1L	down
7973564*	PSME1	down
7979743	RDH11	down
7979757*	ZFYVE26	down
7980146*	NPC2	down
7981824	NA	down
7985959*	GDPGP1	down
7987536*	RMDN3	down
7988124	PPIP5K1	down
7988132*	STRC	down
7989619	PPIB	down
7991323*	PEX11A	down
7993223*	CLEC16A	down
7996725	DUS2	down
7996908	SNTB2	down
7999791	NA	down
8002919	KARS	Down
8005994	ERAL1	down
8006392	PSMD11	down
8006531	SLFN5	down
8006812*	PSMB3	down
8007302*	TUBG1	down
8007312*	TUBG2	down
8007715	NMT1	down
8008139*	UBE2Z	down
8009164*	DCAF7	down
8010924	VPS53	down
8011599	ANKFY1	down
8012856*	ELAC2	down
8013588	POLDIP2	down
8013641*	PIGS	down
8014115	MYOID	down
8014903*	NA	down
8015545	RAB5C	down
8016099*	EFTUD2	down
8021727	CNDP2	down
8026106	CALR	down
8027876	TMEM147	down
8028705	TIMM50	down
8028756	PSMC4	down
8031827	ZNF587	down
8033912	DNMT1	down
8036010*	PEPD	down
8042576*	NAGK	down
8043100	TMSB10	down
8043197	VAMP8	down
8043937*	CNOT11	down
8047403*	CASP10	down
8048926	SP140L	down
8058914	AAMP	down
8059350	AP1S3	down
8059361*	WDFY1	down
8062349	RPN2	down
8062981	PIGT	down
8063211	NCOA3	down
8063369*	RNF114	down
8064522*	IDH3B	Down
8065832*	TRPC4AP	down
8066939*	B4GALT5	down
8075585	RTCB	down
8080938	MITF	down
8086028	GLB1	down
8088247	ARHGEF3	down
8088634	NA	down
8089544	CCDC80	down
8089568	CD200R1	down
8091385	CP	down
8091991	NA	down
8092169*	TNFSF10	down
8092230	ZMAT3	down
8092541	LIPH	down
8093398*	PCGF3	down
8093685	HTT	down
8095139*	SRD5A3	down
8098547	NA	down
8102311*	CASP6	down
8103911	IRF2	down
8105077	CARD6	down
8108558*	SLC35A4	down
8108593*	WDR55	down
8114145	VDAC1	down
8116096	DDX41	down
8117243*	LRRC16A	down
8117321*	TRIM38	down
8122013	L3MBTL3	down
8122803*	NA	down
8123062	TMEM181	down
8123800*	NA	down
8123951*	ADTRP	down
8126588*	XPO5	down
8126729	NA	down
8129254*	MAN1A1	down
8131631*	HDAC9	down
8133690*	MDH2	down
8134091*	CLDN12	down
8135422*	BCAP29	down
8136095	AHCYL2	Down
8136580	RAB19	down
8139392	DDX56	down
8147112*	NA	down
8148059	DEPTOR	down
8153474	TSTA3	down
8154733*	ACO1	down
8156770	GALNT12	down
8159249	MRPS2	down
8160914	VCP	down
8163452	FKBP15	down
8165866	STS	down
8168762	CSTF2	down
8169249	MID2	down
8170882	ATP6AP1	down
8173979*	NOX1	down
8173999*	XKRX	down
8175844	IDH3G	down
8179298*	CSNK2B	down
8180343	RAC1	down
7897728	FBXO6	down
7898799*	C1QC	down
7898805*	C1QB	down
7906355*	CD1E	down
7917561	GBP4	down
7919971*	RFX5	down
7931951	SFMBT2	down
7934215*	SPOCK2	down
7938035*	TRIM22	down
7942569*	SLCO2B1	down
7945962*	TRIM21	down
7948274*	UBE2L6	down
7949340	BATF2	down
7953428	CD4	down
7953993	BCL2L14	down
7960947	A2M	down
7964119	STAT2	down
7978123*	PSME2	down
7980958*	LGMN	down
7981290	WARS	down
7993195*	NA	Down
7995926*	NLRC5	down
8006214*	ADAP2	down
8010426	RNF213	down
8010454	RNF213	down
8026971*	IFI30	down
8029536*	APOC1	down
8034304*	ACP5	down
8057744	STAT1	down
8066214	TGM2	down
8066905	ZNFX1	down
8072710	APOL6	down
8072735	APOL1	down
8075720	APOL2	down
8082075	DTX3L	down
8086125*	TRANK1	down
8090018	PARP9	down
8115147*	CD74	down
8117435*	BTN3A2	down
8117458	BTN3A1	down
8117476*	BTN3A3	down
8117760*	HLA-F	down
8117777*	NA	down
8118556*	NA	down
8118594*	HLA-DPB1	down
8125463*	NA	down
8125483	TAP2	down
8125993	ETV7	down
8140971	SAMD9L	down
8143327	PARP12	down
8145317*	ADAMDEC1	down
8146092*	IDO1	down
8161964	FRMD3	down
8177732*	HLA-A	down
8178193*	HLA-DRA	down
8178205*	HLA-DQA2	down
8179019*	HLA-F	down
8179041*	NA	down
8179049*	HLA-J	down
8179481*	HLA-DRA	down
8179489*	NA	Down
8179495*	PSMB9	down
8179519*	HLA-DPB1	down
8179731*	NA	down
8180003*	NA	down
8180022*	NA	down
8180029*	HLA-DQB2	down
8180034*	TAP2	down
8180049*	PSMB8	down
8180061*	TAP1	down
8180078*	HLA-DMB	down
8180086*	HLA-DMA	down
8180093*	HLA-DOA	down
8180100*	HLA-DPA1	down
7894264	NA	down
7895149	NA	down
7896038	NA	down
7896908	PUSL1	down
7897263*	RNF207	down
7898115*	TMEM51	down
7898161*	EFHD2	down
7903827	STRIP1	down
7904050*	MOV10	down
7905881*	ADAM15	down
7908694	NAV1	down
7908793	NA	down
7909127	MFSD4	down
7909188*	IKBKE	down
7912239	GPR157	down
7912374	SRM	down
7912496	MTHFR	down
7912537	NA	down
7912638*	TMEM51-AS1	down
7913256	DDOST	down
7915184*	NA	down
7915543*	SLC6A9	down
7915659	HECTD3	down
7918394*	EPS8L3	down
7919872*	FAM63A	down
7920271*	S100A4	down
7920291	NA	Down
7920642	MUC1	down
7923662*	PIK3C2B	down
7924150*	TMEM206	down
7924823	JMJD4	down
7925876	NA	down
7929882	SEMA4G	down
7930537*	TCF7L2	down
7931899	NA	down
7934196*	PSAP	down
7934477*	CAMK2G	down
7935058	MYOF	down
7935188	NA	down
7937518	TSPAN4	down
7937713	SYT8	down
7938519*	MICALCL	down
7939546*	CD82	down
7939665*	MDK	down
7939767*	MADD	down
7940160	DTX4	down
7940530	MYRF	down
7940798	MARK2	down
7941621	DPP3	down
7942697	NA	down
7944164	TMPRSS4	down
7945204	ST14	down
7945666	CTSD	down
7946781*	PLEKHA7	down
7947815*	ACP2	down
7948444	TCN1	down
7948588	SYT7	down
7949765	PPP1CA	down
7951309	MMP13	down
7951896	PCSK7	down
7952132*	SLC37A4	down
7952290	TRIM29	down
7953341*	TAPBPL	down
7953483	USP5	down
7955613*	KRT7	down
7958989*	PLBD2	down
7962842	NA	down
7964203*	BAZ2A	down
7969414	KLF5	down
7976000	ADCK1	down
7976567	BDKRB1	down
7977046*	TNFAIP2	down
7977249	INF2	down
7977511	TEP1	down
7978260	DHRS1	down
7983405*	DUOXA2	down
7983478*	C15orf48	down
7983512*	SQRDL	down
7984779	PML	down
7985240	TMED3	down
7985620*	ALPK3	down
7987230*	LPCAT4	down
7988350*	DUOX2	down
7990417	SCAMP2	down
7994737*	NA	down
7997152*	CHST4	down
7997158	NA	down
7997401	BCO1	down
7998222*	MRPL28	down
7999909*	GPRC5B	down
8000375*	ARHGAP17	down
8000543*	NA	down
8000811*	MAPK3	down
8001030*	PYCARD	down
8001552*	CIAPIN1	down
8002133*	PSMB10	down
8002421*	VAC14	down
8005475	TRIM16L	down
8005661*	NA	down
8006984	PSMD3	down
8007188	CNP	down
8007505*	DHX8	down
8007620*	GRN	down
8008664	AKAP1	down
8009666	RAB37	down
8009693*	TMEM104	down
8010354	GAA	Down
8011293	CLUH	down
8011516	ATP2A3	down
8011671*	GGT6	down
8011713*	CXCL16	down
8012126	CLDN7	down
8014768	NA	down
8017867*	FAM20A	down
8018324*	GGA3	down
8019211	NPLOC4	down
8019622	TMEM106A	down
8021301	RAB27B	down
8023043*	PSTPIP2	down
8024687*	TJP3	down
8028524	ACTN4	down
8029086	CEACAM5	down
8029098	CEACAM6	down
8029560	CLPTM1	down
8032789*	STAP2	down
8034420*	MAN2B1	down
8034589*	FARSA	down
8037205	NA	down
8037222	CEACAM8	down
8037794*	PRKD2	down
8038261*	GYS1	down
8039389	PTPRH	down
8040365*	TRIB2	down
8040698	SLC35F6	down
8040753	TMEM214	down
8043657*	CNNM4	down
8045539*	NA	down
8047738	NA	down
8048717	SGPP2	down
8050160	MBOAT2	down
8051298*	GALNT14	down
8051322	XDH	down
8053214	AUP1	down
8053406	RETSAT	down
8054054*	NA	down
8058390*	RAPH1	down
8058973	ZNF142	Down
8059222	DNPEP	down
8060353*	RBCK1	down
8062041*	ACSS2	down
8062251	NA	down
8062927*	PI3	down
8063000*	NA	down
8063078*	CTSA	down
8063351*	SLC9A8	down
8063893	ADRM1	down
8064613	SLC4A11	down
8065612*	NOL4L	down
8065920*	NA	down
8065948*	FER1L4	down
8066513	SDC4	down
8068254*	IL10RB	down
8068810	SLC37A1	down
8069399*	NA	down
8070538	C2CD2	down
8072108	ASPHD2	down
8072926*	H1F0	down
8073605*	BIK	down
8076569	TTLL12	down
8076998*	PLXNB2	down
8077082	LMF2	down
8080100*	RAD54L2	down
8082797	TF	down
8084717*	ST6GAL1	down
8084895*	MUC20	down
8084929	SLC51A	down
8085300	SEC13	down
8087485	NA	down
8088425*	FAM3D	down
8090823	SLCO2A1	down
8092978	MUC4	down
8093230	NA	down
8096070	BMP3	down
8103025*	ZNF827	down
8104079*	FAT1	down
8106170	TMEM171	down
8114050*	8-Sep	Down
8115623*	ATP10B	down
8118833*	UHRF1BP1	down
8119926	TMEM63B	down
8122843	ESR1	down
8123606	SERPINB9P1	down
8125766	BAK1	down
8129677*	SGK1	down
8133721	HSPB1	down
8136849*	GSTK1	down
8136863*	TMEM139	down
8137798	PSMG3	down
8139859*	GUSB	down
8143575	EPHA1	down
8144880*	SH2D4A	down
8145027	FAM160B2	down
8145669*	NA	down
8146921	RDH10	down
8148548	PSCA	down
8148572*	LY6E	down
8149330*	CTSB	down
8150036	KIF13B	down
8150112	GSR	down
8152828	GSDMC	down
8153334	PSCA	down
8153342*	LYPD2	down
8155707	NA	down
8156058	NA	down
8157362*	ZNF618	down
8157381*	ZNF618	down
8158167*	LCN2	down
8158242*	URM1	down
8158671*	NA	down
8158684*	NA	down
8158961*	GTF3C5	down
8160670	AQP3	down
8161174	GNE	down
8162502*	FBP1	down
8162729*	TRIM14	down
8162744*	CORO2A	down
8163505*	HDHD3	Down
8164535*	CRAT	down
8164580*	PTGES	down
8172280*	SLC9A7	down
8175924*	NAA10	down
8178115*	CFB	down
8178561	ABHD16A	down
8179028	LOC554223	down
8179112*	ABCF1	down
8179331*	C2	down
8179351*	CFB	down
8179364*	SKIV2L	down
8179638	TRIM26	down
8180166*	TAPBP	down
7892796	NA	up
7893130	NA	up
7894970*	NA	up
7895574	NA	up
7896160	NA	up
7899502*	RNU11	up
7902043*	DNAJC6	up
7916506*	C1orf168	up
7930612	NA	up
7932498*	SKIDA1	up
7944765	NA	up
7953383*	SCARNA10	up
7961710*	ABCC9	up
7964631	FAM19A2	up
7971165	NA	up
7978407*	PRKD1	up
7985317*	CEMIP	up
7999291	C16orf89	up
8006504*	FNDC8	up
8009380	SNORA38B	up
8013521*	NA	up
8013523	NA	up
8043782*	CNGA3	up
8045287*	NA	up
8049530	LRRFIP1	up
8076223*	NA	up
8089145	ABI3BP	Up
8098604	ANKRD37	up
8101762*	SNCA	up
8104141*	PLEKHG4B	up
8107204*	NA	up
8108180*	NA	up
8127658	NA	up
8132248	NA	up
8147990	NA	up
8156358	NA	up
8165694*	NA	up
8165696	NA	up
8165698*	NA	up
8165700	NA	up
8165707	NA	up
8168868	ARMCX1	up
8175531*	CDR1	up

*indicates leading edge gene

TABLE 22

Term	FDR

antigen processing and presentation of exogenous antigen (GO: 0019884)	2.64E−13
antigen processing and presentation of exogenous peptide antigen (GO: 0002478)	2.64E−13
antigen processing and presentation of peptide antigen (GO: 0048002)	8.84E−13
antigen processing and presentation (GO: 0019882)	1.19E−11
antigen processing and presentation of exogenous peptide antigen via MHC class I	1.74E−08
(GO: 0042590)
antigen processing and presentation of peptide antigen via MHC class I (GO: 0002474)	1.74E−08
antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-	6.31E−08
dependent (GO: 0002479)
antigen processing and presentation of exogenous peptide antigen via MHC class II	3.53E−05
(GO: 0019886)
antigen processing and presentation of peptide antigen via MHC class II (GO: 0002495)	3.53E−05
interferon-gamma-mediated signaling pathway (GO: 0060333)	3.53E−05
antigen processing and presentation of peptide or polysaccharide antigen via MHC class II	3.68E−05
(GO: 0002504)
signal transduction involved in mitotic G1 DNA damage checkpoint (GO: 0072431)	3.68E−05
intracellular signal transduction involved in G1 DNA damage checkpoint (GO: 1902400)	3.68E−05
DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest	3.68E−05
(GO: 0006977)
signal transduction involved in mitotic DNA integrity checkpoint (GO: 1902403)	3.68E−05
signal transduction involved in mitotic cell cycle checkpoint (GO: 0072413)	3.68E−05
signal transduction involved in mitotic DNA damage checkpoint (GO: 1902402)	3.68E−05
signal transduction involved in DNA integrity checkpoint (GO: 0072401)	4.33E−05
signal transduction involved in DNA damage checkpoint (GO: 0072422)	4.33E−05
signal transduction involved in cell cycle checkpoint (GO: 0072395)	4.71E−05
regulation of cellular amino acid metabolic process (GO: 0006521)	7.97E−05
regulation of cellular amine metabolic process (GO: 0033238)	0.000147804
positive regulation of cell cycle arrest (GO: 0071158)	0.000198665
cellular response to interferon-gamma (GO: 0071346)	0.000264094
DNA damage response, signal transduction by p53 class mediator (GO: 0030330)	0.000334076
response to interferon-gamma (GO: 0034341)	0.000334076
negative regulation of G1/S transition of mitotic cell cycle (GO: 2000134)	0.000681069
negative regulation of cell cycle G1/S phase transition (GO: 1902807)	0.000681069
negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle	0.000741508
(GO: 0051436)
regulation of antigen processing and presentation (GO: 0002577)	0.000976729
proteasome-mediated ubiquitin-dependent protein catabolic process (GO: 0043161)	0.000976729
regulation of cell cycle arrest (GO: 0071156)	0.001026492
signal transduction in response to DNA damage (GO: 0042770)	0.001086018
positive regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle	0.001116512
(GO: 0051437)
proteasomal protein catabolic process (GO: 0010498)	0.001272502
negative regulation of ligase activity (GO: 0051352)	0.001272502
negative regulation of ubiquitin-protein transferase activity (GO: 0051444)	0.001272502
regulation of G1/S transition of mitotic cell cycle (GO: 2000045)	0.001303525
regulation of cell cycle G1/S phase transition (GO: 1902806)	0.001475051
signal transduction by p53 class mediator (GO: 0072331)	0.001548279
regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle (GO: 0051439)	0.00173373
regulation of cellular ketone metabolic process (GO: 0010565)	0.001946114
antigen processing and presentation of endogenous antigen (GO: 0019883)	0.002234329
negative regulation of protein modification by small protein conjugation or removal	0.002330593
(GO: 1903321)
anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic	0.002330593
process (GO: 0031145)
post-translational protein modification (GO: 0043687)	0.003201008
positive regulation of ubiquitin-protein transferase activity (GO: 0051443)	0.003523951
proteolysis involved in cellular protein catabolic process (GO: 0051603)	0.003541561
cytokine-mediated signaling pathway (GO: 0019221)	0.004071611
protein catabolic process (GO: 0030163)	0.004704633
positive regulation of ligase activity (GO: 0051351)	0.004980771
regulation of antigen processing and presentation of peptide antigen (GO: 0002583)	0.005288359
negative regulation of protein ubiquitination (GO: 0031397)	0.005404568
modification-dependent protein catabolic process (GO: 0019941)	0.008295631
protein polyubiquitination (GO: 0000209)	0.008295631
modification-dependent macromolecule catabolic process (GO: 0043632)	0.009325558
antigen processing and presentation of endogenous peptide antigen via MHC class I	0.009413569
(GO: 0019885)
regulation of ubiquitin-protein transferase activity (GO: 0051438)	0.010265072
antigen processing and presentation of endogenous peptide antigen (GO: 0002483)	0.012182104
negative regulation of protein modification process (GO: 0031400)	0.012670399
regulation of ligase activity (GO: 0051340)	0.013876213
ubiquitin-dependent protein catabolic process (GO: 0006511)	0.013915779
regulation of apoptotic signaling pathway (GO: 2001233)	0.014172597
positive regulation of antigen processing and presentation (GO: 0002579)	0.018895095
negative regulation of transferase activity (GO: 0051348)	0.019241154
regulation of T cell activation (GO: 0050863)	0.019692533
O-glycan processing (GO: 0016266)	0.019692533
T cell costimulation (GO: 0031295)	0.022242877
protein N-linked glycosylation via asparagine (GO: 0018279)	0.022242877
regulation of I-kappaB kinase/NF-kappaB signaling (GO: 0043122)	0.022924012
lymphocyte costimulation (GO: 0031294)	0.022924012
peptidyl-asparagine modification (GO: 0018196)	0.022924012
positive regulation of T cell activation (GO: 0050870)	0.023552255
T cell receptor signaling pathway (GO: 0050852)	0.025340097
negative regulation of viral release from host cell (GO: 1902187)	0.025340097
tRNA metabolic process (GO: 0006399)	0.028902105
protein N-linked glycosylation (GO: 0006487)	0.033855
regulation of cytokine production (GO: 0001817)	0.03675597
G1/S transition of mitotic cell cycle (GO: 0000082)	0.037323339
cell cycle G1/S phase transition (GO: 0044843)	0.037323339
regulation of type I interferon production (GO: 0032479)	0.04117111
positive regulation of protein modification by small protein conjugation or removal	0.048722949
(GO: 1903322)

TABLE 23

Pathway	FDR

Interferon gamma signaling	9.67E−08
ER-Phagosome pathway	1.34E−07
Antigen processing-Cross presentation	1.74E−06
Interferon Signaling	3.74E−06
MHC class II antigen presentation	3.74E−06
Class I MHC mediated antigen processing & presentation	7.67E−05
Vpu mediated degradation of CD4	8.51E−05
AUF1 (hnRNP D0) destabilizes mRNA	0.00012195
Hh ligand biogenesis disease	0.00019564
Hedgehog ligand biogenesis	0.00019564
Processing-defective Hh variants abrogate ligand secretion	0.00019564
Ubiquitin-dependent degradation of Cyclin D1	0.00019564
Cross-presentation of soluble exogenous antigens (endosomes)	0.00019564
Autodegradation of the E3 ubiquitin ligase COP1	0.00019564
Regulation of activated PAK-2p34 by proteasome mediated degradation	0.00019564
Regulation of Apoptosis	0.00019564
CDK-mediated phosphorylation and removal of Cdc6	0.00019564
p53-Independent DNA Damage Response	0.00019564
Stabilization of p53	0.00019564
Ubiquitin-dependent degradation of Cyclin D	0.00019564
Regulation of ornithine decarboxylase (ODC)	0.00019564
Ubiquitin Mediated Degradation of Phosphorylated Cdc25A	0.00019564
p53-Independent G1/S DNA damage checkpoint	0.00019564
Programmed Cell Death	0.00019564
Vif-mediated degradation of APOBEC3G	0.00021794
degradation of AXIN	0.00023347
SCF-beta-TrCP mediated degradation of Emi1	0.00023347
Apoptosis	0.00024085
degradation of DVL	0.0002789
SCF(Skp2)-mediated degradation of p27/p21	0.0002789
p53-Dependent G1 DNA Damage Response	0.00030002
p53-Dependent G1/S DNA damage checkpoint	0.00030002
CDT1 association with the CDC6:ORC:origin complex	0.00033297
Degradation of GLI2 by the proteasome	0.00033904
GLI3 is processed to GLI3R by the proteasome	0.00033904
Degradation of GLI1 by the proteasome	0.00033904
Antigen Presentation: Folding, assembly and peptide loading of class I MHC	0.00033904
G1/S DNA Damage Checkpoints	0.00036634
Autodegradation of Cdh1 by Cdh1:APC/C	0.00036634
Asymmetric localization of PCP proteins	0.00052044
Cytokine Signaling in Immune system	0.00052735
AMER1 mutants destabilize the destruction complex	0.00052735
Host Interactions of HIV factors	0.00052735
Degradation of beta-catenin by the destruction complex	0.00052735
phosphorylation site mutants of CTNNB1 are not targeted to the proteasome by the	0.00052735
destruction complex
S33 mutants of beta-catenin aren't phosphorylated	0.00052735
truncated APC mutants destabilize the destruction complex	0.00052735
deletions in the AXIN genes in hepatocellular carcinoma result in elevated WNT signaling	0.00052735
APC/C:Cdc20 mediated degradation of Securin	0.00052735
deletions in the AMER1 gene destabilize the destruction complex	0.00052735
Activation of NF-kappaB in B cells	0.00052735
T41 mutants of beta-catenin aren't phosphorylated	0.00052735
Assembly of the pre-replicative complex	0.00052735
Cyclin E associated events during G1/S transition	0.00052735
Cyclin A:Cdk2-associated events at S phase entry	0.00052735
AXIN mutants destabilize the destruction complex, activating WNT signaling	0.00052735
APC truncation mutants have impaired AXIN binding	0.00052735
misspliced GSK3beta mutants stabilize beta-catenin	0.00052735
truncations of AMER1 destabilize the destruction complex	0.00052735
APC truncation mutants are not K63 polyubiquitinated	0.00052735
TCF7L2 mutants don't bind CTBP	0.00052735
S45 mutants of beta-catenin aren't phosphorylated	0.00052735
S37 mutants of beta-catenin aren't phosphorylated	0.00052735
AXIN missense mutants destabilize the destruction complex	0.00052735
Cdc20:Phospho-APC/C mediated degradation of Cyclin A	0.00063305
APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in	0.00063305
late mitosis/early G1
Orel removal from chromatin	0.00067558
Switching of origins to a post-replicative state	0.00067558
APC:Cdc20 mediated degradation of cell cycle proteins prior to satisfation of the cell cycle	0.00067558
checkpoint
Regulation of mRNA stability by proteins that bind AU-rich elements	0.00071424
Translocation of ZAP-70 to Immunological synapse	0.00077576
Removal of licensing factors from origins	0.00079073
APC/C:Cdc20 mediated degradation of mitotic proteins	0.00079073
Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins	0.00086583
PCP/CE pathway	0.00104426
Regulation of DNA replication	0.00104426
Phosphorylation of CD3 and TCR zeta chains	0.00109603
Regulation of APC/C activators between G1/S and early anaphase	0.00122728
Antigen processing: Ubiquitination & Proteasome degradation	0.00165974
M/G1 Transition	0.00186599
DNA Replication Pre-Initiation	0.00186599
PD-1 signaling	0.00186599
Regulation of mitotic cell cycle	0.00199262
APC/C-mediated degradation of cell cycle proteins	0.00199262
Hedgehog ‘on’ state	0.00234744
beta-catenin independent WNT signaling	0.00307868
Post-translational protein modification	0.0045083
Synthesis of DNA	0.00577858
Hedgehog ‘off’ state	0.00577858
Defective ALG14 causes congenital myasthenic syndrome (ALG14-CMS)	0.00577858
Defective DPAGT1 causes DPAGT1-CDG (CDG-1j) and CMSTA2	0.00577858
Defective ALG1 causes ALG1-CDG (CDG-1k)	0.00577858
Diseases associated with N-glycosylation of proteins	0.00577858
Defective MGAT2 causes MGAT2-CDG (CDG-2a)	0.00577858
Asparagine N-linked glycosylation	0.00577858
Defective ALG8 causes ALG8-CDG (CDG-1h)	0.00577858
Defective ALG3 causes ALG3-CDG (CDG-1d)	0.00577858
Defective MAN1B1 causes MRT15	0.00577858
Defective RFT1 causes RFT1-CDG (CDG-1n)	0.00577858
Defective MOGS causes MOGS-CDG (CDG-2b)	0.00577858
Defective ALG12 causes ALG12-CDG (CDG-1g)	0.00577858
Defective ALG11 causes ALG11-CDG (CDG-1p)	0.00577858
Defective MPDU1 causes MPDU1-CDG (CDG-1f)	0.00577858
Defective ALG6 causes ALG6-CDG (CDG-1c)	0.00577858
Defective ALG2 causes ALG2-CDG (CDG-1i)	0.00577858
Defective ALG9 causes ALG9-CDG (CDG-1l)	0.00577858
Generation of second messenger molecules	0.00577858
Cytosolic tRNA aminoacylation	0.00716219
DNA Replication	0.00836342
Metabolism of amino acids and derivatives	0.009109
Costimulation by the CD28 family	0.0094808
G1/S Transition	0.0105585
HIV Infection	0.01259271
Downstream TCR signaling	0.01527904
Signaling by Hedgehog	0.0155106
Cell Cycle Checkpoints	0.01753312
O-linked glycosylation of mucins	0.02063405
S Phase	0.02401362
Downstream signaling events of B Cell Receptor (BCR)	0.02749436
Mitotic G1-G1/S phases	0.04108854
Separation of Sister Chromatids	0.04827289

TABLE 24

Term	FDR

response to magnesium ion (GO: 0032026)	0.01178146
positive regulation of release of sequestered	0.01178146
calcium ion into cytosol (GO: 0051281)
potassium ion transport (GO: 0006813)	0.01178146
cellular potassium ion transport (GO: 0071804)	0.01178146
potassium ion transmembrane transport (GO: 0071805)	0.01178146
regulation of endocytosis (GO: 0030100)	0.01247912
positive regulation of calcium ion transport into cytosol	0.01909394
(GO: 0010524)
regulation of release of sequestered calcium ion into cytosol	0.03574339
(GO: 0051279)
regulation of vesicle-mediated transport (GO: 0060627)	0.04808581
regulation of calcium ion transport into cytosol	0.04808581
(GO: 0010522)

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Claims

What is claimed is:

1. A method of diagnosing lung cancer in a subject comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes; and (b) comparing the expression of the one or more genes to a control sample of those genes from individuals without cancer; wherein the one or more genes are selected from the group consisting of genes in Table 12, Table 13 or Table 21, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having lung cancer.

2. The method of claim 1, wherein non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject not having lung cancer.

3. A method of diagnosing lung cancer in a subject comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes; and (b) comparing the expression of the one or more genes to a control sample of those genes from individuals with cancer; wherein the one or more genes are selected from the group consisting of genes in Table 12, Table 13 or Table 21, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the subject not having lung cancer.

4. The method of claim 3, wherein non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having lung cancer.

5. A method of determining whether a subject has quit smoking comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes selected from the group consisting of genes in Table 5 or Table 6; and (b) comparing the expression of the one or more genes to a control sample of those genes from non-smokers; wherein altered expression of the subject's genes relative to the control sample is indicative of the subject having quit smoking.

6. The method of claim 5, wherein non-altered expression of the subject's one or more genes relative to the control sample is indicative of the subject not having quit smoking.

7. A method of determining whether a subject has quit smoking comprising the steps of: (a) measuring a biological sample comprising nasal epithelial cells of the subject for expression of one or more genes selected from the group consisting of genes in Table 5 or Table 6; and (b) comparing the expression of the one or more genes to a control sample of those genes from smokers; wherein altered expression of the subject's genes relative to the control sample is indicative of the subject not having quit smoking.

8. The method of claim 7, wherein non-altered expression of the subject's one or more genes relative to the control sample is indicative of the subject having quit smoking.

9. A method of determining the likelihood that a subject has lung cancer, the method comprising: (a) subjecting a biological sample comprising nasal epithelial cells to a gene expression analysis, wherein the gene expression analysis comprises comparing gene expression levels of one or more genes selected from the group of genes in Table 12, Table 13 or Table 21 to the expression levels of a control sample of those genes from individuals without cancer; and (b) determining the likelihood that the subject has lung cancer by determining differential expression of the subject's one or more genes relative to the group of genes in Table 12, Table 13 or Table 21, wherein differential expression of the subject's genes relative to the control sample is indicative of the subject having a high likelihood of lung cancer.

10. The method of claim 9, wherein non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having a low likelihood of lung cancer.

11. A method of determining the likelihood that a subject has lung cancer, the method comprising: (a) subjecting a biological sample comprising nasal epithelial cells to a gene expression analysis, wherein the gene expression analysis comprises comparing gene expression levels of one or more genes selected from the group of genes in Table 12, Table 13 or Table 21 to the expression levels of a control sample of those genes from individuals with cancer; and (b) determining the likelihood that the subject has lung cancer by determining differential expression of the subject's one or more genes relative to the group of genes in Table 12, Table 13 or Table 21, wherein differential expression of the subject's genes relative to the control sample is indicative of the subject having a low likelihood of lung cancer.

12. The method of claim 11, wherein non-differential expression of the subject's one or more genes relative to the control sample is indicative of the subject having a high likelihood of lung cancer.

13. The method of claims 1-12, wherein at least two genes are measured.

14. The method of claims 1-12, wherein at least five genes are measured.

15. The method of claims 1-12, wherein at least ten genes are measured.

16. The method of claims 1-12, wherein at least twenty genes are measured.

17. The method of claims 1-12, wherein at least thirty genes are measured.

18. The method of claims 1-12, wherein at least forty genes are measured.

19. The method of claims 1-12, wherein at least fifty genes are measured.

20. The method of claims 1-4 and 9-19, wherein the genes in Table 12 comprise those genes identified in cluster 1.

21. The method of claims 1-4 and 9-19, wherein the genes in Table 12 comprise those genes identified in cluster 2.

22. The method of claims 1-4 and 9-19, wherein the genes in Table 12 comprise those genes identified in cluster 3.

23. The method of claims 1-4 and 9-19, wherein the genes in Table 12 comprise those genes identified in cluster 4.

24. The method of claims 1-23, further comprising determining one or more of the subject's clinical risk factors affecting the subject's risk for having lung cancer.

25. The method of claim 24, wherein the one or more clinical risk factors are selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan, lesion location and time since quitting smoking.

26. The method of claim 25, wherein the subject's positive smoking status is indicative of the subject having lung cancer.

27. The method of claim 25, wherein the subject's advanced age is indicative of the subject having lung cancer.

28. The method of claim 25, wherein the presence of a lung nodule greater than 3 cm on the subject's CT scan is indicative of the subject having lung cancer.

29. The method of claim 25, wherein the subject's time since quitting smoking greater than 15 years is indicative of the subject having lung cancer.

30. The method of claims 5-8 and 13-19, wherein the one or more genes comprise one or more genes from Table 14.

31. The method of claims 5-8 and 13-19, wherein the one or more genes comprise one or more genes from Table 15.

32. The method of claims 1-4 and 9-29, wherein the one or more genes comprise one or more genes from Table 13.

33. The method of claims 1-4 and 9-29, wherein the one or more genes comprise all of the genes from Table 13.

34. The method of claims 1-33, wherein the one or more genes comprise one or more leading edge genes from Table 21.

35. The method of claims 1-34, wherein the one or more genes comprise the genes from Table 13.

36. The method of claims 1-35, wherein the one or more genes further comprise one or more genes from Table 5.

37. The method of claims 1-36, wherein the one or more genes further comprise one or more genes from Table 6.

38. The method of claims 1-37, wherein the expression of the one or more genes is determined using a quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay or a oligonucleotide array assay.

39. The method of claims 1-4 and 9-38, wherein the lung cancer is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, small cell cancer or non-small cell cancer.

40. The method of claims 1-39, wherein the one or more genes comprise mRNA.

41. The method of claim 1-40, wherein the biological sample does not comprise bronchial epithelial cells or bronchial epithelial tissue.

42. The method of claims 1-41, wherein the one or more genes is associated with DNA damage.

43. The method of claims 1-41, wherein the one or more genes is associated with regulation of apoptosis.

44. The method of claims 1-41, wherein the one or more genes is associated with immune system activation.

45. The method of claim 44, wherein the one or more genes is associated with the interferon-gamma signaling pathway.

46. The method of claim 44, wherein the one or more of the genes is associated with antigen presentation.

47. The method of claims 1-4 and 9-46, wherein the method is indicative of the subject having lung cancer or of being at risk of developing lung cancer, wherein the method further comprises administering a treatment to the subject.

48. The method of claim 47, wherein the treatment comprises chemotherapy.

49. The method of claim 47, wherein the treatment comprises radiation therapy.

50. The method of claim 47, wherein the method comprises immunotherapy.

51. The method of claim 47, wherein the method comprises surgical intervention.

52. A composition comprising one or more nucleic acid probes, wherein each of the one or more nucleic acids probes specifically hybridizes with an mRNA expressed from five or more genes selected from the group of genes in Table 5, Table 6, Table 12, Table 13 or Table 21.

53. The composition of claim 52, wherein at least ten or more genes are measured.

54. The composition of claims 52-53, wherein at least fifteen genes are measured.

55. The composition of claims 52-54, wherein at least twenty genes are measured.

56. The composition of claims 52-55, wherein at least thirty genes are measured.

57. The composition of claims 52-56, wherein at least forty genes are measured.

58. The composition of claims 52-57, wherein at least fifty genes are measured.

59. The composition of claims 52-58, wherein at least one hundred genes are measured.

60. The composition of claims 52-59, wherein the genes in Table 12 comprise those genes identified in cluster 1.

61. The composition of claims 52-60, wherein the genes in Table 12 comprise those genes identified in cluster 2.

62. The composition of claims 52-61, wherein the genes in Table 12 comprise those genes identified in cluster 3.

63. The composition of claims 52-62, wherein the genes in Table 12 comprise those genes identified in cluster 4.

64. The composition of claims 52-59, wherein one or more genes in Table 12 comprise at least one gene from each of clusters 1, 2, 3 and 4.

65. The composition of claims 52-64, wherein the one or more genes is associated with DNA damage.

66. The composition of claims 52-64, wherein the one or more genes is associated with regulation of apoptosis.

67. The composition of claims 52-64, wherein the one or more genes is associated with immune system activation.

68. The composition of claim 67, wherein the one or more genes is associated with the interferon-gamma signaling pathway.

69. The composition of claim 67, wherein the one or more of the genes is associated with antigen presentation.

70. The composition of claims 52-69, wherein the one or more of the genes comprise one or more of the leading edge genes identified in Table 21.

71. A minimally-invasive method of determining the likelihood that a subject does not have lung cancer, the method comprising:

(a) detecting, by quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay or a oligonucleotide array assay, mRNA or cDNA expression levels in a sample comprising nasal epithelial cells from a subject;

(b) determining mRNA or cDNA expression levels in the sample of nasal epithelial cells of two or more gene selected from the group consisting of the genes in Table 12, Table 13 or Table 21; and

(c) based on the expression levels determined in (b), determining a lung cancer risk-score that is indicative of the likelihood that the subject does not have lung cancer.

72. The method of claim 71, wherein the subject has undergone an indeterminate or non-diagnostic bronchoscopy procedure.

73. The method of claims 71-72, wherein the genes comprise at least 5 genes from Table 13.

74. The method of claims 71-72, wherein the genes comprise at least 10 genes from Table 13.

75. The method of claims 71-72, wherein the genes comprise at least 15 genes from Table 13.

76. The method of claims 71-72, wherein the genes comprise at least 20 genes from Table 13.

77. The method of claims 71-72, wherein the genes comprise at least 25 genes from Table 13.

78. The method of claims 71-72, wherein the genes comprise all of the genes from Table 13.

79. The method of claims 71-78, further comprising determining one or more of the subject's clinical risk factors affecting the subject's risk for having lung cancer.

80. The method of claim 79, wherein the one or more clinical risk factors are selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan, lesion location and time since quitting smoking.

81. The method of claim 80, wherein the subject's positive smoking status is indicative of the subject having lung cancer.

82. The method of claim 80, wherein the subject's advanced age is indicative of the subject having lung cancer.

83. The method of claim 80, wherein the presence of a lung nodule greater than 3 cm on the subject's CT scan is indicative of the subject having lung cancer.

84. The method of claim 80, wherein the subject's time since quitting smoking greater than 15 years is indicative of the subject having lung cancer.

85. The method of claim 80, wherein the genes comprise at least 5 genes from Table 13, and further comprising determining one or more of the subject's clinical risk factors affecting the subject's risk for having lung cancer, wherein the one or more clinical risk factors are selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan, lesion location and time since quitting smoking

86. The method of claim 80, wherein the subject's smoking status is determined by measuring the expression of one or more genes selected from the group consisting of genes in Table 5 or Table 6 in the epithelial cells of the subject.

87. The method of claim 80, wherein the subject's time since quitting smoking is determined by measuring the expression of one or more genes selected from the group consisting of genes in Table 5 or Table 6 in the epithelial cells of the subject.