# Patent

## Title
Method for determining coronary artery disease risk

## Abstract

Markers and methods useful for assessing coronary artery disease in a subject are provided, along with kits for measuring their expression. Also provided are predictive models, based on the markers, as well as computer systems, and software embodiments of the models for scoring and optionally classifying samples.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Ser. No. 12/816,232, filed Jun. 15, 2010, U.S. Provisional Application No. 61/187,203, filed Jun. 15, 2009, and U.S. Provisional Application No. 61/245,190, filed Sep. 23, 2009, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes.


BACKGROUND
1. Field of the Invention
The invention relates to predictive models for determining the extent of coronary artery disease (CAD) risk based on marker expression measurements, to their methods of use, and to computer systems and software for their implementation.
2. Description of the Related Art
Mortality and morbidity from CAD and myocardial infarction (MI) are a major global health burden. Major determinants of current CAD likelihood are sex, age, and chest-pain type.1, 2 Other risk factors such as diabetes, smoking, dyslipidemia, and family history have been associated with future cardiovascular event risk.3 In addition, atherosclerosis has a systemic inflammatory component including activation and migration of immune cells into the vessel wall.1, 5 In fact, since such cells are derived from and have interactions with circulating blood, quantitative measurements of circulating blood cell gene expression reflects the extent of CAD.6, 7 These observations likely reflect both changes in cell type distributions, which have prognostic value for cardiovascular events8 and gene expression changes within a specific cell type or lineage.
The “gold standard” for detecting CAD is invasive coronary angiography; however, this is costly, and can pose risk to the patient. Prior to angiography, non-invasive diagnostic modalities such as myocardial perfusion imaging (MPI) and CT-angiography may be used, however these have complications including radiation exposure, contrast agent sensitivity, and only add moderately to obstructive CAD identification.9, 10 
Unmet Clinical and Scientific Need
A non-invasive blood test that could reliably identify patients with CAD would have significant clinical utility. As such, a major advancement in the fight against atherosclerosis would be the development of non-invasive diagnostic tests that can aid in the diagnosis and assessment of the extent of CAD in patients. Herein the development and validation of an algorithm using marker expression and clinical factors (e.g., age and gender) for such a purpose is described.
SUMMARY
Disclosed herein is a computer-implemented method for scoring a first sample obtained from a subject, including: obtaining a first dataset associated with the first sample, wherein the first dataset includes quantitative expression data for at least one marker set selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7; wherein term 1 includes marker 1, marker 2, and marker 3, wherein marker 1 includes AF161365, wherein marker 2 includes HNRPF or ACBD5, and wherein marker 3 includes TFCP2 or DDX18; wherein term 2 includes marker 4, marker 5, and marker 6, wherein marker 4 includes AF289562 or CD248, wherein marker 5 includes HNRPF or ACBD5, and wherein marker 6 includes TFCP2 or DDX18; wherein term 3 includes marker 7, marker 8, marker 9, and marker 10 wherein marker 7 includes CD79B or CD19, wherein marker 8 includes SPIB or BLK, wherein marker 9 includes CD3D or LCK, and wherein marker 10 includes TMC8 or CCT2; wherein term 4 includes marker 11, marker 12, marker 13, and marker 14, wherein marker 11 includes S100A12 or MMP9, wherein marker 12 includes CLEC4E or ALOX5AP, wherein marker 13 includes S100A8 or NAMPT, and wherein marker 14 includes RPL28 or SSRP1; wherein term 5 includes marker 15, marker 16, marker 17, marker 18, and marker 19, wherein marker 15 includes S100A12 or MMP9, wherein marker 16 includes CLEC4E or ALOX5AP, wherein marker 17 includes S100A8 or NAMPT, wherein marker 18 includes AQP9 or GLT1D1, and wherein marker 19 includes NCF4 or NCF2; wherein term 6 includes marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, wherein marker 20 includes CASP5 or H3F3B, wherein marker 21 includes IL18RAP or TXN, wherein marker 22 includes TNFAIP6 or PLAUR, wherein marker 23 includes IL8RB or BCL2A1, wherein marker 24 includes TNFRSF10C or PTAFR, wherein marker 25 includes KCNE3 or LAMP2, and wherein marker 26 includes TLR4 or TYROBP; and wherein term 7 includes marker 27, marker 28, marker 29, and marker 30, wherein marker 27 includes SLAMF7 or CX3CR1, wherein marker 28 includes KLRC4 or CD8A, wherein marker 29 includes CD3D or LCK, and wherein marker 30 includes TMC8 or CCT2; and determining, by a computer processor, a first score from the first dataset using an interpretation function, wherein the first score is predictive of CAD in the subject.
In an embodiment, the first dataset includes quantitative expression data for at least two marker sets selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, the first dataset includes quantitative expression data for at least three marker sets selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, the first dataset includes quantitative expression data for at least four marker sets selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, the first dataset includes quantitative expression data for at least five marker sets selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, the first dataset includes quantitative expression data for at least six marker sets selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, the first dataset includes quantitative expression data for the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7.
In an embodiment, the interpretation function is based on a predictive model. In an embodiment, the predictive model is selected from the group consisting of a partial least squares model, a logistic regression model, a linear regression model, a linear discriminant analysis model, a ridge regression model, and a tree-based recursive partitioning model. In an embodiment, the predictive model performance is characterized by an area under the curve (AUC) ranging from 0.68 to 0.70. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.70 to 0.79. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.80 to 0.89. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.90 to 0.99.
In an embodiment, the first dataset further includes a clinical factor. In an embodiment, the clinical factor is selected from the group consisting of: age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, a medical history parameter, a medical symptom parameter, height, weight, a body-mass index, resting heart rate, and smoker/non-smoker status.
In an embodiment, the obtaining the first dataset associated with the first sample includes obtaining the first sample and processing the first sample to experimentally determine the first dataset. In an embodiment, the obtaining the first dataset associated with the first sample includes receiving the first dataset from a third party that has processed the first sample to experimentally determine the first dataset.
In an embodiment, the method includes classifying the first sample according to the first score. In an embodiment, the classifying is predictive of the presence or absence of CAD in the subject. In an embodiment, the classifying is predictive of the extent of CAD in the subject. In an embodiment, the classifying is predictive of the risk of CAD in the subject. In an embodiment, the method includes rating CAD risk based on the first score.
In an embodiment, the first sample includes peripheral blood cells. In an embodiment, the peripheral blood cells include leukocytes. In an embodiment, the first sample includes RNA extracted from peripheral blood cells.
In an embodiment, the quantitative expression data are derived from hybridization data. In an embodiment, the quantitative expression data are derived from polymerase chain reaction data. In an embodiment, the quantitative expression data are derived from an antibody binding assay. In an embodiment, the first dataset is obtained stored on a storage memory.
In an embodiment, the subject is a human. In an embodiment, the subject has stable chest pain. In an embodiment, the subject has typical angina or atypical angina or an anginal equivalent. In an embodiment, the subject has no previous diagnosis of myocardial infarction (MI). In an embodiment, the subject has not had a revascularization procedure. In an embodiment, the subject does not have diabetes. In an embodiment, the subject does not have an inflammatory condition or an infectious condition. In an embodiment, the subject is not currently taking a steroid, an immunosuppressive agent, or a chemotherapeutic agent.
Also described herein is a computer-implemented method for scoring a first sample obtained from a subject, including: obtaining a first dataset associated with the first sample, wherein the first dataset includes quantitative expression data for at least two markers selected from the group consisting of AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, and CD8A; and determining, by a computer processor, a first score from the first dataset using an interpretation function, wherein the first score is predictive of CAD in the subject.
In an embodiment, the first dataset includes a clinical factor. In an embodiment, the clinical factor is age and/or gender. In an embodiment, the clinical factor is selected from the group consisting of: age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, a medical history parameter, a medical symptom parameter, height, weight, a body-mass index, resting heart rate, and smoker/non-smoker status.
In an embodiment, the first dataset includes quantitative expression data for at least three markers. In an embodiment, the first dataset includes quantitative expression data for at least four markers. In an embodiment, the first dataset includes quantitative expression data for at least five markers. In an embodiment, the first dataset includes quantitative expression data for at least six markers.
In an embodiment, the interpretation function is based on a predictive model. In an embodiment, the predictive model is selected from the group consisting of a partial least squares model, a logistic regression model, a linear regression model, a linear discriminant analysis model, a ridge regression model, and a tree-based recursive partitioning model. In an embodiment, the predictive model performance is characterized by an area under the curve (AUC) ranging from 0.68 to 0.70. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.70 to 0.79. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.80 to 0.89. In an embodiment, the predictive model performance is characterized by an AUC ranging from 0.90 to 0.99.
In an embodiment, the obtaining the first dataset associated with the first sample includes obtaining the first sample and processing the first sample to experimentally determine the first dataset. In an embodiment, the obtaining the first dataset associated with the first sample includes receiving the first dataset from a third party that has processed the first sample to experimentally determine the first dataset.
In an embodiment, the method includes classifying the first sample according to the first score. In an embodiment, the classifying is predictive of the presence or absence of CAD in the subject. In an embodiment, the classifying is predictive of the extent of CAD in the subject. In an embodiment, the classifying is predictive of the risk of CAD in the subject. In an embodiment, the method includes rating CAD risk based on the first score.
In an embodiment, the first sample includes peripheral blood cells. In an embodiment, the peripheral blood cells include leukocytes. In an embodiment, the first sample includes RNA extracted from peripheral blood cells.
In an embodiment, the quantitative expression data are derived from hybridization data. In an embodiment, the quantitative expression data are derived from polymerase chain reaction data. In an embodiment, the quantitative expression data are derived from an antibody binding assay. In an embodiment, the first dataset is obtained stored on a storage memory.
In an embodiment, the subject is a human. In an embodiment, the subject has stable chest pain. In an embodiment, the subject has typical angina or atypical angina or an anginal equivalent. In an embodiment, the subject has no previous diagnosis of myocardial infarction (MI). In an embodiment, the subject has not had a revascularization procedure. In an embodiment, the subject does not have diabetes. In an embodiment, the subject does not have an inflammatory condition or an infectious condition. In an embodiment, the subject is not currently taking a steroid, an immunosuppressive agent, or a chemotherapeutic agent.
Also described herein is a system for predicting CAD in a subject, the system including: a storage memory for storing a dataset associated with a sample obtained from the subject, wherein the first dataset includes quantitative expression data for at least one marker set selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7; wherein term 1 includes marker 1, marker 2, and marker 3, wherein marker 1 includes AF161365, wherein marker 2 includes HNRPF or ACBD5, and wherein marker 3 includes TFCP2 or DDX18; wherein term 2 includes marker 4, marker 5, and marker 6, wherein marker 4 includes AF289562 or CD248, wherein marker 5 includes HNRPF or ACBD5, and wherein marker 6 includes TFCP2 or DDX18; wherein term 3 includes marker 7, marker 8, marker 9, and marker 10 wherein marker 7 includes CD79B or CD19, wherein marker 8 includes SPIB or BLK, wherein marker 9 includes CD3D or LCK, and wherein marker 10 includes TMC8 or CCT2; wherein term 4 includes marker 11, marker 12, marker 13, and marker 14, wherein marker 11 includes S100A12 or MMP9, wherein marker 12 includes CLEC4E or ALOX5AP, wherein marker 13 includes S100A8 or NAMPT, and wherein marker 14 includes RPL28 or SSRP1; wherein term 5 includes marker 15, marker 16, marker 17, marker 18, and marker 19, wherein marker 15 includes S100A12 or MMP9, wherein marker 16 includes CLEC4E or ALOX5AP, wherein marker 17 includes S100A8 or NAMPT, wherein marker 18 includes AQP9 or GLT1D1, and wherein marker 19 includes NCF4 or NCF2; wherein term 6 includes marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, wherein marker 20 includes CASP5 or H3F3B, wherein marker 21 includes IL18RAP or TXN, wherein marker 22 includes TNFAIP6 or PLAUR, wherein marker 23 includes IL8RB or BCL2A1, wherein marker 24 includes TNFRSF10C or PTAFR, wherein marker 25 includes KCNE3 or LAMP2, and wherein marker 26 includes TLR4 or TYROBP; and wherein term 7 includes marker 27, marker 28, marker 29, and marker 30, wherein marker 27 includes SLAMF7 or CX3CR1, wherein marker 28 includes KLRC4 or CD8A, wherein marker 29 includes CD3D or LCK, and wherein marker 30 includes TMC8 or CCT2; and a processor communicatively coupled to the storage memory for determining a score with an interpretation function wherein the score is predictive of CAD in the subject.
Also described herein is a computer-readable storage medium storing computer-executable program code, the program code including: program code for storing a dataset associated with a sample obtained from the subject, wherein the first dataset includes quantitative expression data for at least one marker set selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7; wherein term 1 includes marker 1, marker 2, and marker 3, wherein marker 1 includes AF161365, wherein marker 2 includes HNRPF or ACBD5, and wherein marker 3 includes TFCP2 or DDX18; wherein term 2 includes marker 4, marker 5, and marker 6, wherein marker 4 includes AF289562 or CD248, wherein marker 5 includes HNRPF or ACBD5, and wherein marker 6 includes TFCP2 or DDX18; wherein term 3 includes marker 7, marker 8, marker 9, and marker 10 wherein marker 7 includes CD79B or CD19, wherein marker 8 includes SPIB or BLK, wherein marker 9 includes CD3D or LCK, and wherein marker 10 includes TMC8 or CCT2; wherein term 4 includes marker 11, marker 12, marker 13, and marker 14, wherein marker 11 includes S100A12 or MMP9, wherein marker 12 includes CLEC4E or ALOX5AP, wherein marker 13 includes S100A8 or NAMPT, and wherein marker 14 includes RPL28 or SSRP1; wherein term 5 includes marker 15, marker 16, marker 17, marker 18, and marker 19, wherein marker 15 includes S100A12 or MMP9, wherein marker 16 includes CLEC4E or ALOX5AP, wherein marker 17 includes S100A8 or NAMPT, wherein marker 18 includes AQP9 or GLT1D1, and wherein marker 19 includes NCF4 or NCF2; wherein term 6 includes marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, wherein marker 20 includes CASP5 or H3F3B, wherein marker 21 includes IL18RAP or TXN, wherein marker 22 includes TNFAIP6 or PLAUR, wherein marker 23 includes IL8RB or BCL2A1, wherein marker 24 includes TNFRSF10C or PTAFR, wherein marker 25 includes KCNE3 or LAMP2, and wherein marker 26 includes TLR4 or TYROBP; and wherein term 7 includes marker 27, marker 28, marker 29, and marker 30, wherein marker 27 includes SLAMF7 or CX3CR1, wherein marker 28 includes KLRC4 or CD8A, wherein marker 29 includes CD3D or LCK, and wherein marker 30 includes TMC8 or CCT2; and program code for determining a score with an interpretation function wherein the score is predictive of CAD in the subject.
Also described herein is a method for predicting CAD in a subject, including: obtaining a sample from the subject, wherein the sample includes a plurality of analytes; contacting the sample with a reagent; generating a plurality of complexes between the reagent and the plurality of analytes; detecting the plurality of complexes to obtain a dataset associated with the sample, wherein the first dataset includes quantitative expression data for at least one marker set selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7; wherein term 1 includes marker 1, marker 2, and marker 3, wherein marker 1 includes AF161365, wherein marker 2 includes HNRPF or ACBD5, and wherein marker 3 includes TFCP2 or DDX18; wherein term 2 includes marker 4, marker 5, and marker 6, wherein marker 4 includes AF289562 or CD248, wherein marker 5 includes HNRPF or ACBD5, and wherein marker 6 includes TFCP2 or DDX18; wherein term 3 includes marker 7, marker 8, marker 9, and marker 10 wherein marker 7 includes CD79B or CD19, wherein marker 8 includes SPIB or BLK, wherein marker 9 includes CD3D or LCK, and wherein marker 10 includes TMC8 or CCT2; wherein term 4 includes marker 11, marker 12, marker 13, and marker 14, wherein marker 11 includes S100A12 or MMP9, wherein marker 12 includes CLEC4E or ALOX5AP, wherein marker 13 includes S100A8 or NAMPT, and wherein marker 14 includes RPL28 or SSRP1; wherein term 5 includes marker 15, marker 16, marker 17, marker 18, and marker 19, wherein marker 15 includes S100A12 or MMP9, wherein marker 16 includes CLEC4E or ALOX5AP, wherein marker 17 includes S100A8 or NAMPT, wherein marker 18 includes AQP9 or GLT1D1, and wherein marker 19 includes NCF4 or NCF2; wherein term 6 includes marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, wherein marker 20 includes CASP5 or H3F3B, wherein marker 21 includes IL18RAP or TXN, wherein marker 22 includes TNFAIP6 or PLAUR, wherein marker 23 includes IL8RB or BCL2A1, wherein marker 24 includes TNFRSF10C or PTAFR, wherein marker 25 includes KCNE3 or LAMP2, and wherein marker 26 includes TLR4 or TYROBP; and wherein term 7 includes marker 27, marker 28, marker 29, and marker 30, wherein marker 27 includes SLAMF7 or CX3CR1, wherein marker 28 includes KLRC4 or CD8A, wherein marker 29 includes CD3D or LCK, and wherein marker 30 includes TMC8 or CCT2; and determining a score from the dataset using an interpretation function, wherein the score is predictive of CAD in the subject.
Also described herein is a kit for predicting CAD in a subject, including: a set of reagents including a plurality of reagents for determining from a sample obtained from the subject quantitative expression data for at least one marker set selected from the group consisting of the marker sets in term 1, term 2, term 3, term 4, term 5, term 6, and term 7; wherein term 1 includes marker 1, marker 2, and marker 3, wherein marker 1 includes AF161365, wherein marker 2 includes HNRPF or ACBD5, and wherein marker 3 includes TFCP2 or DDX18; wherein term 2 includes marker 4, marker 5, and marker 6, wherein marker 4 includes AF289562 or CD248, wherein marker 5 includes HNRPF or ACBD5, and wherein marker 6 includes TFCP2 or DDX18; wherein term 3 includes marker 7, marker 8, marker 9, and marker 10 wherein marker 7 includes CD79B or CD19, wherein marker 8 includes SPIB or BLK, wherein marker 9 includes CD3D or LCK, and wherein marker 10 includes TMC8 or CCT2; wherein term 4 includes marker 11, marker 12, marker 13, and marker 14, wherein marker 11 includes S100A12 or MMP9, wherein marker 12 includes CLEC4E or ALOX5AP, wherein marker 13 includes S100A8 or NAMPT, and wherein marker 14 includes RPL28 or SSRP1; wherein term 5 includes marker 15, marker 16, marker 17, marker 18, and marker 19, wherein marker 15 includes S100A12 or MMP9, wherein marker 16 includes CLEC4E or ALOX5AP, wherein marker 17 includes S100A8 or NAMPT, wherein marker 18 includes AQP9 or GLT1D1, and wherein marker 19 includes NCF4 or NCF2; wherein term 6 includes marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, wherein marker 20 includes CASP5 or H3F3B, wherein marker 21 includes IL18RAP or TXN, wherein marker 22 includes TNFAIP6 or PLAUR, wherein marker 23 includes IL8RB or BCL2A1, wherein marker 24 includes TNFRSF10C or PTAFR, wherein marker 25 includes KCNE3 or LAMP2, and wherein marker 26 includes TLR4 or TYROBP; and wherein term 7 includes marker 27, marker 28, marker 29, and marker 30, wherein marker 27 includes SLAMF7 or CX3CR1, wherein marker 28 includes KLRC4 or CD8A, wherein marker 29 includes CD3D or LCK, and wherein marker 30 includes TMC8 or CCT2; and instructions for using the plurality of reagents to determine quantitative data from the sample, wherein the instructions include instructions for determining a score from the dataset wherein the score is predictive of CAD in the subject.
In an embodiment, the instructions include instructions for conducting a microarray assay. In an embodiment, the instructions include instructions for conducting a polymerase chain reaction assay.
Also described herein is a system for predicting CAD in a subject, the system including: a storage memory for storing a dataset associated with a sample obtained from the subject, wherein the dataset includes quantitative expression data for at least two markers selected from the group consisting of AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, and CD8A; and a processor communicatively coupled to the storage memory for determining a score with an interpretation function wherein the score is predictive of CAD in the subject.
Also described herein is a computer-readable storage medium storing computer-executable program code, the program code including: program code for storing a dataset associated with a sample obtained from the subject, wherein the dataset includes quantitative expression data for at least two markers selected from the group consisting of AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, and CD8A; and program code for determining a score with an interpretation function wherein the score is predictive of CAD in the subject.
Also described herein is a method for predicting CAD in a subject, including: obtaining a sample from the subject, wherein the sample includes a plurality of analytes; contacting the sample with a reagent; generating a plurality of complexes between the reagent and the plurality of analytes; detecting the plurality of complexes to obtain a dataset associated with the sample, wherein the dataset includes quantitative expression data for at least two markers selected from the group consisting of AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, and CD8A; and determining a score from the dataset using an interpretation function, wherein the score is predictive of CAD in the subject.
Also described herein is a kit for predicting CAD in a subject, including: a set of reagents including a plurality of reagents for determining from a sample obtained from the subject quantitative expression data for at least two markers selected from the group consisting of AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, and CD8A; and instructions for using the plurality of reagents to determine quantitative data from the sample, wherein the instructions include instructions for determining a score from the dataset, wherein the score is predictive of CAD in the subject.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
FIG. 1—Gene Discovery, Algorithm Development, and Validation Patient and Logic Flow Schematic. Initial gene discovery (CATHGEN repository) included both diabetic and non-diabetic patients. Gene discovery from Personalized Risk Evaluation and Diagnosis in the Coronary Tree (PREDICT) involved non-diabetic patients in a paired microarray analysis, that yielded 655 significant genes in common with those from the CATHGEN arrays. For RT-PCR 113 genes were selected and tested on 640 PREDICT patient samples, from which the final algorithm was derived and locked, followed by validation in the PREDICT validation cohort (N=526).
FIG. 2—RT-PCR Analysis of Diabetics vs Non-diabetic Significant Genes from CATHGEN Microarray analysis. Significance of individual genes selected from the CATHGEN microarray cohort in non-diabetic (ND) and diabetic (D) patients is shown. The sex/age adjusted p values from a CAD logistic regression analysis in each subset are plotted (log scale). Significant p values (<0.05) are indicated in red with gene symbols (upper left quadrant and lower right quadrant), non-significant ones in black (upper right quadrant).
FIG. 3—Venn Diagram of microarray, RT-PCR, and algorithm gene sources. A total of 7718 genes were identified, 2438 and 5935, respectively, from the CATHGEN and PREDICT microarray analyses, with an intersection of 655 genes. For the 113 RT-PCR genes, 52 were from PREDICT, 22 from CATHGEN, and 29 from both; 10 were either normalization genes or from previous studies.7 The final algorithm contained 20 informative genes: 10 from both microarray studies, 8 PREDICT alone, and 2 CATHGEN alone.
FIG. 4—Correlation of PCR gene expression to lymphocyte fraction (y-axis) and neutrophil fraction (x-axis) for the 113 PCR genes measured in the PREDICT algorithm development cohort. The range of correlation is up to 0.6 and a total of 42 genes were correlated with neutrophil fraction at >0.2 whereas 39 genes were correlated with lymphocyte count at the same threshold. Genes are identified using the numbering scheme in Table 2.
FIG. 5—Schematic of the Algorithm Structure and Genes. The algorithm consists of overlapping gene expression functions for males and females with a sex-specific linear age function for the former and a non-linear age function for the latter. For the gene expression components, 16/23 genes in 4 terms are gender independent: Term 1—neutrophil activation and apoptosis, Term 3—NK cell activation to T cell ratio, Term 4, B to T cell ratio, and Term 5—AF289562 expression normalized to TFCP2 and HNRPF. In addition, Term 2 consists of 3 sex-independent neutrophil/innate immunity genes (S100A8, S100A12, CLEC4E) normalized to overall neutrophil gene expression (AQP9, NCF4) for females and to RPL28 (lymphocytes) for males. The final male specific term is the normalized expression of TSPAN16. Algorithm score is defined as 1.821−0.755*Term1−0.406*Term3−0.308*Term2*Sex−0.137*Term4−0.548*Term2*(1−Sex)−0.246*Term5−0.481*Term6*Sex+0.851*Sex+0.045*Sex*Age+0.123*(1−Sex)*max(0,Age-55), where Sex is a 0/1 indicator of sex (0=female, 1=male) and age is in years, and is calculated as described (Methods Section below).
FIG. 6—Comparison of Algorithm Performance between Cross-Validation Estimate and Independent Validation. ROC curves of the cross-validation (dashed line) and independent validation (solid line) of the algorithm is shown relative to an AUC of 0.50 (dotted line). The 95% confidence intervals are indicated by the solid areas. The AUC values are: for cross-validation 0.77 (95% CI, 0.73-0.81) and for the independent validation cohort 0.70 (95% CI, 0.65-0.75, p=10−16).
FIG. 7—Allocation of Patients from the PREDICT trial for algorithm development and validation. From a total of 1569 subjects meeting the study inclusion/exclusion criteria 226 were used for gene discovery. The remaining 1343 were divided into independent cohorts for algorithm development (694) and validation (649) as shown; 94% of patients in these cohorts came from the same centers. For algorithm development a total of 640 patient samples were used; 54 were excluded due to incomplete data (Diamond G A, Forrester J S. Analysis of probability as an aid in the clinical diagnosis of coronary-artery disease. N Engl J Med. 1979; 300(24):1350-8.), inadequate blood volume (Stangl V, Witzel V, Baumann G, Stangl K. Current diagnostic concepts to detect coronary artery disease in women. Eur Heart J. 2008; 29(6):707-17.), sex mismatch between experimental and clinical records (Gibbons R J, Abrams J, Chatterjee K, et al. ACC/AHA 2002 guideline update for the management of patients with chronic stable angina—summary article: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines (Committee on the Management of Patients With Chronic Stable Angina). J Am Coll Cardiol. 2003; 41(1):159-68.), or statistical outlier assessment (Cook N R, Ridker P M. Advances in measuring the effect of individual predictors of cardiovascular risk: the role of reclassification measures. Ann Intern Med. 2009; 150(11):795-802.). For the validation cohort a total of 123 samples were excluded based on: inadequate blood volume or RNA yield (43), significant contamination with genomic DNA (78), or prespecified statistical outlier assessment (2).
FIG. 8—The net benefit curve for a diagnostic as a function of pt, a threshold probability that represents the tradeoff between false positives and false negatives. The curve quantifies the net benefit to following the decision rule of score>pt=positive, over a range of possible value for pt. The reference lines reflect the net benefit of a) all subjects positive (lower curve) or b) all subjects negative (line at net benefit=0). The net benefit curve for the gene expression algorithm is shown as the top curve, and is greater than either reference line over clinically relevant range for pt.
FIG. 9—ROC analysis of Validation Cohort Performance For Algorithm and Clinical Variables. Algorithm performance adds to Clinical Factors by Diamond-Forrester. Comparison of the combination of D-F score and algorithm score (heavy solid line) to D-F score alone ( - - - ) in ROC analysis is shown. The AUC=0.50 line (light solid line) is shown for reference. A total of 525 of the 526 validation cohort patients had information available to calculate D-F scores. The AUCs for the two ROC curves are 0.721±0.023 and 0.663±0.025, p=0.003.
FIG. 10—Dependence of Algorithm Score on % Maximum Stenosis in the Validation Cohort. The extent of disease for each patient was quantified by QCA maximum % stenosis and grouped into 5 categories: no measurable disease, 1-24%, 25-49% in ≧1 vessel, 1 vessel ≧50%, and >1 vessel ≧50%. The average algorithm score for each group is illustrated; error bars correspond to 95% confidence intervals.



DETAILED DESCRIPTION
Definitions
In general, terms used in the claims and the specification are intended to be construed as having the plain meaning understood by a person of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In case of conflict between the plain meaning and the provided definitions, the provided definitions are to be used.
The term “acute coronary syndrome” encompasses all forms of unstable coronary artery disease.
The term “coronary artery disease” or “CAD” encompasses all forms of atherosclerotic disease affecting the coronary arteries.
The term “Ct” refers to cycle threshold and is defined as the PCR cycle number where the fluorescent value is above a set threshold. Therefore, a low Ct value corresponds to a high level of expression, and a high Ct value corresponds to a low level of expression.
The term “Cp” refers to the crossing point and is defined as the intersection of the best fit of the log-linear portion of a standard's amplification curve in a real time PCR instrument such as, e.g., a LightCycler, and the noise band (set according to background fluorescence measurements).
The term “FDR” means to false discovery rate. FDR can be estimated by analyzing randomly-permuted datasets and tabulating the average number of genes at a given p-value threshold.
The terms “GL” “GM” and “GU” respectively refer to 1st percentile, median, and 99th percentile of Cp for that gene in the Algorithm Development data set.
The terms “marker” or “markers” encompass, without limitation, lipids, lipoproteins, proteins, cytokines, chemokines, growth factors, peptides, nucleic acids, genes, and oligonucleotides, together with their related complexes, metabolites, mutations, variants, polymorphisms, modifications, fragments, subunits, degradation products, elements, and other analytes or sample-derived measures. A marker can also include mutated proteins, mutated nucleic acids, variations in copy numbers, and/or transcript variants, in circumstances in which such mutations, variations in copy number and/or transcript variants are useful for generating a predictive model, or are useful in predictive models developed using related markers (e.g., non-mutated versions of the proteins or nucleic acids, alternative transcripts, etc.).
The terms “highly correlated gene expression” or “highly correlated marker expression” refer to gene or marker expression values that have a sufficient degree of correlation to allow their interchangeable use in a predictive model of coronary artery disease. For example, if gene x having expression value X is used to construct a predictive model, highly correlated gene y having expression value Y can be substituted into the predictive model in a straightforward way readily apparent to those having ordinary skill in the art and the benefit of the instant disclosure. Assuming an approximately linear relationship between the expression values of genes x and y such that Y=a+bX, then X can be substituted into the predictive model with (Y−a)/b. For non-linear correlations, similar mathematical transformations can be used that effectively convert the expression value of gene y into the corresponding expression value for gene x. The terms “highly correlated marker” or “highly correlated substitute marker” refer to markers that can be substituted into and/or added to a predictive model based on, e.g., the above criteria. A highly correlated marker can be used in at least two ways: (1) by substitution of the highly correlated marker(s) for the original marker(s) and generation of a new model for predicting CAD risk; or (2) by substitution of the highly correlated marker(s) for the original marker(s) in the existing model for predicting CAD risk.
The term “mammal” encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
The term “metagene” refers to a set of genes whose expression values are combined to generate a single value that can be used as a component in a predictive model. (Brunet, J. P., et al. Proc. Natl. Acad. Sciences 2004; 101(12):4164-9)
The term “myocardial infarction” refers to an ischemic myocardial necrosis. This is usually the result of abrupt reduction in coronary blood flow to a segment of the myocardium, the muscular tissue of the heart. Myocardial infarction can be classified into ST-elevation and non-ST elevation MI (also referred to as unstable angina). Myocardial necrosis results in either classification. Myocardial infarction, of either ST-elevation or non-ST elevation classification, is an unstable form of atherosclerotic cardiovascular disease.
The term “sample” can include a single cell or multiple cells or fragments of cells or an aliquot of body fluid, taken from a subject, by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage sample, scraping, surgical incision, or intervention or other means known in the art.
The term “subject” encompasses a cell, tissue, or organism, human or non-human, whether in vivo, ex vivo, or in vitro, male or female.
The term “obtaining a dataset associated with a sample” encompasses obtaining a set of data determined from at least one sample. Obtaining a dataset encompasses obtaining a sample, and processing the sample to experimentally determine the data. The phrase also encompasses receiving a set of data, e.g., from a third party that has processed the sample to experimentally determine the dataset. Additionally, the phrase encompasses mining data from at least one database or at least one publication or a combination of databases and publications. A dataset can be obtained by one of skill in the art via a variety of known ways including stored on a storage memory.
The term “clinical factor” refers to a measure of a condition of a subject, e.g., disease activity or severity. “Clinical factor” encompasses all markers of a subject's health status, including non-sample markers, and/or other characteristics of a subject, such as, without limitation, age and gender. A clinical factor can be a score, a value, or a set of values that can be obtained from evaluation of a sample (or population of samples) from a subject or a subject under a determined condition. A clinical factor can also be predicted by markers and/or other parameters such as gene expression surrogates.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
Methods
Markers and Clinical Factors
The quantity of one or more markers of the invention can be indicated as a value. A value can be one or more numerical values resulting from evaluation of a sample under a condition. The values can be obtained, for example, by experimentally obtaining measures from a sample by an assay performed in a laboratory, or alternatively, obtaining a dataset from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored, e.g., on a storage memory.
In an embodiment, the quantity of one or more markers can be one or more numerical values associated with expression levels of: AF161365, HNRPF, ACBD5, TFCP2, DDX18, AF289562, CD248, HNRPF, ACBD5, TFCP2, DDX18, CD79B, CD19, SPIB, BLK, CD3D, LCK, TMC8, CCT2, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, RPL28, SSRP1, S100A12, MMP9, CLEC4E, ALOX5AP, S100A8, NAMPT, AQP9, GLT1D1, NCF4, NCF2, CASP5, H3F3B, IL18RAP, TXN, TNFAIP6, PLAUR, IL8RB, BCL2A1, TNFRSF10C, PTAFR, KCNE3, LAMP2, TLR4, TYROBP, SLAMF7, CX3CR1, KLRC4, CD8A, CD3D, LCK, TMC8, or CCT2; resulting from evaluation of a sample under a condition. This nomenclature is used to refer to human genes in accordance with guidelines provided by the Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC). Further information about each human gene, such as accession number(s) and aliases, can be found by entering the gene name into the search page on the HGNC Search genenames.org website. For example, entering the term “CD3D” into the Simple Search field of the HGNC website on Jun. 1, 2010 returns the approved gene name of CD3D (CD3d molecule, delta (CD3-TCR complex)), the sequence accession IDs of CD3D (X01451; NM—000732), and the previous symbols of CD3D (T3D). Further human gene names are provided in the Examples section below.
In an embodiment, a condition can include one clinical factor or a plurality of clinical factors. In an embodiment, a clinical factor can be included within a dataset. A dataset can include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, or thirty or more overlapping or distinct clinical factor(s). A clinical factor can be, for example, the condition of a subject in the presence of a disease or in the absence of a disease. Alternatively, or in addition, a clinical factor can be the health status of a subject. Alternatively, or in addition, a clinical factor can be age, gender, chest pain type, neutrophil count, ethnicity, disease duration, diastolic blood pressure, systolic blood pressure, a family history parameter, a medical history parameter, a medical symptom parameter, height, weight, a body-mass index, resting heart rate, and smoker/non-smoker status. Clinical factors can include whether the subject has stable chest pain, whether the subject has typical angina, whether the subject has atypical angina, whether the subject has an anginal equivalent, whether the subject has been previously diagnosed with MI, whether the subject has had a revascularization procedure, whether the subject has diabetes, whether the subject has an inflammatory condition, whether the subject has an infectious condition, whether the subject is taking a steroid, whether the subject is taking an immunosuppressive agent, and/or whether the subject is taking a chemotherapeutic agent. Other examples of clinical factors are listed in the Tables and Figures.
In an embodiment, a marker's associated value can be included in a dataset associated with a sample obtained from a subject. A dataset can include the marker expression value of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, or thirty or more marker(s). For example, a dataset can include the expression values for AF161365, HNRPF, ACBD5; AF161365, HNRPF; or AF161365, ACBD5. Other combinations are described in more detail in the Examples section below.
In an embodiment, one or more markers can be divided into terms. Terms can include one marker, but generally include three or more markers. Terms can be included in a dataset associated with a sample obtained from a subject. The dataset can include one or more terms, two or more terms, three or more terms, four or more terms, five or more terms, six or more terms, seven or more terms, eight or more terms, nine or more terms, or ten or more terms. In an embodiment, a term can include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, or thirty or more marker(s). In an embodiment, the markers are divided into seven distinct terms: term 1, term 2, term 3, term 4, term 5, term 6, and term 7. In an embodiment, term 1 can include marker 1, marker 2, and marker 3, where marker 1 includes AF161365, where marker 2 includes HNRPF or ACBD5, and where marker 3 includes TFCP2 or DDX18. In an embodiment, term 2 can include marker 4, marker 5, and marker 6, where marker 4 includes AF289562 or CD248, where marker 5 includes HNRPF or ACBD5, and where marker 6 includes TFCP2 or DDX18. In an embodiment, term 3 can include marker 7, marker 8, marker 9, and marker 10 where marker 7 includes CD79B or CD19, where marker 8 includes SPIB or BLK, where marker 9 includes CD3D or LCK, and where marker 10 includes TMC8 or CCT2. In an embodiment, term 4 can include marker 11, marker 12, marker 13, and marker 14, where marker 11 includes S100A12 or MMP9, where marker 12 includes CLEC4E or ALOX5AP, where marker 13 includes S100A8 or NAMPT, and where marker 14 includes RPL28 or SSRP1. In an embodiment, term 5 can include marker 15, marker 16, marker 17, marker 18, and marker 19, where marker 15 includes S100A12 or MMP9, where marker 16 includes CLEC4E or ALOX5AP, where marker 17 includes S 100A8 or NAMPT, where marker 18 includes AQP9 or GLT1D1, and where marker 19 includes NCF4 or NCF2. In an embodiment, term 6 can include marker 20, marker 21, marker 22, marker 23, marker 24, marker 25, and marker 26, where marker 20 includes CASP5 or H3F3B, where marker 21 includes IL18RAP or TXN, where marker 22 includes TNFAIP6 or PLAUR, where marker 23 includes IL8RB or BCL2A1, where marker 24 includes TNFRSF10C or PTAFR, where marker 25 includes KCNE3 or LAMP2, and where marker 26 includes TLR4 or TYROBP. In an embodiment, term 7 can include marker 27, marker 28, marker 29, and marker 30, where marker 27 includes SLAMF7 or CX3CR1, where marker 28 includes KLRC4 or CD8A, where marker 29 includes CD3D or LCK, and where marker 30 includes TMC8 or CCT2.
In another embodiment, the invention includes obtaining a sample associated with a subject, where the sample includes one or more markers. The sample can be obtained by the subject or by a third party, e.g., a medical professional. Examples of medical professionals include physicians, emergency medical technicians, nurses, first responders, psychologists, medical physics personnel, nurse practitioners, surgeons, dentists, and any other obvious medical professional as would be known to one skilled in the art. A sample can include peripheral blood cells, isolated leukocytes, or RNA extracted from peripheral blood cells or isolated leukocytes. The sample can be obtained from any bodily fluid, for example, amniotic fluid, aqueous humor, bile, lymph, breast milk, interstitial fluid, blood, blood plasma, cerumen (earwax), Cowper's fluid (pre-ejaculatory fluid), chyle, chyme, female ejaculate, menses, mucus, saliva, urine, vomit, tears, vaginal lubrication, sweat, serum, semen, sebum, pus, pleural fluid, cerebrospinal fluid, synovial fluid, intracellular fluid, and vitreous humour. In an example, the sample is obtained by a blood draw, where the medical professional draws blood from a subject, such as by a syringe. The bodily fluid can then be tested to determine the value of one or more markers using an assay. The value of the one or more markers can then be evaluated by the same party that performed the assay using the methods of the invention or sent to a third party for evaluation using the methods of the invention.
Interpretation Functions
In an embodiment, an interpretation function can be a function produced by a predictive model. An interpretation function can also be produced by a plurality of predictive models. In an embodiment, an interpretation function can include terms Norm1, Norm2, NKup, Tcell, Bcell, Neut, Nup, Ndown, SCA1, AF2, TSPAN, SEX, and INTERCEPT. In a related embodiment, Norm1=RPL28, Norm2=(0.5*HNRPF+0.5*TFCP2), NKup=(0.5*SLAMF7+0.5*KLRC4), Tcell=(0.5*CD3D+0.5*TMC8), Bcell=(2⁄3*CD79B+1⁄3*SPIB), Neut=(0.5*AQP9+0.5*NCF4), Nup=(1⁄3*CASP5+1⁄3*IL18RAP+1⁄3*TNFAIP6), Ndown=(0.25*IL8RB+0.25*TNFRSF10C+0.25*TLR4+0.25*KCNE3), SCA1=(1⁄3*S100A12+1⁄3*CLEC4E+1⁄3*S100A8), AF2=AF289562, TSPAN=1 if (AF161365-Norm2>6.27 or AF161365=NoCall), 0 otherwise, SEX=1 for Males, 0 for Females. In a related embodiment, for Males, INTERCEPT=Intercept+SEX+MAGE*Age, with Age in years, and for Females, INTERCEPT=Intercept+OFAGE2*max(0,Age-60), with Age in years. In a related embodiment, coefficients Intercept=1.82120871, SEX=0.851181, OFAGE2=0.123283, MAGE=0.044868, TSPAN=−0.48182, AF2=—0.24592, Bcell=−0.13717, SCA1M=−0.30754, NeutF=−0.54778, Nupdown=−0.75514, and NK=−0.40579. In a related embodiment, a score is determined according to INTERCEPT−Nupdown*(Nup−Ndown)−NK*(NKup−Tcell)−SCA1M*SEX*(SCA1−Norm1)−Bcell*(Bcell−Tcell)−NeutF*(1−SEX)*(SCA1−Neut)−TSPANcoef*SEX*(TSPAN)−AF2*(AF2−Norm2). In an embodiment, an interpretation function can include any linear combination of age, gender (i.e., sex), and one or more terms.
In an embodiment, a predictive model can include a partial least squares model, a logistic regression model, a linear regression model, a linear discriminant analysis model, a ridge regression model, and a tree-based recursive partitioning model. In an embodiment, a predictive model can also include Support Vector Machines, quadratic discriminant analysis, or a LASSO regression model. See Elements of Statistical Learning, Springer 2003, Hastie, Tibshirani, Friedman; which is herein incorporated by reference in its entirety for all purposes. Predictive model performance can be characterized by an area under the curve (AUC). In an embodiment, predictive model performance is characterized by an AUC ranging from 0.68 to 0.70. In an embodiment, predictive model performance is characterized by an AUC ranging from 0.70 to 0.79. In an embodiment, predictive model performance is characterized by an AUC ranging from 0.80 to 0.89. In an embodiment, predictive model performance is characterized by an AUC ranging from 0.90 to 0.99.
Assays
Examples of assays for one or more markers include DNA assays, microarrays, polymerase chain reaction (PCR), RT-PCR, Southern blots, Northern blots, antibody-binding assays, enzyme-linked immunosorbent assays (ELISAs), flow cytometry, protein assays, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, immunoassays, including, by way of example, but not limitation, RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, or competitive immunoassays, immunoprecipitation, and the assays described in the Examples section below. The information from the assay can be quantitative and sent to a computer system of the invention. The information can also be qualitative, such as observing patterns or fluorescence, which can be translated into a quantitative measure by a user or automatically by a reader or computer system. In an embodiment, the subject can also provide information other than assay information to a computer system, such as race, height, weight, age, gender, eye color, hair color, family medical history and any other information that may be useful to a user, such as a clinical factor described above.
Informative Marker Groups
In addition to the specific, exemplary markers identified in this application by name, accession number, or sequence, included within the scope of the invention are all operable predictive models of CAD and methods for their use to score and optionally classify samples using expression values of variant sequences having at least 90% or at least 95% or at least 97% or greater identity to the exemplified sequences or that encode proteins having sequences with at least 90% or at least 95% or at least 97% or greater identity to those encoded by the exemplified genes or sequences. The percentage of sequence identity may be determined using algorithms well known to those of ordinary skill in the art, including, e.g., BLASTn, and BLASTp, as described in Stephen F. Altschul et al., J. Mol. Biol. 215:403-410 (1990) and available at the National Center for Biotechnology Information website maintained by the National Institutes of Health. As described below, in accordance with an embodiment of the present invention, are all operable predictive models and methods for their use in scoring and optionally classifying samples that use a marker expression measurement that is now known or later discovered to be highly correlated with the expression of an exemplary marker expression value in addition to or in lieu of that exemplary marker expression value. For the purposes of the present invention, such highly correlated genes are contemplated either to be within the literal scope of the claimed inventions or alternatively encompassed as equivalents to the exemplary markers. Identification of markers having expression values that are highly correlated to those of the exemplary markers, and their use as a component of a predictive model is well within the level of ordinary skill in the art. The Examples section below provides numerous examples of methods for identifying highly correlated markers and substituting them for algorithm markers in predictive models of CAD and methods for their use to score and optionally classify samples.
Computer Implementation
In one embodiment, a computer comprises at least one processor coupled to a chipset. Also coupled to the chipset are a memory, a storage device, a keyboard, a graphics adapter, a pointing device, and a network adapter. A display is coupled to the graphics adapter. In one embodiment, the functionality of the chipset is provided by a memory controller hub and an I/O controller hub. In another embodiment, the memory is coupled directly to the processor instead of the chipset.
The storage device is any device capable of holding data, like a hard drive, compact disk read-only memory (CD-ROM), DVD, or a solid-state memory device. The memory holds instructions and data used by the processor. The pointing device may be a mouse, track ball, or other type of pointing device, and is used in combination with the keyboard to input data into the computer system. The graphics adapter displays images and other information on the display. The network adapter couples the computer system to a local or wide area network.
As is known in the art, a computer can have different and/or other components than those described previously. In addition, the computer can lack certain components. Moreover, the storage device can be local and/or remote from the computer (such as embodied within a storage area network (SAN)).
As is known in the art, the computer is adapted to execute computer program modules for providing functionality described herein. As used herein, the term “module” refers to computer program logic utilized to provide the specified functionality. Thus, a module can be implemented in hardware, firmware, and/or software. In one embodiment, program modules are stored on the storage device, loaded into the memory, and executed by the processor.
The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
Embodiments of the entities described herein can include other and/or different modules than the ones described here. In addition, the functionality attributed to the modules can be performed by other or different modules in other embodiments. Moreover, this description occasionally omits the term “module” for purposes of clarity and convenience.
EXAMPLES
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).
Materials and Methods
General Study Design
The overall study design is shown in FIG. 1. This study had four distinct, consecutive phases. The PREDICT clinical trial registration information is available on the clinicaltrials.gov website at NCT00500617 on May 28, 2010.
Phase 1—Cathgen Discovery.
Phase 1 was Initial Gene Discovery from the Duke University CATHGEN registry, a retrospective blood repository.11 Briefly, 198 subjects (88 cases, 110 controls) from this repository were enrolled between August 2004 and November, 2005. Clinical inclusion and exclusion criteria were described previously and included both diabetic and non-diabetic patients.7 All CATHGEN patients gave written informed consent and the study protocol was approved by the Duke University IRB. Microarrays were performed to identify CAD sensitive genes, and a subset of genes was selected for RT-PCR replication. Given the phase I findings, only non-diabetic subjects were included subsequently.
Phase H—Predict Discovery.
Phase 2 was a prospective gene discovery phase with subjects from the PREDICT study, where 198 patients (99 case: control pairs, matched for age and sex) underwent microarray analysis to identify differentially expressed genes.
Phase III—Predict Development.
Phase 3 was prospective algorithm development with 640 patients (210 cases, 430 controls) to determine the inter-relationships between clinical factors, blood cell counts, gene expression, and CAD.
Phase IV—Predict Validation.
After Phase III was completed the locked algorithm was prospectively validated in an independent cohort of 526 patients (192 cases, 334 controls).
Subjects from PREDICT were eligible if they had a history of chest pain, suspected anginal-equivalent symptoms, or a high risk of CAD with no known prior MI, revascularization, or CAD. Detailed inclusion/exclusion criteria have been described.12 Diabetic status was defined by clinical identification, blood glucose (non-fasting ≧200 or fasting ≧126), rorhemoglobin A1c, (≧6.5), or diabetic medication prescription. Complete blood counts with differentials were obtained for all patients. PREDICT patients gave written informed consent, and the study protocol was approved by the Western Institutional Review Board.
Blood Collection, RNA Purification, and RT-PCR
Whole blood samples were collected in PAXgene® tubes prior to coronary angiography, according to the manufacturer's instructions, and then frozen at −20° C. For the CATHGEN samples RNA was purified as described (PreAnalytix, Franklin Lakes, N.J.), followed by quantitative analysis (Ribogreen, Molecular Probes, Eugene, Oreg.). For the PREDICT samples an automated method using the Agencourt RNAdvance system was employed. Microarray samples were labeled and hybridized to 41K Human Whole Genome Arrays (Agilent, PN #G4112A) using the manufacturer's protocol. For PREDICT microarrays all matched pairs were labeled and hybridized together to minimize microarray batch effects. Microarray data sets have been deposited in GEO (GSE 20686).
Amplicon design, cDNA synthesis, and RT-PCR were performed as previously described.7, 12 All PCR reactions were run in triplicate and median values used for analysis. The primers and probes are shown in the Informal Sequence Listing below. The primers and probe for marker CD3D were obtained commercially from Applied Biosystems, Inc. (Assay ID: Hs00174158_ml; Part No. 4331182).
Fractionation of Whole Blood Cells for Cell-Type Specific Gene Expression Measurements
Cell fractionation was performed on fresh blood collected in EDTA tubes. 120 ml blood pooled from 4 different donors was 1:1 diluted with 1×PBS. 15% of the blood was used for granulocyte isolation by density centrifugation and 85% of the blood was used for PBMC isolation prior to T cells, B cells, NK cells, and monocytes fractionation.
Peripheral Blood Mononuclear Cell (PBMC) Isolation
PBMC was isolated by density centrifugation. 20 ml diluted blood was layered on 20 ml Histopaque 1077 (Sigma Cat No. 10771) in 50 ml conical tubes and was centrifuged at room temperature for 30 min at 400×g. The PBMC layer was carefully aspirated into new tubes and washed with 1× phosphate-buffered saline (PBS) twice and centrifuged at 200×g for 10 min. The washed PBMC was re-suspended in cold bufferl (1×PBS, 0.1% BSA and 2 mMEDTA) and stored on ice. 5% of the cells were lysed in RLT buffer (Qiagen RNeasy Mini kit, Cat No. 74104) for pre-selection RNA isolation.
Granulocyte Isolation
Granulocytes (neutrophils, eosinophils, basophils) were purified by density centrifugation using two different density mediums. In 15 ml conical tube, 3 ml Hisopaque 1077 was layered on 3 ml Histopaque 1119 (Sigma Cat No. 11191) and 6 ml of the diluted blood was then layered on Histopaque 1077. The tube was centrifuged at room temperature (RT) for 30 min at 700×g. The granulocyte layer was then aspirated into a new tube and washed twice. The pellet was re-suspended in RLT buffer for granulocyte RNA isolation.
Positive Cell Isolation with Magnetic Beads
The subsequent cell types (T cells, B cells, natural killer (NK) cells, monocytes) were positively selected from PBMC used the following reagents and the recommended procedures.
CD8+ T cells—Dynal® CD8 positive isolation kit (Invitrogen Cat. No. 113.33D)
CD3+ T cells—Dynabeads® CD3 (Invitrogen Cat. No. 111.51D)
CD19+ B cells—Dynabeads® CD19 pan B (Invitrogen Cat. No. 111.43D)
CD14+ Monocytes—Dynabeads® CD14 (monocytes/macrophages) (Invitrogen Cat. No. 111.49D)
CD56+NK cells—Dynabeads® Pan Mouse IgG (Invitrogen Cat. No. 110.41) cross-linked with mouse anti-human CD56 antibodies (BD bioscience Cat No. 556325)
Briefly, PBMC were incubated with antibody-coupled magnetic beads at 4° C. for 20 min and washed 3 times with buffer 1 on the magnet. The selected cells were then re-suspended in RLT buffer for RNA isolation.
RNA Isolation
The RNA samples in RLT buffer were purified using the Qiagen RNeasy Mini kit following the manufacturer's instructions.
Coronary Angiographic Analysis and Case:Control Definition
All patients were clinically referred for angiography and angiograms were performed based on local, institutional protocols. For CATHGEN patients, clinical angiographic interpretation defined cases as ≧75% maximum stenosis in one major vessel or ≧50% in two vessels and controls as <25% stenosis in all major vessels.
For PREDICT patients, core laboratory QCA reads (Cardiovascular Research Foundation New York) were used for case: control classification. Cases had ≧50% stenosis in at least one major coronary vessel and controls <50% stenosis in all major vessels.
Correlation between Gene Expression and Cell Type Distributions
Correlations with complete blood counts and database gene expression analysis (SymAtlas) were used to identify highly cell-type selective genes. In addition, whole blood cell fractionation by density centrifugation or through positive antibody selection followed by RT-PCR was performed on specific cell fractions.
Statistical Methods
All statistical methods were performed using the R software package. The statistical methods used are described and referenced in greater detail below.
Array Normalization
Agilent processed signal values for array normalization were scaled to a trimmed mean of 100 and then log 2 transformed. Standard array QC metrics (percent present, pairwise correlation, and signal intensity) were used for quality assessment, resulting in 3 of 198 CATHGEN and 12 of 210 PREDICT samples being excluded.
Array Analysis
For the CATHGEN array, logistic regression (unadjusted and sex/age adjusted) was used to assess gene expression association with case: control status. For the PREDICT array, given the paired design, conditional logistic regression was used. False discovery rates were used to account for multiple comparisons. GOEAST was used to determine over-representation of Gene Ontology (GO) terms.13 
Gene Selection
Genes for RT-PCR were selected based on significance, fold-change, pathway analysis, and literature support. Hierarchical clustering based on gene: gene correlations ensured that RT-PCR genes represented multiple clusters. Normalization genes were selected based on low variance, moderate to high expression, and no significant association with case: control status, sex, age, or cell counts.
PCR Statistical Analysis
Clinical/demographic factors were assessed for CAD association using univariate and multivariate logistic regression. Gene expression association with CAD and other clinical/demographic factors was assessed by robust logistic regression (unadjusted and sex/age adjusted).7 
Algorithm Development and Validation
Hierarchical clustering was used to group genes using a correlation cutoff Clusters were reduced to meta-genes14 and normalization genes based on correlation structure, known biology, and cell count correlation. For meta-gene pairs with high correlation and opposite disease regulation, ratio terms (differences on the log scale) were defined. Meta-genes independently associated with outcome were selected by the LASSO method, with sex by meta-gene interactions allowed during variable selection.15 
The final algorithm was fit using Ridge regression16, where the outcome variable was case:control status and the predictors the LASSO-selected meta-genes and sex-specific age terms. Sex was a binary predictor, and age a linear predictor with separate slopes for males, females >60, and females <60. Gene expression term penalization was based on cross-validation and prior evidence. Model performance was estimated using leave-one-out cross-validation. Algorithm performance was validated in an independent patient cohort with ROC analysis as primary endpoint.
Algorithm Calculation and Transformation
Data Preprocessing and QC Steps

    
    
        1) Compute median of triplicate wells for each algorithm gene/sample
        
            a. If one well has a no call, take the median of the two remaining wells
            b. If two or three wells have a no call, the algorithm gene receives a no call for that sample
        
        
        2) If AF161365 (TSPAN16) receives a no call, impute the value of 38 as the median value for that gene.
        3) If any algorithm gene other than AF161365 receives a no call, the sample fails for Missing Gene Cp. None of the 640 samples in Algorithm Development would fail this metric.
        4) Compute the median of the algorithm gene SD's, excluding AF161365. If this value is greater than 0.15, the sample fails for High Replicate SD.
        5) For each algorithm gene i, floor the Cp value by replacing values less than GLi with GLi This value represents the 1st percentile of Cp for that gene in the Algorithm Development set.
        6) For each algorithm gene i, ceiling the Cp value by replacing values greater than GUi with GUi. This value represents the 99th percentile of Cp for that gene in the Algorithm Development set.
        7) For each algorithm gene i, compute the absolute value of the difference between its Cp value and GMi, where GMi represents the median Cp for that gene in the Algorithm Development set. Sum this value across the algorithm genes (excluding AF161365). If the sum is greater than 27.17, the sample fails for Expression Profile Out of Range. 27.17 represents the largest value of this metric within the Algorithm Development set.
    
    


In certain cases, an algorithm score will not be calculated for a subject. Reasons for this include low PAXgene® tube blood volume, lab QC failure, etc. The frequency of occurrence of these failures will be tabulated, though these subjects will not be included in the analysis set. Subjects with missing Diamond Forrester scores will not be included in the analysis set.
Algorithm Calculation

    
    
        1) Define Norm1=RPL28
        2) Define Norm2=(0.5*HNRPF+0.5*TFCP2)
        3) Define NKup=(0.5*SLAMF7+0.5*KLRC4)
        4) Define Tcell=(0.5*CD3D+0.5*TMC8)
        5) Define Bcell=(2⁄3*CD79B+1⁄3*SPIB)
        6) Define Neut=(0.5*AQP9+0.5*NCF4)
        7) Define Nup=(1⁄3*CASP5+1⁄3*IL18RAP+1⁄3*TNFAIP6)
        8) Define Ndown=(0.25*IL8RB+0.25*TNFRSF10C+0.25*TLR4+0.25*KCNE3)
        9) Define SCA1=(1⁄3*S100A12+1⁄3*CLEC4E+1⁄3*S100A8)
        10) Define AF2=AF289562
        11) Define TSPAN=1 if (AF161365-Norm2>6.27 or AF161365=NoCall), 0 otherwise
        12) Define SEX=1 for Males, 0 for Females
        13) Define Intercept
        
            a. For Males, INTERCEPT=2.672+0.0449*Age
            b. For Females, INTERCEPT=1.821+0.123*(Age-60), if negative set to 0
        
        
        14) Define Score=INTERCEPT−0.755*(Nup−Ndown)−0.406*(NKup−Tcell)−0.308*SEX*(SCA1−Norm1)−0.137*(Bcell−Tcell)−0.548*(1−SEX)*(SCA1−Neut)−0.482*SEX*(TSPAN)−0.246*(AF2−Norm2)
    
    


Score Transformation
The endpoint analyses defined were performed using raw algorithm scores. For clinical reporting purposes, as well as ease of presentation, raw scores may be transformed into a transformed score with a scale designed for ease of clinical use as follows:
Input is Raw Score
If Raw Score<−2.95, set RawScore=−2.95
If Raw Score>1.57, set RawScore=1.57
Raw Score=2.95+RawScore
Final Score=RawScore*40/4.52
Round Final Score up to nearest integer
If Final Score is greater than 40, set to 40
If Final Score is less than 1, set to 1
Value obtained is the Final Transformed Score
Estimation of Score Variability
A total of 41 replicate samples were tested from a large PAXgene® blood pool. The standard deviation of the raw score for these replicates was 0.13. The confidence interval around a given raw score was then the raw score plus or minus 1.96*0.13. The upper and lower bounds of this confidence interval were linearly transformed to the 0 to 40 scale, and then transformed to a confidence interval around the likelihood using the score to likelihood function described above.
Example 1
Demographic Data
Baseline demographic characteristics of the CATHGEN registry and PREDICT study patient cohorts are shown in Table 1. In general, CAD cases were more frequently men, older, had higher SBP, and more dyslipidemia.
Example 2
Phase I: Initial Gene Discovery (Cathgen)
A total of 2438 genes showed significant CAD association (p<0.05) in a 195 subject case:control analysis (FIG. 1). Clinical and demographic factor analysis of gene expression showed diabetes as the most significant (p=0.0006, Table 3). Based on statistical significance and biological relevance, 88 genes (Table 4) were selected for RT-PCR analysis on these same samples. CAD-gene expression analysis in non-diabetic and diabetic subsets (N=124 and 71, respectively), showed 42 and 12 significant genes, respectively (p<0.05), with no intersection (FIG. 2). Further work was thus limited to non-diabetics.
We observed a strong diabetes-gene expression interaction effect on CAD risk in the CATHGEN cohort, and thus restricted algorithm development to PREDICT non-diabetics. The CATHGEN diabetic subjects encompassed a range of disease severity and a variety of medications, some of which modulate gene expression and affect cardiovascular disease.17 
Example 3
Phase II: Non-Diabetic Gene Discovery (Predict)
Microarray CAD gene discovery on 210 PREDICT patient samples used a paired case:control experimental design, to reduce confounding effects of age, sex, and microarray batch processing. CAD analysis on the 99 case:control pairs after QC exclusions yielded 5935 significant genes (p<0.05) with 655 genes in common with the CATHGEN results (FIG. 3, Table 5).
Pathway Analysis of Discovery Genes
Gene Ontology (GO) analysis of these 655 genes identified 189 significant biological process terms (p<0.05, Table 6), largely reflecting inflammation, cellular and stress response, cell death, and apoptosis. The cellular and molecular ontologies showed enrichment of 32 and 49 terms respectively, including mitochondrial function, apoptotic protease activator activity, and antigen binding.
Gene Selection
A total of 113 genes (Table 2) were selected by statistical significance, biological relevance, and prior association with CAD and gene expression measured by RT-PCR in the PREDICT development cohort. Known cell-type specific markers, those correlated with cell counts in PREDICT, and candidate normalization genes, were also represented.
Example 4
Phase III: Prospective Algorithm Development (Predict)
The algorithm was derived using the RT-PCR and clinical data from the PREDICT development cohort. The most significant clinical factors for CAD:gene expression association were age, sex, chest pain type, and neutrophil count. Age and sex were independent risk factors for CAD (Table 1) and showed significant gene expression correlation. Chest pain type was also a significant independent risk factor (p=0.005), but was gene expression independent. Neutrophil count was significantly correlated (positively or negatively) to expression of 93 of 113 RT-PCR genes, and was significantly associated with CAD in males (p=0.049), but not females (p=0.77). Gene expression correlations for all genes to neutrophil and lymphocyte fraction were computed (FIG. 4). A correlation cut-off of >0.2 yielded 39 genes as lymphocyte-associated and 42 genes as neutrophil-associated. Neutrophil-associated genes showed both up and down regulation with CAD status, whereas lymphocyte-associated genes were generally down-regulated. There was significant gender-specific regulation of neutrophil correlated genes (males 40/42 genes up-regulated, females, 41/42 down-regulated) whereas lymphocyte gene down-regulation was gender independent.
Hierarchical clustering of the 113 PCR genes resulted in 18 correlated clusters (Table 2), with finer correlation substructure within the lymphocyte and neutrophil associated genes. There were 3 lymphocyte subgroups representing T-cells (clusters 1,2,3), B-cells (cluster 3), and NK cells (cluster 12). Three neutrophil subgroups were also identified: previously described neutrophil genes (IL8RB, S100A8, S100A12, TXN, BCL2A1; cluster 13, 16); newly identified up-regulated neutrophil genes (CLEC4E, CASP5, TNFAIP6; cluster 16) and down-regulated neutrophil genes (KCNE3, TLR4, TNFRSF10C; clusters 13, 14).7 The 29 genes in clusters 4-11 did not have clear cell-type association.
Algorithm Derivation
Based on the correlation and cell-type analyses, 15 meta-genes and 3 normalization genes were defined as inputs for model variable selection. Selection by the LASSO method, and weight penalization by Ridge regression resulted in the final, locked algorithm, comprising 20 CAD-associated genes and 3 normalization genes in 6 meta-genes (FIG. 5). The algorithm score was defined as the predicted regression model value.
Summary
The PCR algorithm development set was sufficiently powered to investigate the relationship between CAD, clinical factors, and gene expression. The most significant independent clinical risk factors for CAD were age, gender, and chest pain type, the components of the Diamond-Forrester risk model for CAD likelihood,1 supporting its use as a reference to assess algorithm performance.12 
The relationships between age, gender, CAD, and gene expression are complex. Increasing age and male gender are well-known risk-factors for CAD which affects gene expression in circulating cells.18, 19 The majority of genes measured by RT-PCR in this study correlated with lymphocyte or neutrophil fraction (FIG. 4; r>0.2 for 39 and 42 genes respectively). Genes in the neutrophil-associated group include many we previously identified (clusters 6,13,14; Table 2).7 Lymphocyte group genes include those known to be expressed in T-cells (CD3, TMC8), B-cells (SPIB, CD79B), and NK-cells (SLAMF7, KLRC4) (Clusters 1,3, and 12, respectively). Lymphocyte-associated gene expression decreases with CAD in a gender-independent fashion, consistent with decreased lymphocyte counts being correlated with increased cardiovascular risk.8 In contrast, neutrophil-associated genes display significant sex-specific expression differences with CAD: in males 95% of the neutrophil genes were up-regulated whereas 98% were down-regulated in females, consistent with increased granulocyte counts in males being associated with higher CAD risk, with smaller effects in females.20 
Biological Significance of Algorithm Terms
The use of correlated meta-genes as building blocks for the algorithm is significantly reflective of gene expression cell-type specificity. The algorithm genes are expressed selectively in multiple types of circulating cells including neutrophils, NK cells, B and T-lymphocytes21, supporting roles for both adaptive and innate immune responses in atherosclerosis.4 
Algorithm term 1 genes (FIG. 5) preferentially expressed in neutrophils, may reflect neutrophil apoptosis, as caspase-5 is increased with CAD, whereas TNFRSF10C, an anti-apoptotic decoy receptor of TRAIL, is decreased.22 Term 2 genes up-regulated with CAD likely reflect both innate immune activation (S100A8 and S100A12),23 and a cellular necrosis response (CLEC4E).24 S100A8 and S100A12 are up-regulated in chronic inflammatory conditions, perhaps reflecting a more general pathophysiological signal, consistent with increased CAD in disorders such as rheumatoid arthritis.25, 26 
Term 2 is normalized in a gender specific manner. In males normalization to RPL28, which is strongly expressed in lymphocytes, reflects the neutrophil to lymphocyte ratio, which is prognostic for death or MI in a CAD population.8 In females normalization to AQP9 and NCF4, two CAD insensitive neutrophil genes, permits assessment of neutrophil up-regulation of the S100s and CLEC4E.
Term 3 consists of 2 NK cell receptors, SLAMF7 and KLRC4, normalized to T-cell specific genes (TMC8 and CD3D). SLAMF7 may specifically activate NK cell function, while inhibiting B and T cells.27 KLRC4 is also likely involved in NK cell activation.28 NK cells have been associated with atherosclerosis in both mouse models and humans, and reduced lymphocyte counts associated with cardiac events.8, 29 
Term 4 is a gene expression based measure of the B/T-cell ratio. The role of T cells is complex, whereas B cells have been shown in mouse models to be athero-protective.30, 31 In this study apparent up-regulation of B-cell specific genes is correlated with CAD, perhaps indicating an immunological response to disease. The last two terms, based on AF289562 (AF2) and TSPAN16 are genes of unknown function.
Example 5
Phase IV: Prospective Algorithm Validation (Predict)
The estimated cross-validated algorithm AUC in ROC analysis in the PREDICT development set was 0.77 (95% CI 0.73 to 0.81); prospective validation in the independent PREDICT validation set of 526 patients (192 cases, 334 controls) yielded an AUC of 0.70 (95% CI=0.65 to 0.75) (FIG. 6).
For algorithm development in Phases III and IV, we used a robust approach, which minimized the effect of any single gene, by using meta-genes as building blocks.14, 32 Penalized stepwise logistic regression (LASSO) selected significant meta-genes from a 640 patient data set which greatly exceeded the number of candidate variables (15 meta-genes), reducing the likelihood of over-fitting. Further, in order to minimize over-weighting of individual terms, meta-gene coefficients were penalized using Ridge regression.
The cross-validated model AUC was 0.77 (95% CI 0.73 to 0.81), suggesting the algorithm score was a significant CAD predictor, and the validation cohort AUC was 0.70, with overlapping confidence intervals (95% CI=0.65 to 0.75). This modest decrease may reflect an over-optimistic cross-validation estimate, as we did not re-select terms during each iteration.
Thus, using a series of microarray and RT-PCR data sets, comprising more than 1,500 patients, we have derived and validated an algorithm, consisting of the expression levels of 23 genes, sex, and age, which assesses the likelihood of obstructive CAD in non-diabetic patients.
Example 6
Summary of Above Examples
This study presents the development and validation of a whole blood derived RT-PCR based gene-expression algorithm for assessment of obstructive CAD likelihood in non-diabetic patients, and includes several key findings. First, gene expression patterns that differentiate diabetic patients with and without CAD were very different from those for study patients without diabetes. In the initial Gene Discovery Cohort, 2438 genes were differentially expressed in cases versus controls. In the second, PREDICT gene discovery cohort in non-diabetic patients, 5935 genes were differentially expressed and 655 overlapped with the initial gene discovery genes. Based on overall correlations and biological significance, 113 of these 655 genes, were selected for RT-PCR analysis in the independent algorithm development cohort (Phase III), which also identified relationships between clinical factors, cell counts, and gene expression. The algorithm, including 23 gene expression levels, age, and sex, was derived from these data and locked. It was then prospectively shown to have significant diagnostic accuracy in Phase IV, the prospective PREDICT validation cohort, with an AUC of 0.70 (95% CI=0.65 to 0.75; p=10−16).
We consider our results robust, due to at least two factors. First, we used a carefully designed, serial, four-phase study comprising >1,500 patients, with initial microarray-based gene discovery confirmed by quantitative RT-PCR measurements in independent patients. Second, we used QCA to define CAD cases and controls, yielding a more accurate gold standard.
Example 7
Removal of One Term from the Algorithm
In the following series of examples (7-47), we examined the sensitivity of the algorithm and the algorithm development process to differences in terms, markers, and statistical methods. Each example follows the same general procedure: 1) identify a plausible alternative model approach (e.g., fewer terms, alternate markers, etc.); 2) rebuild the algorithm based on that alternative approach, including re-weighting the terms and/or markers as appropriate; and 3) assess whether the new model retains significant predictive accuracy.
The ability of the algorithm to determine the likelihood of CAD in the absence of one out of the seven terms was assessed. A single term was removed sequentially from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, term 1 was removed from the algorithm while terms 2-7 and the clinical factors (age and gender) remained in the algorithm. The markers in terms 1-7 are shown in the table below. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. For example, when term 1 was removed from the algorithm the altered algorithm was as follows:
Algorithm Calculation (Ridge Regression; Removal of Term 1)

    
    
        1) Define Norm1=RPL28
        2) Define Norm2=(0.5*HNRPF+0.5*TFCP2)
    
    


3) Define NKup=(0.5*SLAMF7+0.5*KLRC4)

    
    
        4) Define Tcell=(0.5*CD3D+0.5*TMC8)
        5) Define Bcell=(2⁄3*CD79B+1⁄3*SPIB)
        6) Define Neut=(0.5*AQP9+0.5*NCF4)
        7) Define Nup=(1⁄3*CASP5+1⁄3*IL18RAP+1⁄3*TNFAIP6)
        8) Define Ndown=(0.25*IL8RB+0.25*TNFRSF10C+0.25*TLR4+0.25*KCNE3)
        9) Define SCA1=(1⁄3*MMP9+1⁄3*CLEC4E+1⁄3*S100A8)
        10) Define AF2=AF289562
        11) Define SEX=1 for Males, 0 for Females
        12) Define Intercept
        
            a. For Males, INTERCEPT=0.70+0.044*Age
            b. For Females, INTERCEPT=0.38+0.126*(Age-60), if negative set to 0
        
        
        13) Define Score=INTERCEPT−0.39*(Nup−Ndown)−0.26*(NKup−Tcell)−0.33*SEX*(SCA1−Norm1)−0.06*(Bcell−Tcell)−0.07*(1−SEX)*(SCA1−Neut)−0.26*(AF2−Norm2)
    
    


A similar algorithm development procedure was used for the sequential removal of the other terms in this example as well as examples below. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 7. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All six-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of one term.











Term
Markers







Term 1
AF161365, HNRPF, TFCP2


Term 2
AF289562, HNRPF, TFCP2


Term 3
CD79B, SPIB, CD3D, TMC8


Term 4
S100A12, CLEC4E, S100A8, RPL28


Term 5
S100A12, CLEC4E, S100A8, AQP9, NCF4


Term 6
CASP5, IL18RAP, TNFAIP6, IL8RB, TNFRSF10C, KCNE3,



TLR4


Term 7
SLAMF7, KLRC4, CD3D, TMC8









Example 8
Removal of Two Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of two out of the seven terms was assessed. Two distinct terms were removed from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, terms 6-7 were removed from the algorithm while terms 1-5 and the clinical factors remained in the algorithm. All possible five term combinations were assessed. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 9. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All five-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of two terms.
Example 9
Removal of Three Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of three out of the seven terms was assessed. Three distinct terms were removed from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, terms 5-7 were removed from the algorithm while terms 1-4 and the clinical factors remained in the algorithm. All possible four term combinations were assessed. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 10. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All four-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of three terms.
Example 10
Removal of Four Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of four out of the seven terms was assessed. Four distinct terms were removed from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, terms 4-7 were removed from the algorithm while terms 1-3 and the clinical factors remained in the algorithm. All possible three term combinations were assessed. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 11. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All three-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of four terms.
Example 11
Removal of Five Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of five out of the seven terms was assessed. Five distinct terms were removed from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, terms 3-7 were removed from the algorithm while terms 1-2 and the clinical factors remained in the algorithm. All possible two term combinations were assessed. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 12. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All two-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of five terms.
Example 12
Removal of Six Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of six out of the seven terms was assessed. Six distinct terms were removed from the algorithm while maintaining the other terms and the clinical factors of age and gender. For example, terms 2-7 were removed from the algorithm while term 1 and the clinical factors remained in the algorithm. Two statistical methods were used for the assessment: logistic regression and ridge regression. For all analyses, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for each of the calculations as well as the mean and standard deviation of the results are shown in Table 13. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. All one-term sets tested were significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of six terms.
Example 13
Removal of all Seven Terms from the Algorithm
The ability of the algorithm to determine the likelihood of CAD in the absence of seven out of the seven marker expression terms was assessed. Seven distinct terms were removed from the algorithm while maintaining the clinical factors of age and gender. Two statistical methods were used for the assessment: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric used. AUC was computed using cross validation. Summary statistics for the calculations are shown in Table 14. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 8. The age plus gender plus zero-marker expression term set tested was significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after removal of all seven marker expression terms. This indicates that the algorithm weighting of gender and age is superior to the weighting of clinical factors in the DF model.
Example 14
Replacement of S100A12 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above (See Table 1b and Table 2). For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, MMP9 was substituted for S100A12 in all relevant terms of the algorithm, here MMP9 was substituted for S100A12 in terms 4 and 5. For example, when S100A12 was replaced in the algorithm with MMP9, the altered algorithm was as follows:
Algorithm Calculation (Logistic Regression, Substitution of MMP9 for S100A12

    
    
        1) Define Normi=RPL28
        2) Define Norm2=(0.5*HNRPF+0.5*TFCP2)
        3) Define NKup=(0.5*SLAMF7+0.5*KLRC4)
        4) Define Tcell=(0.5*CD3D+0.5*TMC8)
        5) Define Bcell=(2⁄3*CD79B+1⁄3*SPIB)
        6) Define Neut=(0.5*AQP9+0.5*NCF4)
        7) Define Nup=(1⁄3*CASP5+1⁄3*IL18RAP+1⁄3*TNFAIP6)
        8) Define Ndown=(0.25*IL8RB+0.25*TNFRSF10C+0.25*TLR4+0.25*KCNE3)
        9) Define SCA1=(1⁄3*MMP9+1⁄3*CLEC4E+1⁄3*S100A8)
        10) Define AF2=AF289562
        11) Define TSPAN=1 if (AF161365-Norm2>6.27 or AF161365=NoCall), 0 otherwise
        12) Define SEX=1 for Males, 0 for Females
        13) Define Intercept
        
            a. For Males, INTERCEPT=5.28+0.047*Age
            b. For Females, INTERCEPT=4.44+0.120*(Age-60), if negative set to 0
        
        
        14) Define Score=INTERCEPT−1.05*(Nup−Ndown)−0.56*(NKup−Tcell)−0.35*SEX*(SCA1−Norm1)−0.30*(Bcell−Tcell)−0.89*(1−SEX)*(SCA1−Neut)−0.87*SEX*(TSPAN)−0.38*(AF2−Norm2)
    
    


A similar algorithm development procedure was used in examples below. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 15
Replacement of CLEC4E with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, ALOX5AP was substituted for CLEC4E in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 16
Replacement of S100A8 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, NAMPT was substituted for S100A8 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 17
Replacement of CASP5 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, H3F3B was substituted for CASP5 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 18
Replacement of IL18RAP with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, TXN was substituted for IL18RAP in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 19
Replacement of TNFAIP6 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, PLAUR was substituted for TNFAIP6 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 20
Replacement of AQP9 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, GLT1D1 was substituted for AQP9 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 21
Replacement of NCF4 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, NCF2 was substituted for NCF4 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 22
Replacement of CD3D with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, LCK was substituted for CD3D in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 23
Replacement of TMC8 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, CCT2 was substituted for TMC8 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 24
Replacement of CD79B with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, CD19 was substituted for CD79B in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 25
Replacement of SPIB with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, BLK was substituted for SPIB in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 26
Replacement of HNRPF with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, ACBD5 was substituted for HNRPF in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 27
Replacement of TFCP2 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, DDX18 was substituted for TFCP2 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 28
Replacement of RPL28 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, SSRP1 was substituted for RPL28 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 29
Replacement of AF289562 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, CD248 was substituted for AF289562 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 30
Replacement of SLAMF7 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, CX3 CR1 was substituted for SLAMF7 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 31
Replacement of KLRC4 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, CD8A was substituted for KLRC4 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 32
Replacement of IL8RB with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, BCL2A1 was substituted for IL8RB in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 33
Replacement of TNFRSF10C with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, PTAFR was substituted for TNFRSF10C in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 34
Replacement of KCNE3 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, LAMP2 was substituted for KCNE3 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 35
Replacement of TLR4 with a Highly Correlated Substitute Marker
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. The correlation value for this particular replacement is shown in Table 15. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that substituted one gene at a time. In this example, TYROBP was substituted for TLR4 in all relevant terms of the algorithm. Summary statistics for the calculations are shown in Table 15. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) were considered significantly better than the DF model. See Table 16 for DF AUC. The algorithm with the substitute marker remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of the algorithm marker with the highly correlated substitute marker.
Example 36
Random Replacement of Five Algorithm Markers with Five Distinct, highly correlated substitute markers
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. See Table 15 for the highly correlated substitute markers. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that randomly substituted five highly correlated markers at a time for five distinct algorithm markers. For the random marker substitutions, 100 iterations each were run and the mean and the standard deviation were calculated. Summary statistics for the calculations are shown in Table 16. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) are considered significantly better than the DF model. The algorithm with the substitute markers remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of five algorithm markers with five highly correlated substitute markers.
Example 37
Random Replacement of Ten Algorithm Markers with Ten Distinct, Highly Correlated Substitute Markers
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. See Table 15 for the highly correlated substitute markers. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that randomly substituted ten highly correlated markers at a time for ten distinct algorithm markers. For the random marker substitutions, 100 iterations each were run and the mean and the standard deviation were calculated. Summary statistics for the calculations are shown in Table 16. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) are considered significantly better than the DF model. The algorithm with the substitute markers remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of ten algorithm markers with ten highly correlated substitute markers.
Example 38
Random Replacement of Fifteen Algorithm Markers with Fifteen Distinct, Highly Correlated Substitute Markers
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. See Table 15 for the highly correlated substitute markers. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that randomly substituted fifteen highly correlated markers at a time for fifteen distinct algorithm markers. For the random marker substitutions, 100 iterations each were run and the mean and the standard deviation were calculated. Summary statistics for the calculations are shown in Table 16. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) are considered significantly better than the DF model. The algorithm with the substitute markers remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of fifteen algorithm markers with fifteen highly correlated substitute markers.
Example 39
Random Replacement of Twenty Algorithm Markers with Twenty Distinct, Highly Correlated Substitute Markers
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. See Table 15 for the highly correlated substitute markers. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that randomly substituted twenty highly correlated markers at a time for twenty distinct algorithm markers. For the random marker substitutions, 100 iterations each were run and the mean and the standard deviation were calculated. Summary statistics for the calculations are shown in Table 16. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) are considered significantly better than the DF model. The algorithm with the substitute markers remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of twenty algorithm markers with twenty highly correlated substitute markers.
Example 40
Random Replacement of all Algorithm Markers with Distinct, Highly Correlated Substitute Markers
For each algorithm marker, a highly correlated, non-algorithm substitute marker was identified from the Phase III PCR data set described above. For each marker, a Pearson correlation value between that marker and all other markers was computed and then we picked the substitute marker with maximal correlation to the algorithm marker of interest. This substitute was the marker with the highest correlation to the algorithm marker, subject to the restriction that a substitute marker was not used more than once in the terms of the algorithm. See Table 15 for the highly correlated substitute markers. Two statistical methods were used for the analysis: logistic regression and ridge regression. For the analysis, the area under the ROC curve (AUC) was the primary accuracy metric. AUC was computed using cross validation. Accuracy was computed for models that randomly substituted highly correlated markers at a time for all algorithm markers. For the random marker substitutions, 100 iterations each were run and the mean was calculated. Summary statistics for the calculations are shown in Table 16. AUC's greater than the upper bound of the confidence interval for the AUC of Diamond Forrester (DF) are considered significantly better than the DF model. The algorithm with the substitute markers remained significantly better than the DF model indicating that the algorithm remains predictive of the likelihood of CAD even after replacement of all algorithm markers with highly correlated substitute markers.
Example 41
Removal of Markers from Term 1
Term 1 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers.
We found that all reduced terms produced in this analysis remained predictive of CAD. See Table 17.
Example 42
Removal of Markers from Term 2
Term 2 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers.
We found that all reduced terms produced in this analysis remained predictive of CAD. See Table 18.
Example 43
Removal of Markers from Term 3
Term 3 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers. In addition, for the two marker reduced sets, the same process was repeated again where one correlated replacement marker was used along with one original algorithm marker.
We found that all reduced terms produced in this analysis remained predictive of CAD, except for: LCK/CCT2/CD19/BLK; LCK/CD19/BLK; CCT2/CD19/BLK; LCK/CCT2/CD 19; LCK/CD 19; CCT2/CD 19; CD3D/CD 19; LCK/CD 19; and CCT2/CD 19. See Table 19. TMC8/CD19 was predictive of CAD when AUC using Ridge regression was calculated, but not when AUC using Logistic Regression was calculated. See Table 19.
Example 44
Removal of Markers from Term 4
Term 4 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers.
We found that all reduced terms produced in this analysis remained predictive of CAD. See Table 20.
Example 45
Removal of Markers from Term 5
Term 5 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers. In addition, for the two marker reduced sets, the same process was repeated again where one correlated replacement marker was used along with one original algorithm marker.
We found that all reduced terms produced in this analysis remained predictive of CAD, except for: MMP9/ALOX5AP/GLT1D1/NCF2; MMP9/ALOX5AP/NAMPT/NCF2; MMP9/GLT1D1/NCF2; MMP9/ALOX5AP/NCF2; MMP9/NAMPT/NCF2; MMP9/GLT1D1; ALOX5AP/NCF2; MMP9/NCF2; ALOX5AP/AQP9; and ALOX5AP/NCF2. See Table 21. ALOX5AP/NCF4 was predictive of CAD when AUC using Ridge regression was calculated, but not when AUC using Logistic Regression was calculated. See Table 21.
Example 46
Removal of Markers from Term 6
Term 6 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers. In addition, for the two marker reduced sets, the same process was repeated again where one correlated replacement marker was used along with one original algorithm marker.
We found that all reduced terms produced in this analysis remained predictive of CAD, except for: H3F3B/TXN/BCL2A1/LAMP2/TYROBP; H3F3B/TXN/BCL2A1/LAMP2; H3F3B/TXN/BCL2A1/TYROBP; TXN/PLAUR/BCL2A 1/TYROBP; H3F3B/TXN/PLAUR/BCL2A1; H3F3B/BCL2A1/TYROBP; TXN/BCL2A1/TYROBP; H3F3B/TXN/BCL2A1; H3F3B/TXN/TYROBP; TXN/PLAUR/BCL2A1; TXN/PLAUR/BCL2A1; H3F3B/BCL2A1; H3F3B/TYROBP; TXN/BCL2A1; TXN/TYROBP; TXN/IL8RB; and TXN/TNFRSF10C. See Table 22.
Example 47
Removal of Markers from Term 7
Term 7 algorithm and highly correlated substitute markers were sequentially removed from the algorithm to determine whether the algorithm would remain predictive of the likelihood of CAD in their absence. All other terms and their associated markers were removed from the algorithm, thus in this analysis each term was considered on its own. Each term on the model is a delta term, with n_i markers on the left side of the delta term and m_i markers on the right side of the delta term. We examined two marker ‘reduced terms’ where only one of the n_i left-hand side markers and one of the possible m_i right-hand side markers was used in the term. There were thus n_i*m_i possible two marker reduced terms. We also examined ‘reduced terms’ produced by the sequential removal of markers from the full term for both the algorithm markers as well as the substitute markers.
For each of the reduced terms, models were fit including gender, age, and the reduced term, and cross-validated AUC's were estimated. These cross validated AUC's were compared to the AUC's from a model that included gender, age, and the full term. For each reduced term, we tested whether there was still a statistically significant predictive effect of the term, i.e., whether the decrease in AUC was sufficient to render the marker reduced set not beneficial in prediction of CAD. The same process was repeated for all reduced marker sets where correlated replacement markers were used in place of original algorithm markers. In addition, for the two marker reduced sets, the same process was repeated again where one correlated replacement marker was used along with one original algorithm marker.
We found that all reduced terms produced in this analysis remained predictive of CAD, except for: LCK/CCT2/CX3CR1/CD8A; LCK/CX3CR1/CD8A; CCT2/CX3CR1/CD8A; LCK/CCT2/CD8A; LCK/CD8A; CCT2/CD8A; TMC8/CD8A; and CD3D/CD8A. See Table 23.
Example 48
Validation of the Diagnostic Accuracy of the Algorithm for Assessment of CAD in Non-Diabetic Patients
Herein we report initial prospective validation of a gene expression algorithm for the likelihood of obstructive CAD, defined as one or more coronary atherosclerotic lesions causing ≧50% luminal diameter stenosis, in non-diabetic patients with suspected CAD.
Methods
General Study Design and Study Population
Subjects were enrolled in PREDICT, a 39 center (US) prospective study, between July 2007 and April 2009. The study was approved at the institutional review board at all participating centers and all patients gave written informed consent. Subjects referred for diagnostic coronary angiography were eligible if they had a history of chest pain, suspected anginal-equivalent symptoms, or a high risk of CAD, and no known prior myocardial infarction (MI), revascularization, or obstructive CAD. Subjects were ineligible if at catheterization, they had acute MI, high risk unstable angina, severe non-coronary heart disease (congestive heart failure, cardiomyopathy or valve disease), systemic infectious or inflammatory conditions, or were taking immunosuppressive or chemotherapeutic agents.
From 2418 enrolled subjects who met inclusion criteria, 606 diabetic patients were excluded, as this initial algorithm development and validation was focused on non-diabetics. Of the remaining 1812 patients, 237 had angiographic images unsuitable for QCA and 6 had unusable blood samples. For the remaining 1569 subjects, 226 were used in gene discovery (Elashoff M R, Wingrove J A, Beineke P, et al. Development of a Blood-based Gene Expression Algorithm for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients, submitted. Circulation: Cardiovascular Genetics. 2010); the remaining 1343 were divided into independent algorithm development and validation cohorts (FIG. 7) sequentially based on date of enrollment.
Clinical Evaluation and Quantitative Coronary Angiography
Pre-specified clinical data, including demographics, medications, clinical history and presentation, and MPI results were obtained by research study coordinators at study sites using standardized data collection methods and data were verified by independent study monitors.
Coronary angiograms were analyzed by computer-assisted QCA. Specifically, clinically-indicated coronary angiograms performed according to site protocols were digitized, de-identified and analyzed with a validated quantitative protocol at Cardiovascular Research Foundation, New York, N.Y. (Lansky A J, Popma J J. Qualitative and quantitative angiography Philadelphia, Pa.: Saunders; 1998 Text Book of Interventional Cardiology)). Trained technicians, blinded to clinical and gene expression data, visually identified all lesions >10% diameter stenosis (DS) in vessels with diameter >1.5 mm. Using the CMS Medis system, (Medis, version 7.1, Leiden, the Netherlands), technicians traced the vessel lumen across the lesion between the nearest proximal and distal non-diseased locations. The minimal lumen diameter (MLD), reference lumen diameter (RLD=average diameter of normal segments proximal and distal of lesion) and % DS (% DS=(1−MLD/RLD)×100) were then calculated.
The Diamond-Forrester (D-F) risk score, comprised of age, sex, and chest pain type, was prospectively chosen to evaluate the added value of the gene expression score to clinical factors (Diamond G A, Forrester J S. Analysis of probability as an aid in the clinical diagnosis of coronary-artery disease. N Engl J Med. 1979; 300(24):1350-8). D-F classifications of chest pain type (typical angina, atypical angina and non-anginal chest pain) were assigned based on subject interviews (Diamond G A, Forrester J S. Analysis of probability as an aid in the clinical diagnosis of coronary-artery disease. N Engl J Med. 1979; 300(24):1350-8), and D-F scores assigned (Chaitman B R, Bourassa M G, Davis K, et al. Angiographic prevalence of high-risk coronary artery disease in patient subsets (CASS). Circulation. 1981; 64(2):360-7). Subjects without chest pain symptoms were classified as non-anginal chest pain. MPIs were performed as clinically indicated, according to local protocols, and interpreted by local readers with access to clinical data but not gene expression or catheterization data. MPIs were defined as positive if ≧1 reversible or fixed defect consistent with obstructive CAD was reported. Indeterminate or intermediate defects were considered negative.
Obstructive CAD and Disease Group Definitions
Patients with obstructive CAD (N=192) were defined prospectively as subjects with 1 atherosclerotic plaque in a major coronary artery (1.5 mm lumen diameter) causing 50% luminal diameter stenosis by QCA; non-obstructive CAD (N=334) had no lesions >50%.
Blood Samples
Prior to coronary angiography, venous blood samples were collected in PAXgene® RNA-preservation tubes. Samples were treated according to manufacturer's instructions, then frozen at −20° C.
RNA Purification and RT-PCR
Automated RNA purification from whole blood samples using the Agencourt RNAdvance system, cDNA synthesis, and RT-PCR were performed as described (Elashoff M R, Wingrove J A, Beineke P, et al. Development of a Blood-based Gene Expression Algorithm for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients, submitted. Circulation: Cardiovascular Genetics. 2010.). All PCR reactions were run in triplicate and median values used for analysis. Genomic DNA contamination was detected by comparison of expression values for splice-junction spanning and intronic ADORA3 assays normalized to expression values of TFCP2 and HNRPF. The RPS4Y1 assay was run as confirmation of sex for all patients; patients were excluded if there was an apparent mismatch with clinical data. Sample QC metrics and pass-fail criteria were pre-defined and applied prior to evaluation of results as described (Elashoff M R, Wingrove J A, Beineke P, et al. Development of a Blood-based Gene Expression Algorithm for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients, submitted. Circulation: Cardiovascular Genetics. 2010.).
Statistical Methods
Analyses for Table 24 used SAS Version 9.1 (SAS Institute Inc, Cary, N.C., USA). All other analysis was performed using R Version 2.7 (R Foundation for Statistical Computing, Vienna, Austria). Unless otherwise specified, univariate comparisons for continuous variables were done by t-test and categorical variables by Chi-square test. All reported p-values are two-sided.
Gene Expression Algorithm Score
The algorithm was locked prior to the validation study. Raw algorithm scores were computed from median expression values for the 23 algorithm genes, age and sex as described and used in all statistical analyses; scores were linearly transformed to a 0-40 scale for ease of reporting.
ROC Estimation and AUC Comparison
ROC curves were estimated for the a) gene expression algorithm score, b) the D-F risk score, c) a combined model of algorithm score and D-F risk score, d) MPI, and e) a combined model of algorithm score and MPI. Standard methods (Newson R. Confidence intervals for rank statistics: Somers' D and extensions. Stata Journal. 2006; 6:309-334.) were used to estimate the empirical ROC curves and associated AUCs and AUC standard errors. The Z-test was used to test AUCs versus random (AUC=0.50).
Paired AUC comparisons: i) gene expression algorithm score plus D-F risk score vs D-F risk score, and ii) gene expression algorithm score plus MPI vs MPI; were performed by bootstrap. For each comparison, 10,000 bootstrap iterations were run, and the observed AUC difference computed. The median bootstrapped AUC difference was used to estimate the AUC difference, and the p-value estimated using the empirical distribution of bootstrapped AUC differences (i.e. the observed quantile for 0 AUC difference in the empirical distribution).
Logistic Regression
A series of logistic regression models were fit with disease status as the binary dependent variable, and compared using a likelihood ratio test between nested models. Comparisons were: i) gene expression algorithm score plus D-F risk score versus D-F risk score alone; ii) gene expression algorithm score plus MPI versus MPI alone; iii) gene expression algorithm score versus the demographic component of the gene expression algorithm score.
Correlation of Algorithm Score with Maximum Percent Stenosis
The correlation between algorithm score and percent maximum stenosis as continuous variables was assessed by linear regression. Stenosis values were grouped into five increasing categories (no measurable disease, 1-24%, 25-49% in ≧1 vessel, 1 vessel ≧50%, and >1 vessel ≧50%) and ANOVA was used to test for a linear trend in algorithm score across categories.
Reclassification of Disease Status
Gene expression algorithm score and D-F risk scores were defined as low (0% to <20%), intermediate (≧20%, <50%), and high risk (≧50%) obstructive CAD likelihoods. MPI results were classified as negative (no defect/possible fixed or reversible defect) or positive (fixed or reversible defect). For the D-F risk score analysis, a reclassified subject was defined as i) D-F intermediate risk to low or high algorithm score, ii) D-F high risk to algorithm low risk, or iii) D-F low risk to algorithm high. For the MPI analysis, a reclassified subject included i) MPI positive to low risk based on algorithm score, or ii) MPI negative to high risk based on algorithm score. Net reclassification improvement (NRI) of the gene expression algorithm score (and associated p-value) compared to either the D-F risk score or MPI was computed as described in (Pencina M J, D'Agostino R B, Sr., D'Agostino R B, Jr., Vasan R S. Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. Stat Med. 2008; 27(2):157-72; discussion 207-12.). NRI is a measure of reclassification clinical benefit, and is sensitive to both the fraction and accuracy of reclassification.
NRI Formula
NRI considers as positive reclassifications those patients whose classification moves in the ‘correct’ direction (disease subjects moving to a higher risk classification and non-disease subjects moving to a lower risk classification). Similarly, NRI considers as negative reclassifications those patients whose classification moves in the incorrect direction (disease subjects moving to a lower risk classification and non-disease subjects moving to a higher risk classification). The NRI formula is then the difference between the fraction of positive reclassifications and the fraction of negative reclassifications.

NRI=(pup,events−pdown,events)−(pup,nonevents−pdown,nonevents)

where:

    
    
        pup, events=# events moving up/# events
        pdown, events=# events moving down# events
        pup, nonevents=# nonevents moving up/# nonevents
        pdown,nonevents=#nonevents moving down/# nonevents for significance testing,

z=NRI/(ve+vne)1/2 

    
    


where:

    
    
        ve=(pup, events+pdown, events)/#events
        vne=(pup, nonevents+pdown, nonevents)/#nonevents
        (formulas from {Pencina et al., 2008})
    
    


Logistic Regression Analyses
D-F Risk Score Model














Model Term
Odds Ratio
95% CI
p-value
Model AIC







D-F risk score
1.018
1.012 to 1.023
<.001
652.53









Gene Expression Algorithm Score+D-F Risk Score














Model Term
Odds Ratio
95% CI
p-value
Model AIC



















D-F risk score
1.012
1.007 to 1.018
<.001



Gene
1.64
1.37 to 1.96
<.001
622.3


expression


algorithm


score









MPI Model














Model Term
Odds Ratio
95% CI
p-value
Model AIC







MPI
1.52
0.88 to 2.67
.14
388.53









Gene Expression Algorithm Score+MPI














Model Term
Odds Ratio
95% CI
p-value
Model AIC



















MPI
1.04
0.57 to 1.90
.90



Gene
1.85
1.45 to 2.37
<.001
362.15


expression


algorithm


score









Net Benefit Analysis
Vickers {Vickers et al., 2008} defines the net benefit curve for a diagnostic as a function of pt, a threshold probability that represents the tradeoff between false positives and false negatives. The curve quantifies the net benefit to following the decision rule of score>pt=positive, over a range of possible value for pt. The reference lines reflect the net benefit of a) all subjects positive (lower curve in FIG. 8) or b) all subjects negative (line at net benefit=0). The net benefit curve for the gene expression algorithm is the upper curve in FIG. 8, and is greater than either reference line over clinically relevant range for pt.
Full Clinical Model
Methods
To further assess the added value of the gene expression algorithm a ‘full’ clinical factor model was developed that incorporated the 11 clinical factors that showed univariate significance (p<0.05) between obstructive disease and no obstructive disease patients in the development set. The 11 factors were:

    
    
        sex
        age
        chest pain type
        race
        statin use
        aspirin use
        anti-platelet use
        ACE inhibitor use
        systolic blood pressure
        hypertension
        dyslipidemia
    
    


A logistic regression model was then fit using disease status as the dependent variable and these 11 factors as predictor variables. A subject's ‘full clinical model score’ was the subject's predicted value from this model.
Results
Results are reported for the validation set. The AUC of the full clinical model was 0.732, and the AUC for the gene expression algorithm plus the full clinical model was 0.745 (p=0.09). The nested logistic regression comparison of the gene expression algorithm plus the full clinical model versus the full clinical model alone gave a p-value of 0.014.
The NRI of the gene expression algorithm plus the full clinical model versus the full clinical model alone was 10% (p=0.02).
Discussion
The full clinical model evaluated here further supports the concept that the algorithm score adds to known or apparent clinical factors in the PREDICT population. This model suffers from the lack of independent validation, as has been done for the Diamond-Forrester formulation, hence it's role as primary comparator.
Statistical Outlier Assessment
Samples were classified as gene expression outliers based on the following criterion: Σ|gi−mi|>27, where gi is the expression value for the i'th gene, and mi is the median expression value for the i'th gene across the development set.
Results
A total of 1343 non-diabetic patients from the PREDICT trial, enrolled between July 2007 and April 2009, were sequentially allocated to independent development (N=694) and validation (N=649) sets. The limitation to non-diabetic patients was based on the significant differences observed in CAD classifier gene sets dependent on diabetic status (Elashoff M R, Wingrove J A, Beineke P, et al. Development of a Blood-based Gene Expression Algorithm for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients, submitted. Circulation: Cardiovascular Genetics. 2010.). The patient flow, set assignment, and exclusions are shown in FIG. 7. The demographic and clinical characteristics of these sets by disease status, after exclusions, are summarized in Table 24. The clinical characteristics of the development and validation sets were similar. Overall, subjects were 57% male, 37% had obstructive CAD and 26% had no detectable CAD. Significant clinical or demographic variables that were associated with obstructive CAD in both cohorts were increased age, male sex, chest pain type, elevated systolic blood pressure (all p<0.001), hypertension (p=0.001), and white ethnicity (p=0.015).
The gene expression algorithm was developed as described above, with obstructive CAD defined by QCA as ≧50% stenosis in ≧1 major coronary artery. This corresponds approximately to 65-70% stenosis based on clinical angiographic read. The 23 algorithm genes, grouped in the 6 terms, 4 sex-independent and 2 sex-specific, are shown schematically in the figures. The subsequent analyses are for the independent validation set only.
ROC Analysis
The prospectively defined primary endpoint was the area under the ROC curve for algorithm score prediction of disease status. The AUC was 0.70±0.02, (p<0.001) with independently significant performance in male (0.66) and female subsets (0.65) (p<0.001 for each). As a clinical comparator, we used the Diamond-Forrester (D-F) risk score, which was developed to quantify likelihood of current CAD and validated in a large cohort (Diamond G A, Forrester J S. Analysis of probability as an aid in the clinical diagnosis of coronary-artery disease. N Engl J Med. 1979; 300(24):1350-8; Chaitman B R, Bourassa M G, Davis K, et al. Angiographic prevalence of high-risk coronary artery disease in patient subsets (CASS). Circulation. 1981; 64(2):360-7.). ROC analysis showed a higher AUC for the combination of algorithm score and D-F risk score, compared to D-F risk score alone (AUC 0.72 versus 0.66, p=0.003, FIG. 9).
The most prevalent form of non-invasive imaging in PREDICT was MPI. In the validation set 310 patients had clinically-indicated MPIs performed, of which 72% were positive. Comparative ROC analysis showed an increased AUC for the combined algorithm score and MPI versus MPI alone (AUC 0.70 versus 0.54, p<0.001).
Sensitivity, Specificity
Sensitivity and specificity were determined at an algorithm score threshold of 14.75, corresponding to a disease likelihood of 20%, with 33% of patients having scores below this value. At this threshold, the sensitivity was 85% with a specificity of 43%, corresponding to negative and positive predictive values of 83% and 46%, respectively.
Regression Analysis
A series of nested logistic regression models (see methods) were used to assess the independent contribution of the algorithm score and other predictors. Algorithm score added to the D-F risk score (p<0.001), and to MPI (p<0.001), and the algorithm gene expression terms added (p=0.003) to the algorithm demographic terms (see methods).
Association with Disease Severity
The algorithm score was correlated with maximum percent stenosis (R=0.34, p<0.001), and the average algorithm score increased monotonically with increasing percent maximum stenosis (p<0.001, FIG. 10). The average scores for patients with and without obstructive CAD were 25 and 17, respectively.
Reclassification
Reclassification may be a more clinically relevant measure of a predictor's comparative performance than standard measures such as AUC (Cook N R, Ridker P M. Advances in measuring the effect of individual predictors of cardiovascular risk: the role of reclassification measures. Ann Intern Med. 2009; 150(11):795-802.). Tables 25A and 25B show reclassification results for the gene expression algorithm compared to D-F risk score and MPI. In this study the net reclassification improvement for the gene expression algorithm score compared to the D-F risk score was 20% (p<0.001), and to MPI was 21% (p<0.001).
In subjects with intermediate D-F risk scores, 78% (75/96) of patients were reclassified by the gene expression algorithm. Specifically, for the intermediate D-F group, 22% (21/96) were correctly and 8% ( 7/96) incorrectly reclassified as low risk; 27% (26/96) were correctly and 22% (21/96) incorrectly reclassified as high risk. An additional 38 D-F low risk subjects (15%) were reclassified as high risk (22 correctly, 16 incorrectly), and 28 D-F high risk subjects (16%) reclassified as low risk (22 correctly, 6 incorrectly). Overall, when reclassification errors occurred, they were to a higher risk category, consistent with the gene expression algorithm having a higher NPV than PPV.
Discussion
This study prospectively validates in non-diabetic patients a non-invasive test for obstructive CAD defined by QCA that is based on gene expression in circulating whole blood cells, age and gender. This study extends our previous work on correlation of gene expression changes in blood with CAD (Wingrove J A, Daniels S E, Sehnert A J, et al. Correlation of Peripheral-Blood Gene Expression With the Extent of Coronary Artery Stenosis. Circulation: Cardiovascular Genetics. 2008; 1(1):31-38.) to prospective validation of a classifier for non-diabetic patients with obstructive CAD by ROC analysis (Elashoff M R, Wingrove J A, Beineke P, et al. Development of a Blood-based Gene Expression Algorithm for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients, submitted. Circulation: Cardiovascular Genetics. 2010.). The test yields a numeric score (0-40) with higher scores corresponding to higher likelihood of obstructive CAD and higher maximum percent stenosis.
It has been suggested that reclassification of patient clinical risk or status, as captured by the NRI, may be a more appropriate measure than comparative ROC analysis for evaluating potential biomarkers (Pencina M J, D'Agostino R B, Sr., D'Agostino R B, Jr., Vasan R S. Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. Stat Med. 2008; 27(2):157-72; discussion 207-12; Cook N R, Ridker P M. Advances in measuring the effect of individual predictors of cardiovascular risk: the role of reclassification measures. Ann Intern Med. 2009; 150(11):795-802.). The gene expression algorithm score improves the accuracy of clinical CAD assessment as shown by an NRI of 20% relative to the D-F score. For the most prevalent non-invasive test, MPI, the NRI was 21%, although these results are likely confounded by the referral bias inherent in this angiographically referred population. Overall, independent of MPI result or D-F risk category, increasing gene expression score leads to monotonically increased risk of obstructive CAD (Table 25A,B).
This gene-expression test could have clinical advantages over current non-invasive CAD diagnostic modalities since it requires only a standard venous blood draw, and no need for radiation, intravenous contrast, or physiologic and pharmacologic stressors. One potential clinical benefit of improving non-invasive assessment of CAD is to reduce invasive diagnostic coronary angiograms in patients without obstructive CAD. In the validation cohort, for example, only 37% of patients undergoing invasive angiography had obstructive CAD and the rate was particularly low in women (26%). A similar overall rate of obstructive CAD on angiography for patients without prior known CAD in a very large registry was recently reported, with little sensitivity to the exact definition of obstructive CAD (Patel M R, Peterson E D, Dai D, et al. Low diagnostic yield of elective coronary angiography. N Engl J Med. 2010; 362(10):886-95.). The gene-expression test described here identified a low-likelihood (<20%) of obstructive CAD in 33% of patients referred for invasive angiography, although the majority of these patients were also at low risk by clinical factor analysis (Table 25A).
CONCLUSIONS
We describe the prospective multi-center validation of a peripheral blood-based gene expression test to determine the likelihood of obstructive CAD in non-diabetic patients as defined by invasive angiography. This test provides additional information to clinical factors and non-invasive imaging as measured by patient CAD status classification. Clinical use of this test may reduce further testing of patients with suspected CAD.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.
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TABLE 1a







Phase 1 and 11 Microarray Cohorts










Phase I - CATHGEN Microarray Cohort
Phase II - PREDICT Paired Microarray Cohort














Controls
Cases
p.
Controls
Cases
p.


Variable
(N = 108)
(N = 87)
value
(N = 99)
(N = 99)
value
















Sex (% Male)
55 (50.9%)
58 (66.7%)
0.039
75 (75.8%)
75 (75.8%)
0.868


Age (yrs)
55 ± 11
63 ± 10
<.001
55 ± 12
62 ± 11
<.001


Caucasian
56 (51.9%)
60 (69%)  
0.023
85 (85.9%)
92 (92.9%)
0.166


BMI
32 ± 7 
30 ± 6 
0.098
30 ± 7 
30 ± 6 
0.722


Current Smoker
41 (38%)  
45 (51.7%)
0.075
14 (14.1%)
25 (25.3%)
0.074


Systolic BP
144 ± 22 
153 ± 25 
0.007
132 ± 17 
138 ± 18 
0.009


Diastolic BP
83 ± 13
87 ± 15
0.077
82 ± 11
80 ± 12
0.271


Hypertension
67 (62%)  
65 (74.7%)
0.084
55 (55.6%)
65 (65.7%)
0.191


Dyslipidemia
55 (50.9%)
58 (66.7%)
0.039
50 (50.5%)
69 (69.7%)
0.009


Neutrophil Count
3.8 ± 1.2
  4 ± 1.3
0.392
3.9 ± 1.2
4.3 ± 1.5
0.037


Lymphocyte Count
1.8 ± 0.7
1.9 ± 0.7
0.87
 2 ± 0.7
1.9 ± 0.6
0.239
















TABLE 1b







Phase III and IV Algorithm Development and Validation Cohorts











Phase IV - PREDICT Algorithm



Phase III - PREDICT Algorithm
Validation Cohort












Development Cohort
Controls
Cases














Variable
Controls (N = 410)
Cases (N = 230)
p. value
(N = 334)
(N = 192)
p. value
















Sex (% Male)
193 (47.1%)
180 (78.3%)
<.001
165 (49.4%)
134 (69.8%)
<.001


Age (yrs)
57 ± 12
64 ± 11
<.001
57.7 ± 11.7
64.7 ± 9.8 
<.001


Caucasian
347 (84.6%)
210 (91.3%)
0.022
293 (87.7%)
181 (94.3%)
0.015


BMI
31 ± 8 
30 ± 6 
0.348
31.3 ± 7.0 
29.8 ± 5.5 
0.010


Current Smoker
 87 (21.2%)
 45 (19.6%)
0.693
 68 (20.4%)
 38 (19.8%)
0.703


Systolic BP
133 ± 18 
138 ± 18 
<.001
132 (18.1)   
140 (17.7)   
<.001


Diastolic BP
80 ± 12
80 ± 11
0.944
77.5 (10.9)    
79.2 (11.3)    
0.086


Hypertension
248 (60.5%)
167 (72.6%)
0.003
203 (60.8%)
142 (74.0%)
0.001


Dyslipidemia
225 (54.9%)
170 (73.9%)
<.001
208 (62.3%)
133 (69.3%)
0.110


Neutrophil Count
  4 ± 1.2
4.3 ± 1.4
0.054
4.0 ± 1.2
4.1 ± 1.3
0.171


Lymphocyte Count
  2 ± 0.6
1.9 ± 0.6
0.007
1.9 ± 0.6
1.9 ± 0.6
0.411





Microarray cohorts omit subjects whose array data was excluded based on QC analysis (3 CATHGEN, 12 PREDICT)













TABLE 2







Markers Evaluated by RT-PCR in the Algorithm Development Cohort












Marker
MicroArray


Metagene
Algorithm


Symbol
Evidence1
Cell-Type2
Cluster
Term
Term















DDX18
3

1.1




SSRP1
3

1.2


CCT2
3
2
1.3


RPL28
N
2
1.4
Norm
2b


XIST
2
1, 4, 5
1.5


RASSF7
3

1.6


PKD1
3

1.7


AGPAT5
3
2, 7
1.8


GLS
3

1.9


TMC8
3

1.10
 1
3b, 4b


RPS4Y1
2
3
1.11


KLF12
3
4
1.12


LCK
2, 3
3, 4, 8
1.13


CD3D
2, 3
3, 4, 8
1.14
 1
3b, 4b


AES
3

1.15


ZAP70
3
3, 4, 8
1.16


CD81
3
7, 8
1.17


QDPR
3
2, 5
1.18


FXN
2
2
1.19


CORO2A
3

1.20


TCEA1
3
7
1.21


KMO
3
5, 7
2.1


TLR7
3
5
2.2


RHOC
3

2.3


CX3CR1
3
6, 8
2.4


IL11RA
1, 2
3, 4
3.1


IL7R
1, 2, 3
3, 4, 8
3.2
 3


FAIM3
2, 3
3, 4, 7
3.3


TCF7
2, 3
3, 4, 8
3.4
 3


CD79B
2, 3
7
3.5
 2
4a


SPIB
2, 3
2, 5, 7
3.6
 2
4a


CD19
3
5, 7
3.7


BLK
3
5, 7
3.8


PI16
2

3.9


LRRN3
3
3, 4
3.10
 4


HNRNPF
N

4.1
Norm
5b, 6b


TFCP2
N

4.2
Norm
5b, 6b


ACBD5
3

4.3


DIAPH1
3

4.4


CD37
3
7
4.5


PLAGL2
3
1
4.6


SRA1
3

5.1


CD300A
2
8
5.2


ELMO2
3
5, 8
5.3


CD33
2
1, 6
6.1


CSPG2
1, 2

6.2


CAT
2
2, 5
6.3


NOD2
1, 3
1, 6
6.4


KCNMB1
2

6.5
 5


TCF7L2
3
1, 6, 8
6.6
 5


PDK4
3

6.7
 5


TBC1D8
3
1, 5, 6
6.8


NR4A1
3
5
7.1


CDKN1C
3
6, 8
7.2


C2
2

7.3


CLC
2
1, 2
8.1
 6


OLIG2
2

8.2


ADORA3
2

8.3
 6


MMD
1, 2, 3
7
9.1


HIST1H2AE
1, 3
4, 7
9.2
 7


AMFR
2

10.1


CD34
N
2
10.2


A_24_P128361
3

11.1
 8
5a


(AF289562)


CD248
2, 3
4
11.2


KLRC4
2
4, 8
12.1
 9
3a


TARP
2, 3
4, 8
12.2


CCR5
2
4, 5
12.3


CD8A
1
3, 4, 8
12.4


SLAMF7
2
5, 8
12.5
 9
3a


KLRC2
2
3, 4, 8
12.6


PRSS23
2
8
12.7


NCAM1
N
8
12.8


TNFRSF10C
3

13.1
11
1b


IL8RB
1, 3
1, 6, 8
13.2
11
1b


TLR4
3
1, 6
13.3
11
1b


NAMPT
3
1, 5, 6
13.4


AQP9
3
1, 6
13.5
10
2c


S100A8
1, 2, 3
1, 5, 6
13.6
12
2a


NCF4
2, 3
1, 6
13.7
10
2c


GLT1D1
1, 2, 3

13.8


TXN
2, 3
2, 5
13.9


GABARAPL1
3

13.10


SIRPB2
1, 3

13.11


TRPM6
3

13.12


CD93
1, 2, 3
1, 5, 6
13.13


ASPRV1
3

13.14


ALOX5AP
2, 3
5
13.15


BCL2A1
1, 2, 3
1, 6, 8
13.16


F11R
3

14.1


PTAFR
3
1, 6
14.2


H3F3B
3
7
14.3


TYROBP
2, 3
1, 6, 8
14.4


NCF2
3
1, 5, 6
14.5


KCNE3
2, 3
1, 6
14.6
11
1b


LAMP2
2, 3
1
14.7


PLAUR
3
1, 6
14.8


CD14
1
1, 5, 6
14.9


HK3
1, 2
1, 6, 8
14.10


IL18
1

14.11


RGS18
1, 2
1, 6
15.1


BMX
2, 3

16.1


MMP9
2, 3

16.2


S100A12
1, 2, 3
1, 5, 6
16.3
12
2a


CLEC4E
2, 3

16.4
12
2a


CLEC4D
2, 3
1, 6
16.5


CASP5
2, 3

16.6
13
1a


TNFAIP6
2, 3
1
16.7
13
1a


IL18RAP
1, 3
3, 4, 8
16.8
13
1a


ARG1
2, 3

17.1
14


HP
1
1, 2
17.2


CBS
2, 3

17.3
14


AF161365
3

17.4
15
6a


ALAS2
N

18.1





1Microarray Evidence: 1 = Wingrove et al, 2 = CATHGEN, 3 = PREDICT, N = normalization Marker


2Cell Type: 1 = CD33+, 2 = CD34+, 3 = CD4+, 4 = CD8+, 5 = Dendritic, 6 = CD14+, 7 = CD19+, 8 = CD56+













TABLE 3







Significance of Clinical Variables in


CATHGEN Marker discovery set










Clinical Variable
p-value














Diabetes
0.000560741



Anti Hypertensive Use
0.012462227



HDL
0.088459908



Neutrophil Count
0.129686671



Antidiabetic Use
0.140870844



LDL
0.146873756



Total Cholesterol
0.172382024



WBC Count
0.189994635



Lipid Lowering Agent Use
0.200078333



Triglycerides
0.207728761



Diastolic BP
0.21703689



Chest Pain
0.219704278



Monocyte Count
0.23769698



Platelet Count
0.238534146



Smoker
0.257352165



Lymphocyte Count
0.261169567



Anticoagulant Use
0.321044006



Anti Inflammatory Use
0.332101624



Antiplatelet Use
0.336359859



Statin Use
0.390097042



Calcium Channel Blocker Use
0.401676568



Sex
0.409669446



Postmenopausal
0.418849343



Alcohol Use
0.495208348



NSAID Use
0.536650232



ACE Inhibitor Use
0.687539195



Vasodilator Use
0.715979777



Systolic BP
0.716766737



Antiarrhythmic Use
0.763504492



Salicylates
0.805576705



Beta Blocker Use
0.819779733



Hypertension
0.834786056



Black
0.847458733



Age
0.984504316

















TABLE 4







RT-PCR Results on CATHGEN cohort Markers











Marker
Non-Diabetic p
Diabetic p















KLRG1
0.933635139
0.000313584



GZMK
0.176629393
0.002075813



CCR5
0.524551866
0.002796076



RPS4Y1
0.641924002
0.003924492



TUBB2A
0.905726045
0.012164059



TARP
0.855579011
0.013579949



IGHA1
0.427023322
0.015653596



CACNA2D2
0.579670417
0.021884775



ADRB2
0.14583996
0.035331896



DB097529
0.739638806
0.037474362



CB853344
0.924313185
0.042530621



RHOH
0.914493918
0.045421079



GPR114
0.113792718
0.082926442



RPS27A
0.127518837
0.085484803



CD3E
0.114159341
0.090230797



RELA
0.800147639
0.124184492



HDC
0.611947115
0.124749411



NR1D1
0.08855384
0.140309177



RRN3
0.883475152
0.14306721



MARCO
0.000742446
0.162858627



ARL17P1
0.009929764
0.163503477



POLR2L
0.110001621
0.169570816



RPL10A
0.372025559
0.176554229



TLR5
5.31034E−05
0.187801635



RPL34
0.047258313
0.194514225



CARKL
0.796426726
0.197876342



DPM3
0.100527185
0.210155758



C11orf2
0.279960963
0.21235462



LIF
0.319291
0.220377076



DHFR
0.005845519
0.227352382



BU540282
0.855833364
0.253041264



CDC42SE2
0.303933209
0.27279888



OLIG2
9.8531E−05
0.291441723



DERL3
0.009989003
0.311630921



SLK
0.022499454
0.315243668



MBOAT2
7.53321E−07
0.32533079



ST3GAL1
0.555439718
0.329090787



FOLR3
0.293485861
0.330960224



NDUFS7
0.510992855
0.362739986



SLC29A1
0.000196258
0.370006714



TCF7
0.139201093
0.384656786



BQ130147
0.005433882
0.39124831



SPSB2
0.710554126
0.392430072



REEP3
0.003636115
0.39572088



CBS
8.54923E−05
0.414841711



GSTO1
0.000439166
0.421164955



VSIG4
0.03654483
0.436274059



OLIG1
0.000739337
0.438928192



RPL8
0.420798397
0.441110854



CR609588
0.829179104
0.44827808



ARG1
9.77852E−05
0.454989416



JAK2
6.14999E−05
0.462535965



CLC
8.43913E−05
0.478209075



PAPSS1
0.002660178
0.497255641



HSPB1
0.011649931
0.503891496



MPZL1
0.069994815
0.504344915



BC032451
0.015738039
0.505628786



BCL2A1
2.81815E−05
0.50979301



CKLF
8.76337E−06
0.515802792



S100A9
1.04727E−07
0.5350388



MAPK8IP1
0.000267919
0.558711324



LOXL2
0.153997075
0.559866641



GSTP1
0.802223179
0.622441442



SLC22A1
0.000127897
0.626928629



HGF
0.001272015
0.63284641



EPOR
0.918974368
0.633466985



ETFB
0.143878666
0.645850919



SSNA1
0.103788889
0.6470392



IRF2
0.018278933
0.665824694



ASMTL
0.311592758
0.681691103



ST6GALNAC3
0.000812432
0.686396961



CSTA
3.1114E−06
0.707081235



SMN1
0.473451351
0.714837746



REEP5
0.000215833
0.733733395



FCGBP
0.074075812
0.796385743



S100A12
4.72256E−06
0.804439181



CAT
4.59232E−08
0.81384176



LOC644246
2.85943E−06
0.820487985



FRAT1
3.39803E−05
0.859050707



ATP11B
6.96563E−05
0.882770629



LGALS1
0.039299421
0.918250705



YWHAZ
0.023358903
0.927846666



MMD
0.153204886
0.941639541



CD33
0.101691174
0.950753885



CD248
0.186672242
0.973814259



ADORA3
0.000150846
0.975200559



TXN
3.22949E−08
0.99228328



LPGAT1
1.58563E−06
0.995574922

















TABLE 5





Marker Symbol

















AA303143



AA601031



ABCC2



ABHD2



ABHD5



ABLIM1



ACO2



ACOX1



ACSL1



ACTB



ACVR2B



ADA



ADNP



AF034187



AF085968



AF161353



AF471454



AI276257



AIM1L



AK021463



AK022268



AK023663



AK024956



AK056689



AK092942



AK098835



AK124192



ALOX12



ALOX5



ALOX5AP



ALS2CR13



AMBN



AMFR



AMICA1



ANXA2



ANXA3



AOAH



AP1S2



APBA2



APBB1



APEH



APH1A



APOBEC3G



APRT



AQP2



AQP8



ARG1



ARHGAP24



ARHGAP9



ARHGDIA



ARID5B



ARPC1B



ASCL2



ATG3



ATP1B2



ATP5D



ATP6V0B



ATP7B



AW076051



AW579245



AX721252



AY003763



AY062331



A_23_P158868



A_23_P335398



A_23_P348587



A_23_P44053



A_24_P101960



A_24_P144383



A_24_P221375



A_24_P238427



A_24_P384604



A_24_P417996



A_24_P418712



A_24_P745883



A_24_P84408



A_24_P916228



A_24_P929533



A_32_P28158



A_32_P62137



B2M



B4GALT5



BACH2



BAGE



BAZ1A



BBS2



BC024289



BC031973



BC038432



BC043173



BC062739



BC073935



BCL2A1



BCL3



BCL6



BCL7A



BG777521



BI024548



BI026064



BM703463



BMX



BOP1



BQ365891



BRF1



BRI3



BST1



BTBD14A



BTNL8



BU633383



BX110908



BYSL



C10orf54



C11orf2



C12orf35



C14orf156



C15orf38



C16orf24



C16orf57



C1orf96



C20orf24



C20orf3



C20orf77



C2orf39



C6orf129



C6orf32



C7orf34



C8orf31



C9orf19



CALM3



CAMKK2



CAPNS1



CASP4



CASP5



CBS



CCDC108



CCDC92



CCL3L3



CCPG1



CD200



CD248



CD302



CD3D



CD3E



CD5



CD58



CD6



CD7



CD79B



CD93



CD96



CDKL5



CDKN1A



CEACAM4



CEBPB



CEBPD



CFLAR



CFP



CHI3L2



CIB3



CKLF



CLEC12A



CLEC2D



CLEC4D



CLEC4E



CLIC1



CMTM2



CNTNAP2



COL14A1



COMMD6



COP1



COX6B2



COX6C



CPD



CR2



CR593845



CR610181



CR613361



CR613944



CREB5



CRIP1



CRISPLD2



CSF2RA



CSF2RB



CSTA



CTBP2



CYB5D2



CYP1A2



CYP4F2



CYP4F3



CYP4F8



DCXR



DDX11



DDX3Y



DEDD2



DEFA4



DEK



DENND3



DHRS3



DHRS7B



DHRSX



DKFZP434B0335



DKFZp434F142



DKFZp547E087



DOCK10



DOCK8



DOK3



DPF3



DPPA5



DRAP1



DUOX2



DUSP13



DUSP3



DYNLT1



ECH1



ECHDC3



EEF2



EIF1AX



EIF2AK2



EIF2C4



EIF4B



EIF5A



EMP3



EMR3



ENST00000337102



ENST00000360102



ENTPD1



ETS1



EXOC6



EXOSC6



F5



FAIM3



FAM108A1



FAM113B



FAM26B



FAM44A



FAU



FBXL5



FCAR



FCER1A



FGD4



FIBP



FKBP5



FKBP9



FLJ22662



FLJ40092



FNDC3B



FOS



FOXJ1



FOXP1



FPR1



FRAT1



FRAT2



FRS2



FRS3



FTH1



FXYD5



FYB



GADD45GIP1



GAMT



GBP2



GCA



GLRX



GLT1D1



GLUL



GMFG



GNB1



GPA33



GPBAR1



GPC1



GPD1



GPR160



GPR172A



GPR37L1



GRB10



GSTT1



GTF2I



GYG1



H2AFZ



H3F3A



HAL



HAP1



HDAC4



HDDC2



HDGFL1



HEBP2



HIST1H2AC



HIST1H2AJ



HIST1H2AM



HIST1H2BC



HIST2H2AC



HLA-DRB5



HLA-E



HLA-F



HMGB2



HOMER3



HOXB7



HSBP1



HSDL2



HSPA1A



HSPB1



HTATIP2



ID2



ID3



IFITM4P



IGF2R



IGHA1



IGHD



IGHM



IL13RA1



IL18R1



IL1R2



IL23A



IL7R



IMPA2



IMPDH1



INCA



IRAK3



ISG20



ITM2C



JDP2



KCNE3



KCNG1



KCNJ15



KIAA0319L



KIAA1430



KIAA1833



KLF6



KLHL3



KLRC4



KSR1



LAG3



LAMP2



LAT2



LCK



LHPP



LILRA2



LILRB3



LILRP2



LIMS2



LIN7A



LIN7B



LOC137886



LOC149703



LOC150166



LOC153546



LOC220433



LOC389641



LOC401233



LOC401357



LOC439949



LOC440104



LOC440348



LOC440731



LOC497190



LOC644246



LOXL2



LPGAT1



LRRK2



LSM10



LSM7



LST1



LTBP2



LTBP3



LY96



MACF1



MAGED1



MAGED2



MAGEH1



MAK



MAN1C1



MAN2A2



MAP1LC3B



MAP3K2



MAP3K3



MAP4K4



MAPK14



MAPK8IP1



MAX



MBOAT2



MCL1



MEA1



MEGF10



METTL9



MGAM



MGC14425



MLKL



MLSTD2



MMD



MME



MMP9



MNDA



MORC3



MOSC1



MOSPD2



MPZL1



MRLC2



MRPL42P5



MRPL53



MSRB2



MST150



MUC20



MUM1



MXD1



MYBPH



MYC



MYH14



MYL6



MYO15B



MYO1F



MYO1G



NAPSA



NAPSB



NBPF11



NCF4



NDRG2



NDUFB3



NDUFS8



NFATC1



NFIL3



NGFRAP1



NIN



NMI



NMT2



NOVA1



NPIP



NRBF2



NRIP3



NRP1



NRSN2



NUDT16



OLIG1



OR4C15



OR52B2



OSBPL2



OSBPL6



OSTF1



OXNAD1



PACSIN2



PADI4



PARP1



PDCD7



PDE9A



PDK2



PDLIM7



PELI1



PFDN5



PFKFB3



PGD



PHB



PHC2



PHF5A



PHGDH



PIK3C2B



PIM2



PISD



PITPNA



PLA2G4A



PLA2G7



PLAG1



PLD3



PLEKHA1



PLEKHM1



PLXNC1



POLR2A



PPP1R12B



PPP4R2



PRAP1



PRKAR1A



PRKAR1B



PRKCA



PRKCD



PRKDC



PRKY



PRSS23



PSMB9



PSMD8



PTEN



PTOV1



PTPRCAP



PTPRK



PTPRM



PXK



PYCARD



PYGL



QPCT



QPRT



RAB24



RAB27A



RAB31



RAB32



RABGAP1L



RABIF



RAC1



RAC2



RAI1



RALB



RALGDS



RARA



RASSF2



RBP7



RCC2



REEP5



REPS2



RFWD2



RGS16



RGS2



RHOG



RHOH



RIMS4



RIT1



RMND5A



RNF130



RNF182



RNF24



ROCK2



ROPN1L



RPL17



RPL18A



RPL22



RPL31



RPL34



RPL36A



RPL37



RPL39



RPS10



RPS15



RPS21



RPS27



RPS27A



RPS28



RPS4X



RPUSD2



RRN3



RTN3



S100A11



S100A12



S100A8



S100A9



S100P



SAMSN1



SAP30



SCRN2



SDCBP



SEC14L1



SEC22B



SEPX1



SERINC1



SERPINB1



SERPINB8



SERPINE1



SF3B14



SFT2D1



SGCE



SH2D5



SLA



SLC16A3



SLC1A7



SLC22A15



SLC22A4



SLC25A37



SLC2A10



SLC2A14



SLC2A8



SLC35B4



SLC37A3



SLC40A1



SLC45A2



SLC8A1



SLIT3



SMARCD3



SMC1A



SMUG1



SOD2



SP100



SPIB



SPRR2C



SRM



SRPK1



SSBP4



ST6GAL1



STAT5A



STC1



STK17B



STMN1



STX10



STX3



SULT1B1



SYNCRIP



SYT15



TAF9B



TALDO1



TANK



TARP



TAX1BP1



TBCD



TBL1XR1



TCEAL1



TCF3



TCF7



THBD



TLR2



TLR8



TM7SF2



TMEM102



TMEM48



TMEM49



TMEM68



TMEM86A



TNFAIP6



INFRSF10A



TP53I11



TP53TG3



TPST1



TRA@



TRAPPC2L



TREM1



TRIB1



TRIM7



TSEN34



TSPAN13



TSPAN16



TSPAN33



TUFM



TXN



TYROBP



U2AF1



UBC



UBE2D3



UBE2G2



UBL5



UBQLN1



UCP2



UPF3A



URG4



USP11



USP53



USP6



VKORC1



VWCE



WDFY3



WDR18



XKR8



XPR1



YOD1



YPEL4



ZBED1



ZCCHC6



ZNF135



ZNF234



ZNF346



ZNF438



ZNF550



ZNF618





















TABLE 6





GOID
Ontology
Term
Log odds ratio
p value



















GO: 0009987
bp
cellular process
0.537
5.55E−19


GO: 0002376
bp
immune system process
1.728
9.64E−16


GO: 0050896
bp
response to stimulus
1.118
2.63E−15


GO: 0006955
bp
immune response
1.796
7.62E−12


GO: 0008152
bp
metabolic process
0.537
1.64E−09


GO: 0065007
bp
biological regulation
0.545
2.34E−09


GO: 0006952
bp
defense response
1.732
1.02E−08


GO: 0050789
bp
regulation of biological process
0.538
2.16E−08


GO: 0043067
bp
regulation of programmed cell death
1.508
1.52E−07


GO: 0010941
bp
regulation of cell death
1.507
1.55E−07


GO: 0044238
bp
primary metabolic process
0.515
2.05E−07


GO: 0007165
bp
signal transduction
0.784
2.09E−07


GO: 0050794
bp
regulation of cellular process
0.520
2.50E−07


GO: 0042981
bp
regulation of apoptosis
1.493
3.04E−07


GO: 0006950
bp
response to stress
1.096
3.47E−07


GO: 0007154
bp
cell communication
0.727
5.29E−07


GO: 0045321
bp
leukocyte activation
2.190
6.88E−07


GO: 0046649
bp
lymphocyte activation
2.307
8.27E−07


GO: 0044237
bp
cellular metabolic process
0.484
4.42E−06


GO: 0006690
bp
icosanoid metabolic process
3.260
9.29E−06


GO: 0001775
bp
cell activation
1.968
9.96E−06


GO: 0043068
bp
positive regulation of programmed cell death
1.746
1.47E−05


GO: 0048519
bp
negative regulation of biological process
0.976
1.64E−05


GO: 0010942
bp
positive regulation of cell death
1.737
1.64E−05


GO: 0002684
bp
positive regulation of immune system process
2.153
2.09E−05


GO: 0033559
bp
unsaturated fatty acid metabolic process
3.120
2.23E−05


GO: 0019538
bp
protein metabolic process
0.702
2.24E−05


GO: 0002521
bp
leukocyte differentiation
2.473
3.07E−05


GO: 0006414
bp
translational elongation
2.100
3.49E−05


GO: 0043065
bp
positive regulation of apoptosis
1.706
4.03E−05


GO: 0009611
bp
response to wounding
1.522
4.38E−05


GO: 0009605
bp
response to external stimulus
1.260
4.61E−05


GO: 0006954
bp
inflammatory response
1.781
4.61E−05


GO: 0007242
bp
intracellular signaling cascade
1.009
6.55E−05


GO: 0006917
bp
induction of apoptosis
1.843
7.07E−05


GO: 0006691
bp
leukotriene metabolic process
3.699
7.33E−05


GO: 0012502
bp
induction of programmed cell death
1.831
7.99E−05


GO: 0030098
bp
lymphocyte differentiation
2.586
8.30E−05


GO: 0002682
bp
regulation of immune system process
1.761
9.77E−05


GO: 0043449
bp
cellular alkene metabolic process
3.603
0.00012


GO: 0044267
bp
cellular protein metabolic process
0.697
0.00024


GO: 0048523
bp
negative regulation of cellular process
0.899
0.00046


GO: 0042110
bp
T cell activation
2.334
0.00051


GO: 0050776
bp
regulation of immune response
2.018
0.00057


GO: 0055114
bp
oxidation reduction
1.237
0.00057


GO: 0042221
bp
response to chemical stimulus
1.082
0.00068


GO: 0043066
bp
negative regulation of apoptosis
1.625
0.00069


GO: 0030097
bp
hemopoiesis
1.833
0.00078


GO: 0043069
bp
negative regulation of programmed cell death
1.608
0.00082


GO: 0060548
bp
negative regulation of cell death
1.608
0.00082


GO: 0002694
bp
regulation of leukocyte activation
2.148
0.00083


GO: 0043170
bp
macromolecule metabolic process
0.431
0.00101


GO:0050865
bp
regulation of cell activation
2.114
0.00102


GO:0043412
bp
macromolecule modification
0.804
0.00130


GO:0051249
bp
regulation of lymphocyte activation
2.174
0.00139


GO:0048583
bp
regulation of response to stimulus
1.526
0.00177


GO:0045619
bp
regulation of lymphocyte differentiation
2.692
0.00245


GO:0051707
bp
response to other organism
1.750
0.00252


GO:0048534
bp
hemopoietic or lymphoid organ development
1.673
0.00284


GO:0048518
bp
positive regulation of biological process
0.786
0.00285


GO:0002696
bp
positive regulation of leukocyte activation
2.297
0.00301


GO:0050867
bp
positive regulation of cell activation
2.297
0.00301


GO:0006793
bp
phosphorus metabolic process
0.928
0.00377


GO:0006796
bp
phosphate metabolic process
0.928
0.00377


GO:0019221
bp
cytokine-mediated signaling pathway
2.387
0.00426


GO:0006464
bp
protein modification process
0.767
0.00461


GO:0045621
bp
positive regulation of lymphocyte
3.046
0.00499




differentiation


GO:0002820
bp
negative regulation of adaptive immune
3.972
0.00499




response


GO:0002823
bp
negative regulation of adaptive immune
3.972
0.00499




response based on somatic recombination of




immune receptors built from immunoglobulin




superfamily domains


GO:0044260
bp
cellular macromolecule metabolic process
0.413
0.00561


GO:0045580
bp
regulation of T cell differentiation
2.724
0.00561


GO:0019370
bp
leukotriene biosynthetic process
3.387
0.00561


GO:0043450
bp
alkene biosynthetic process
3.387
0.00561


GO:0002520
bp
immune system development
1.580
0.00565


GO:0009607
bp
response to biotic stimulus
1.477
0.00577


GO:0031347
bp
regulation of defense response
2.154
0.00638


GO:0043101
bp
purine salvage
4.650
0.00644


GO:0008285
bp
negative regulation of cell proliferation
1.413
0.00689


GO:0001817
bp
regulation of cytokine production
1.908
0.00710


GO:0016310
bp
phosphorylation
0.971
0.00713


GO:0043687
bp
post-translational protein modification
0.840
0.00713


GO:0042113
bp
B cell activation
2.272
0.00713


GO:0051251
bp
positive regulation of lymphocyte activation
2.258
0.00764


GO:0006928
bp
cellular component movement
1.253
0.00800


GO:0043433
bp
negative regulation of transcription factor
2.902
0.00800




activity


GO:0090048
bp
negative regulation of transcription regulator
2.902
0.00800




activity


GO:0030183
bp
B cell differentiation
2.627
0.00807


GO:0002252
bp
immune effector process
1.981
0.00816


GO:0050863
bp
regulation of T cell activation
2.098
0.00821


GO:0070887
bp
cellular response to chemical stimulus
1.562
0.00962


GO:0048522
bp
positive regulation of cellular process
0.757
0.00972


GO:0006412
bp
translation
1.220
0.01068


GO:0043299
bp
leukocyte degranulation
3.650
0.01102


GO:0030091
bp
protein repair
4.387
0.01112


GO:0006916
bp
anti-apoptosis
1.656
0.01149


GO:0007264
bp
small GTPase mediated signal transduction
1.344
0.01149


GO:0042127
bp
regulation of cell proliferation
1.035
0.01275


GO:0007243
bp
protein kinase cascade
1.332
0.01275


GO:0030217
bp
T cell differentiation
2.491
0.01322


GO:0031349
bp
positive regulation of defense response
2.491
0.01322


GO:0006468
bp
protein amino acid phosphorylation
0.992
0.01363


GO:0002698
bp
negative regulation of immune effector
3.557
0.01363




process


GO:0043392
bp
negative regulation of DNA binding
2.709
0.01508


GO:0043603
bp
cellular amide metabolic process
2.449
0.01564


GO:0007166
bp
cell surface receptor linked signal transduction
0.727
0.01692


GO:0008625
bp
induction of apoptosis via death domain
3.470
0.01716




receptors


GO:0009163
bp
nucleoside biosynthetic process
4.165
0.01730


GO:0042451
bp
purine nucleoside biosynthetic process
4.165
0.01730


GO:0042455
bp
ribonucleoside biosynthetic process
4.165
0.01730


GO:0046129
bp
purine ribonucleoside biosynthetic process
4.165
0.01730


GO:0033152
bp
immunoglobulin V(D)J recombination
4.165
0.01730


GO:0051100
bp
negative regulation of binding
2.650
0.01793


GO:0045582
bp
positive regulation of T cell differentiation
2.972
0.01808


GO:0006959
bp
humoral immune response
2.210
0.01808


GO:0042035
bp
regulation of cytokine biosynthetic process
2.210
0.01808


GO:0007162
bp
negative regulation of cell adhesion
2.539
0.02085


GO:0051591
bp
response to cAMP
3.235
0.02085


GO:0042036
bp
negative regulation of cytokine biosynthetic
3.235
0.02085




process


GO:0045727
bp
positive regulation of translation
3.235
0.02085


GO:0051098
bp
regulation of binding
1.839
0.02085


GO:0051101
bp
regulation of DNA binding
1.984
0.02085


GO:0032944
bp
regulation of mononuclear cell proliferation
2.309
0.02085


GO:0050670
bp
regulation of lymphocyte proliferation
2.309
0.02085


GO:0070663
bp
regulation of leukocyte proliferation
2.309
0.02085


GO:0045581
bp
negative regulation of T cell differentiation
3.972
0.02085


GO:0002703
bp
regulation of leukocyte mediated immunity
2.513
0.02085


GO:0002706
bp
regulation of lymphocyte mediated immunity
2.594
0.02085


GO:0018193
bp
peptidyl-amino acid modification
1.779
0.02085


GO:0019321
bp
pentose metabolic process
3.972
0.02085


GO:0045055
bp
regulated secretory pathway
3.235
0.02085


GO:0010310
bp
regulation of hydrogen peroxide metabolic
3.972
0.02085




process


GO:0002822
bp
regulation of adaptive immune response
2.487
0.02155




based on somatic recombination of immune




receptors built from immunoglobulin




superfamily domains


GO:0019748
bp
secondary metabolic process
2.079
0.02180


GO:0006631
bp
fatty acid metabolic process
1.541
0.02416


GO:0046688
bp
response to copper ion
3.802
0.02497


GO:0045628
bp
regulation of T-helper 2 cell differentiation
3.802
0.02497


GO:0002704
bp
negative regulation of leukocyte mediated
3.802
0.02497




immunity


GO:0002707
bp
negative regulation of lymphocyte mediated
3.802
0.02497




immunity


GO:0046456
bp
icosanoid biosynthetic process
2.724
0.02597


GO:0010033
bp
response to organic substance
1.157
0.02601


GO:0080134
bp
regulation of response to stress
1.524
0.02611


GO:0042180
bp
cellular ketone metabolic process
1.016
0.02815


GO:0002712
bp
regulation of B cell mediated immunity
3.098
0.02864


GO:0002889
bp
regulation of immunoglobulin mediated
3.098
0.02864




immune response


GO:0002819
bp
regulation of adaptive immune response
2.387
0.02920


GO:0008624
bp
induction of apoptosis by extracellular signals
2.387
0.02920


GO:0050778
bp
positive regulation of immune response
1.864
0.02930


GO:0030031
bp
cell projection assembly
1.998
0.02943


GO:0002443
bp
leukocyte mediated immunity
1.998
0.02943


GO:0051250
bp
negative regulation of lymphocyte activation
2.650
0.03116


GO:0000122
bp
negative regulation of transcription from RNA
1.843
0.03194




polymerase II promoter


GO:0043094
bp
cellular metabolic compound salvage
3.650
0.03249


GO:0045749
bp
negative regulation of S phase of mitotic cell
3.650
0.03249




cycle


GO:0050777
bp
negative regulation of immune response
3.034
0.03249


GO:0080010
bp
regulation of oxygen and reactive oxygen
3.650
0.03249




species metabolic process


GO:0006968
bp
cellular defense response
2.131
0.03328


GO:0045087
bp
innate immune response
1.716
0.03404


GO:0006739
bp
NADP metabolic process
2.972
0.03786


GO:0045088
bp
regulation of innate immune response
2.580
0.03789


GO:0002697
bp
regulation of immune effector process
2.082
0.04021


GO:0009617
bp
response to bacterium
1.783
0.04172


GO:0006636
bp
unsaturated fatty acid biosynthetic process
2.546
0.04198


GO:0006101
bp
citrate metabolic process
3.513
0.04231


GO:0002828
bp
regulation of T-helper 2 type immune
3.513
0.04231




response


GO:0046777
bp
protein amino acid autophosphorylation
2.066
0.04231


GO:0060263
bp
regulation of respiratory burst
3.513
0.04231


GO:0006082
bp
organic acid metabolic process
0.971
0.04277


GO:0019752
bp
carboxylic acid metabolic process
0.980
0.04277


GO:0043436
bp
oxoacid metabolic process
0.980
0.04277


GO:0009058
bp
biosynthetic process
0.423
0.04277


GO:0030155
bp
regulation of cell adhesion
1.763
0.04277


GO:0050870
bp
positive regulation of T cell activation
2.034
0.04277


GO:0050727
bp
regulation of inflammatory response
2.228
0.04277


GO:0007265
bp
Ras protein signal transduction
1.659
0.04277


GO:0010629
bp
negative regulation of Marker expression
1.186
0.04277


GO:0042742
bp
defense response to bacterium
2.018
0.04277


GO:0002695
bp
negative regulation of leukocyte activation
2.481
0.04277


GO:0009146
bp
purine nucleoside triphosphate catabolic
3.387
0.04455




process


GO:0045620
bp
negative regulation of lymphocyte
3.387
0.04455




differentiation


GO:0050869
bp
negative regulation of B cell activation
3.387
0.04455


GO:0050853
bp
B cell receptor signaling pathway
3.387
0.04455


GO:0050864
bp
regulation of B cell activation
2.449
0.04504


GO:0018212
bp
peptidyl-tyrosine modification
2.449
0.04504


GO:0048584
bp
positive regulation of response to stimulus
1.526
0.04534


GO:0019079
bp
viral genome replication
2.802
0.04639


GO:0009892
bp
negative regulation of metabolic process
0.997
0.04764


GO:0006357
bp
regulation of transcription from RNA
0.950
0.04827




polymerase II promoter


GO:0010605
bp
negative regulation of macromolecule
1.020
0.04827




metabolic process


GO:0010558
bp
negative regulation of macromolecule
1.106
0.04890




biosynthetic process


GO:0005623
cc
cell
0.465
2.15E−24


GO:0044464
cc
cell part
0.465
2.15E−24


GO:0005622
cc
intracellular
0.434
1.13E−11


GO:0044424
cc
intracellular part
0.430
1.26E−10


GO:0005737
cc
cytoplasm
0.537
7.50E−10


GO:0016020
cc
membrane
0.551
1.18E−08


GO:0005829
cc
cytosol
1.207
2.30E−07


GO:0005886
cc
plasma membrane
0.744
7.43E−07


GO:0043229
cc
intracellular organelle
0.400
1.46E−06


GO:0043226
cc
organelle
0.399
1.54E−06


GO:0016021
cc
integral to membrane
0.583
4.13E−06


GO:0044444
cc
cytoplasmic part
0.583
5.65E−06


GO:0031224
cc
intrinsic to membrane
0.559
1.09E−05


GO:0044425
cc
membrane part
0.516
1.44E−05


GO:0022626
cc
cytosolic ribosome
2.173
0.00034


GO:0044445
cc
cytosolic part
1.912
0.00040


GO:0043231
cc
intracellular membrane-bounded organelle
0.339
0.00111


GO:0043227
cc
membrane-bounded organelle
0.339
0.00116


GO:0033279
cc
ribosomal subunit
1.837
0.00223


GO:0022627
cc
cytosolic small ribosomal subunit
2.532
0.00501


GO:0044459
cc
plasma membrane part
0.659
0.00975


GO:0043228
cc
non-membrane-bounded organelle
0.588
0.00986


GO:0043232
cc
intracellular non-membrane-bounded
0.588
0.00986




organelle


GO:0044422
cc
organelle part
0.435
0.01029


GO:0044446
cc
intracellular organelle part
0.433
0.01149


GO:0005887
cc
integral to plasma membrane
0.777
0.01842


GO:0031226
cc
intrinsic to plasma membrane
0.751
0.02085


GO:0032991
cc
macromolecular complex
0.516
0.02085


GO:0005840
cc
ribosome
1.329
0.02085


GO:0005634
cc
nucleus
0.348
0.02601


GO:0016461
cc
unconventional myosin complex
3.650
0.03249


GO:0015935
cc
small ribosomal subunit
1.960
0.03404


GO:0005488
mf
binding
0.469
0.00000


GO:0005515
mf
protein binding
0.601
0.00000


GO:0003824
mf
catalytic activity
0.554
0.00000


GO:0003823
mf
antigen binding
3.025
0.00051


GO:0046983
mf
protein dimerization activity
1.350
0.00085


GO:0004871
mf
signal transducer activity
0.738
0.00223


GO:0060089
mf
molecular transducer activity
0.738
0.00223


GO:0004197
mf
cysteine-type endopeptidase activity
2.223
0.00456


GO:0016491
mf
oxidoreductase activity
1.060
0.00553


GO:0004872
mf
receptor activity
0.763
0.00990


GO:0017070
mf
U6 snRNA binding
4.387
0.01112


GO:0005536
mf
glucose binding
4.387
0.01112


GO:0016208
mf
AMP binding
3.018
0.01639


GO:0030234
mf
enzyme regulator activity
0.895
0.01730


GO:0008113
mf
peptide-methionine-(S)-S-oxide reductase
4.165
0.01730




activity


GO:0043169
mf
cation binding
0.420
0.01977


GO:0043167
mf
ion binding
0.408
0.02085


GO:0046872
mf
metal ion binding
0.419
0.02085


GO:0005529
mf
sugar binding
1.586
0.02085


GO:0016165
mf
lipoxygenase activity
3.972
0.02085


GO:0047485
mf
protein N-terminus binding
2.291
0.02085


GO:0005509
mf
calcium ion binding
0.845
0.02138


GO:0030528
mf
transcription regulator activity
0.733
0.02364


GO:0001848
mf
complement binding
3.802
0.02497


GO:0005527
mf
macrolide binding
3.802
0.02497


GO:0005528
mf
FK506 binding
3.802
0.02497


GO:0019838
mf
growth factor binding
1.776
0.02637


GO:0003735
mf
structural constituent of ribosome
1.348
0.02664


GO:0019210
mf
kinase inhibitor activity
2.412
0.02732


GO:0005198
mf
structural molecule activity
0.901
0.02868


GO:0019899
mf
enzyme binding
1.176
0.02868


GO:0005351
mf
sugar:hydrogen symporter activity
2.387
0.02920


GO:0005402
mf
cation:sugar symporter activity
2.387
0.02920


GO:0004672
mf
protein kinase activity
0.930
0.02979


GO:0004888
mf
transmembrane receptor activity
0.813
0.02981


GO:0019207
mf
kinase regulator activity
1.853
0.03047


GO:0015144
mf
carbohydrate transmembrane transporter
2.317
0.03624




activity


GO:0051119
mf
sugar transmembrane transporter activity
2.317
0.03624


GO:0003700
mf
transcription factor activity
0.928
0.04231


GO:0004859
mf
phospholipase inhibitor activity
3.513
0.04231


GO:0019887
mf
protein kinase regulator activity
1.885
0.04277


GO:0004896
mf
cytokine receptor activity
2.050
0.04277


GO:0008603
mf
cAMP-dependent protein kinase regulator
2.857
0.04277




activity


GO:0015295
mf
solute:hydrogen symporter activity
2.250
0.04277


GO:0055102
mf
lipase inhibitor activity
3.387
0.04455


GO:0016840
mf
carbon-nitrogen lyase activity
3.387
0.04455


GO:0016671
mf
oxidoreductase activity, acting on sulfur group
3.387
0.04455




of donors, disulfide as acceptor


GO:0000166
mf
nucleotide binding
0.514
0.04639


GO:0016787
mf
hydrolase activity
0.502
0.04653




























TABLE 7












Number of
AUC Ridge
AUC Logistic

AUC Ridge
AUC Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
Terms
Regression
Regression

Regression
Regression



























TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
7
0.781188
0.785011

0.781188
0.785011


TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
6
0.775592
0.779372
Mean
0.775346
0.778701


TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
6
0.764453
0.768001
SD
0.00497
0.004876


TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
6
0.778834
0.781621


TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
6
0.777071
0.781157


TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
6
0.778887
0.782202


TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
6
0.776268
0.779626


FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
6
0.776321
0.778929


















TABLE 8






AUC Ridge
AUC Logistic


Term
Regression
Regression







7
0.781188
0.785011


6
0.775346
0.778701


5
0.769504
0.772411


4
0.763641
0.766090


3
0.757567
0.759481


2
0.751191
0.752634


1
0.744420
0.745125


0
0.736732
0.736732









DF Model
0.677495 ± .042437




























TABLE 9












Number
AUC Ridge
AUC Logistic

AUC Ridge
AUC Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression

Regression
Regression



























TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
5
0.756671
0.761212
Mean
0.769504
0.772411


TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
5
0.772815
0.774463
SD
0.006329
0.006033


TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
FALSE
5
0.771306
0.775846


TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
5
0.774389
0.777155


TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
5
0.772889
0.775508


FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
5
0.770334
0.772668


TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
5
0.761507
0.763841


TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
5
0.759744
0.764443


TRUE
TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
5
0.762542
0.765424


TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
5
0.760124
0.76345


FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
5
0.760684
0.763249


TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
5
0.7748
0.77819


TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
5
0.776659
0.779921


TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
5
0.773523
0.775466


FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
5
0.773766
0.775529


TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
5
0.775835
0.779731


TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
5
0.771633
0.775613


FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
5
0.771084
0.773702


TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
5
0.775191
0.776279


FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
5
0.773914
0.776289


FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
5
0.770165
0.772657




























TABLE 10














AUC


AUC









Number
AUC Ridge
Logistic

AUC Ridge
Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression

Regression
Regression



























TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
4
0.753409
0.755542
Mean
0.763641
0.76609


TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
4
0.752269
0.757854
SD
0.007095
0.00666


TRUE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
4
0.75475
0.759132


TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
4
0.754644
0.758973


FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
4
0.752923
0.755964


TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
4
0.769014
0.771379


TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
4
0.772245
0.772932


TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
4
0.769289
0.770514


FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
4
0.76742
0.768296


TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
4
0.770746
0.7748


TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
4
0.768423
0.771601


FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
FALSE
4
0.765129
0.768434


TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
4
0.771042
0.773185


FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
4
0.769152
0.771116


FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
4
0.767262
0.768592


TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
4
0.75684
0.759332


TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
4
0.759744
0.762014


TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
4
0.75722
0.75872


FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
4
0.757622
0.759311


TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
4
0.758392
0.76194


TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
4
0.754232
0.757463


FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
4
0.754739
0.758625


TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
4
0.757696
0.76081


FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
4
0.759448
0.760821


FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
4
0.756228
0.757865


TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
4
0.773956
0.777345


TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
4
0.768824
0.771253


FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
4
0.768391
0.770482


TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
4
0.77252
0.773998


FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
4
0.772277
0.77365


FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
4
0.767536
0.769299


TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
4
0.770376
0.77328


FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
4
0.769743
0.77196


FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
4
0.764875
0.767167


FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
4
0.769057
0.769489




























TABLE 11












Number
AUC Ridge
AUC Logistic

AUC Ridge
AUC Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression

Regression
Regression



























TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
3
0.749217
0.752026
Mean
0.757567
0.759481


TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
3
0.751783
0.754158
SD
0.007376
0.006937


TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
3
0.750474
0.752448


FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
3
0.749492
0.749734


TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
3
0.751223
0.755732


TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
3
0.748985
0.752913


FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
3
0.746725
0.750442


TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
3
0.751804
0.755362


FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
3
0.750864
0.753388


FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
3
0.74946
0.752047


TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
3
0.76856
0.770535


TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
3
0.765245
0.766628


FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
3
0.762711
0.764052


TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
3
0.767916
0.768687


FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
3
0.766618
0.766702


FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
3
0.763767
0.764432


TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
3
0.767388
0.770134


FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
3
0.76458
0.766734


FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
3
0.760884
0.762669


FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
3
0.766005
0.766618


TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
3
0.756133
0.758456


TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
3
0.751213
0.752891


FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
3
0.751899
0.753747


TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
3
0.755404
0.75702


FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
3
0.756904
0.758213


FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
3
0.753166
0.753082


TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
3
0.75268
0.756143


FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
3
0.753155
0.755996


FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
3
0.748932
0.750484


FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
3
0.753103
0.755119


TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
3
0.767969
0.77063


FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
3
0.767832
0.76969


FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
3
0.762099
0.763112


FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
3
0.766544
0.76723


FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
3
0.764105
0.76458




























TABLE 12












Number
AUC Ridge
AUC Logistic

AUC Ridge
AUC Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression

Regression
Regression



























TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
2
0.748341
0.750526
Mean
0.751191
0.752634


TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
2
0.745247
0.747485
SD
0.007213
0.006971


FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
2
0.743589
0.744508


TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
2
0.747945
0.749254


FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
2
0.748203
0.748151


FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
2
0.745722
0.745268


TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
2
0.74682
0.75117


FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
2
0.745522
0.748225


FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
2
0.742333
0.74455


FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
2
0.746968
0.749618


TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
2
0.764516
0.765794


FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
2
0.762225
0.762563


FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
2
0.75759
0.758561


FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
2
0.762415
0.762985


FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
2
0.760145
0.761486


TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
2
0.750526
0.752057


FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
2
0.751012
0.752701


FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
2
0.74568
0.746092


FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
2
0.750811
0.751888


FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
2
0.747739
0.749608


FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
2
0.761655
0.762827




























TABLE 13












Number
AUC Ridge
AUC Logistic

AUC Ridge
AUC Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression

Regression
Regression



























TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
1
0.742993
0.744978
Mean
0.74442
0.745125


FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
1
0.742555
0.743273
SD
0.006498
0.006455


FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
1
0.738732
0.738437


FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
1
0.743288
0.743288


FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
1
0.740939
0.742903


FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
1
0.757125
0.757442


FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
1
0.74531
0.745553



























TABLE 14
















AUC









Number
AUC Ridge
AUC Logistic
AUC Ridge
Logistic


Term 1
Term 2
Term 3
Term 4
Term 5
Term 6
Term 7
of Terms
Regression
Regression
Regression
Regression







FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
0
0.736732
0.736732
0.736732
0.736732






















TABLE 15






Substitute

AUC Ridge
AUC Logistic
RefSeq
RefSeq


Algorithm Marker
Marker
Correlation
Regression
egression
Algorithm Marker
Substitute Marker





















S100A12
MMP9
0.77
0.781
0.784
NM_005621
NM_004994


CLEC4E
ALOX5AP
0.74
0.780
0.783
NM_014358
NM_001629


S100A8
NAMPT
0.90
0.781
0.786
NM_002964
NM_005746


CASP5
H3F3B
0.63
0.783
0.787
NM_001136112
NM_005324


IL18RAP
TXN
0.52
0.774
0.778
NM_003853
NM_003329


TNFAIP6
PLAUR
0.66
0.779
0.783
NM_007115
NM_001005376


AQP9
GLT1D1
0.93
0.781
0.785
NM_020980
NM_144669


NCF4
NCF2
0.91
0.780
0.784
NM_000631
NM_000433


CD3D
LCK
0.95
0.779
0.784
NM_000732
NM_001042771


TMC8
CCT2
0.85
0.781
0.785
NM_152468
NM_006431


CD79B
CD19
0.95
0.796
0.809
NM_000626
NM_001770


SPIB
BLK
0.89
0.780
0.784
NM_003121
NM_001715


HNRPF
ACBD5
0.88
0.779
0.783
NM_001098204
NM_001042473


TFCP2
DDX18
0.88
0.781
0.786
NM_005653
NM_006773


RPL28
SSRP1
0.91
0.782
0.786
NM_000991
NM_003146


AF161365
AF161365
1.00
0.781
0.785
AF161365
AF161365


AF289562
CD248
0.53
0.779
0.783
AF289562
NM_020404


SLAMF7
CX3CR1
0.83
0.778
0.783
NM_021181
NM_001171171


KLRC4
CD8A
0.79
0.794
0.805
NM_013431
NM_001145873


IL8RB
BCL2A1
0.82
0.780
0.785
NM_001557
NM_001114735


TNFRSF10C
PTAFR
0.84
0.781
0.785
NM_003841
NM_000952


KCNE3
LAMP2
0.90
0.781
0.784
NM_005472
NM_001122606


TLR4
TYROBP
0.84
0.780
0.783
NM_138554
NM_003332



Mean
0.82
0.781
0.786



SD
0.13
0.005
0.007





Markers are replaced with the most highly correlated non-algorithm marker in the PCR data set, while ensuring that the set of Substitute Markers has no duplicates.

















TABLE 16








AUC Ridge
AUC Logistic



Genomic Terms
Regression
Regression









Full Model
0.781
0.785



1 Marker Replaced
0.781 ± .009
0.786 ± .014



5 Markers Replaced
0.781 ± .014
0.788 ± .021



10 Markers Replaced
0.778 ± .015
0.785 ± .020



15 Markers Replaced
0.779 ± .014
0.787 ± .020



20 Markers Replaced
0.771 ± .010
0.779 ± .013



All Markers Replaced
0.770
0.775











DF Model
0.677 ± .042







For the 5, 10, 15, 20 Markers replaced analyses, markers were selected at random 100 times for each of the analyses.



















TABLE 17






Delta AUC
Delta AUC







Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Type
Predictive





















AF161365.HNRPF.TFCP2
0
0
3
1
Original
Yes


AF161365.TFCP2
0.003495
0.004097
2
1
Original
Yes


AF161365.HNRPF
−0.00327
−0.00391
2
1
Original
Yes


AF161365.ACBD5.DDX18
0.00473
0.004762
3
1
Substitute
Yes


AF161365.DDX18
4.22E−05
0.000243
2
1
Substitute
Yes


AF161365.ACBD5
−0.00278
−0.0031
2
1
Substitute
Yes






















TABLE 18






Delta AUC
Delta AUC







Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Type
Predictive





















AF289562.HNRPF.TFCP2
0
0
3
2
Original
Yes


AF289562.TFCP2
−6.34E−05
−0.00073
2
2
Original
Yes


AF289562.HNRPF
0.000549
0.000306
2
2
Original
Yes


CD248.ACBD5.DDX18
−0.00505
−0.00625
3
2
Substitute
Yes


CD248.DDX18
−0.00535
−0.00654
2
2
Substitute
Yes


CD248.ACBD5
−0.00506
−0.00588
2
2
Substitute
Yes





















TABLE 19






Delta AUC
Delta AUC






Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Predictive




















CD3D.TMC8.CD79B.SPIB
0
0
4
3
Yes


CD3D.CD79B.SPIB
−0.00039
−0.00056
3
3
Yes


TMC8.CD79B.SPIB
0
9.50E−05
3
3
Yes


CD3D.TMC8.CD79B
0.000612
0.000697
3
3
Yes


CD3D.TMC8.SPIB
−0.00053
−0.00041
3
3
Yes


CD3D.CD79B
−0.00016
8.45E−05
2
3
Yes


CD3D.SPIB
−0.00058
−0.0008
2
3
Yes


TMC8.CD79B
0.00038
0.000676
2
3
Yes


TMC8.SPIB
−0.00048
−0.00023
2
3
Yes


LCK.CCT2.CD19.BLK
−0.00846
−0.00441
4
3
No


LCK.CD19.BLK
−0.00838
−0.00536
3
3
No


CCT2.CD19.BLK
−0.00729
−0.00463
3
3
No


LCK.CCT2.CD19
−0.00619
−0.00338
3
3
No


LCK.CCT2.BLK
−0.00043
−0.00016
3
3
Yes


LCK.CD19
−0.00692
−0.00316
2
3
No


LCK.BLK
−0.00027
−8.45E−05
2
3
Yes


CCT2.CD19
−0.00605
−0.0017
2
3
No


CCT2.BLK
−0.00036
−0.00012
2
3
Yes


CD3D.CD79B
−0.00016
8.45E−05
2
3
Yes


CD3D.SPIB
−0.00058
−0.0008
2
3
Yes


CD3D.CD19
−0.00729
−0.00222
2
3
No


CD3D.BLK
−0.00058
−0.00034
2
3
Yes


TMC8.CD79B
0.00038
0.000676
2
3
Yes


TMC8.SPIB
−0.00048
−0.00023
2
3
Yes


TMC8.CD19
−0.00561
−0.00134
2
3
Mixed


TMC8.BLK
−0.00042
−0.0007
2
3
Yes


LCK.CD79B
−0.00073
−0.00045
2
3
Yes


LCK.SPIB
−0.00109
−0.00118
2
3
Yes


LCK.CD19
−0.00692
−0.00316
2
3
No


LCK.BLK
−0.00027
−8.45E−05
2
3
Yes


CCT2.CD79B
0.000106
0.000116
2
3
Yes


CCT2.SPIB
−0.00057
−0.0007
2
3
Yes


CCT2.CD19
−0.00605
−0.0017
2
3
No


CCT2.BLK
−0.00036
−0.00012
2
3
Yes






















TABLE 20






Delta AUC
Delta AUC







Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Type
Predictive





















S100A12.CLEC4E.S100A8.RPL28
0
0
4
4
Original
Yes


S100A12.CLEC4E.RPL28
−0.00079
−0.00079
3
4
Original
Yes


S100A12.S100A8.RPL28
−0.00068
−0.00068
3
4
Original
Yes


CLEC4E.S100A8.RPL28
0.000528
0.000528
3
4
Original
Yes


S100A12.RPL28
−0.00166
−0.00166
2
4
Original
Yes


CLEC4E.RPL28
−0.00183
−0.00183
2
4
Original
Yes


S100A8.RPL28
0.000538
0.000538
2
4
Original
Yes


MMP9.ALOX5AP.NAMPT.SSRP1
−0.0003
−0.0003
4
4
Substitute
Yes


MMP9.ALOX5AP.SSRP1
−0.00082
−0.00082
3
4
Substitute
Yes


MMP9.NAMPT.SSRP1
−0.00052
−0.00052
3
4
Substitute
Yes


ALOX5AP.NAMPT.SSRP1
0.000169
0.000169
3
4
Substitute
Yes


MMP9.SSRP1
−0.00186
−0.00186
2
4
Substitute
Yes


ALOX5AP.SSRP1
−3.17E−05
−3.17E−05
2
4
Substitute
Yes


NAMPT.SSRP1
−0.0002
−0.0002
2
4
Substitute
Yes





















TABLE 21






Delta AUC
Delta AUC






Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Predictive




















S100A12.CLEC4E.S100A8.AQP9.NCF4
0
0
5
5
Yes


S100A12.CLEC4E.AQP9.NCF4
−0.00021
0.000317
4
5
Yes


S100A12.S100A8.AQP9.NCF4
−0.00173
−0.00269
4
5
Yes


CLEC4E.S100A8.AQP9.NCF4
0.001014
0.001499
4
5
Yes


S100A12.CLEC4E.S100A8.AQP9
−0.00091
−0.00105
4
5
Yes


S100A12.CLEC4E.S100A8.NCF4
0.000348
−0.00013
4
5
Yes


S100A12.AQP9.NCF4
−0.00249
−0.00298
3
5
Yes


CLEC4E.AQP9.NCF4
−0.00016
−0.00042
3
5
Yes


S100A12.CLEC4E.AQP9
−0.00103
−0.00073
3
5
Yes


S100A12.CLEC4E.NCF4
0.000243
9.50E−05
3
5
Yes


S100A8.AQP9.NCF4
−0.00108
−0.00207
3
5
Yes


S100A12.S100A8.AQP9
−0.00226
−0.00336
3
5
Yes


S100A12.S100A8.NCF4
−0.00141
−0.00262
3
5
Yes


CLEC4E.S100A8.AQP9
3.17E−05
0.000338
3
5
Yes


CLEC4E.S100A8.NCF4
0.000771
0.000813
3
5
Yes


S100A12.AQP9
−0.0026
−0.00363
2
5
Yes


S100A12.NCF4
−0.00227
−0.00284
2
5
Yes


CLEC4E.AQP9
−0.00053
−0.0009
2
5
Yes


CLEC4E.NCF4
−0.00024
−0.00076
2
5
Yes


S100A8.AQP9
−0.00246
−0.00325
2
5
Yes


S100A8.NCF4
−0.00091
−0.00302
2
5
Yes


MMP9.ALOX5AP.NAMPT.GLT1D1.NCF2
−0.00325
−0.00535
5
5
Yes


MMP9.ALOX5AP.GLT1D1.NCF2
−0.00498
−0.00693
4
5
No


MMP9.NAMPT.GLT1D1.NCF2
−0.00311
−0.00518
4
5
Yes


ALOX5AP.NAMPT.GLT1D1.NCF2
−0.00376
−0.0057
4
5
Yes


MMP9.ALOX5AP.NAMPT.GLT1D1
−0.00339
−0.0054
4
5
Yes


MMP9.ALOX5AP.NAMPT.NCF2
−0.00509
−0.00703
4
5
No


MMP9.GLT1D1.NCF2
−0.00523
−0.0071
3
5
No


ALOX5AP.GLT1D1.NCF2
−0.00402
−0.00594
3
5
Yes


MMP9.ALOX5AP.GLT1D1
−0.00344
−0.00538
3
5
Yes


MMP9.ALOX5AP.NCF2
−0.00488
−0.00691
3
5
No


NAMPT.GLT1D1.NCF2
−0.00296
−0.00516
3
5
Yes


MMP9.NAMPT.GLT1D1
−0.0033
−0.00529
3
5
Yes


MMP9.NAMPT.NCF2
−0.00537
−0.00736
3
5
No


ALOX5AP.NAMPT.GLT1D1
−0.00362
−0.00534
3
5
Yes


ALOX5AP.NAMPT.NCF2
−0.0036
−0.0056
3
5
Yes


MMP9.GLT1D1
−0.00518
−0.00711
2
5
No


MMP9.NCF2
−0.00516
−0.00706
2
5
No


ALOX5AP.GLT1D1
−0.00404
−0.00613
2
5
Yes


ALOX5AP.NCF2
−0.00433
−0.00623
2
5
No


NAMPT.GLT1D1
−0.00266
−0.00437
2
5
Yes


NAMPT.NCF2
−0.00303
−0.00566
2
5
Yes


S100A12.AQP9
−0.0026
−0.00363
2
5
Yes


S100A12.NCF4
−0.00227
−0.00284
2
5
Yes


S100A12.GLT1D1
−0.00245
−0.00356
2
5
Yes


S100A12.NCF2
−0.00359
−0.00476
2
5
Yes


CLEC4E.AQP9
−0.00053
−0.0009
2
5
Yes


CLEC4E.NCF4
−0.00024
−0.00076
2
5
Yes


CLEC4E.GLT1D1
−0.00023
−0.001
2
5
Yes


CLEC4E.NCF2
−0.00113
−0.00249
2
5
Yes


S100A8.AQP9
−0.00246
−0.00325
2
5
Yes


S100A8.NCF4
−0.00091
−0.00302
2
5
Yes


S100A8.GLT1D1
−0.00209
−0.00289
2
5
Yes


S100A8.NCF2
−0.00297
−0.00497
2
5
Yes


MMP9.AQP9
−0.00341
−0.00537
2
5
Yes


MMP9.NCF4
−0.00317
−0.00544
2
5
Yes


MMP9.GLT1D1
−0.00518
−0.00711
2
5
No


MMP9.NCF2
−0.00516
−0.00706
2
5
No


ALOX5AP.AQP9
−0.00481
−0.00669
2
5
No


ALOX5AP.NCF4
−0.00386
−0.00645
2
5
Mixed


ALOX5AP.GLT1D1
−0.00404
−0.00613
2
5
Yes


ALOX5AP.NCF2
−0.00433
−0.00623
2
5
No


NAMPT.AQP9
−0.00221
−0.00344
2
5
Yes


NAMPT.NCF4
−0.00186
−0.00385
2
5
Yes


NAMPT.GLT1D1
−0.00266
−0.00437
2
5
Yes


NAMPT.NCF2
−0.00303
−0.00566
2
5
Yes





















TABLE 22






Delta AUC
Delta AUC






Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Predictive




















CASP5.IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.KCNE3.TLR4
0
0
7
6
Yes


CASP5.IL18RAP.IL8RB.TNFRSF10C.KCNE3.TLR4
−0.00726
−0.00732
6
6
Yes


CASP5.TNFAIP6.IL8RB.TNFRSF10C.KCNE3.TLR4
−0.00772
−0.00773
6
6
Yes


IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.KCNE3.TLR4
0.00226
0.002999
6
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.KCNE3
−0.00147
−0.00101
6
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.TLR4
0.001077
0.001045
6
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.KCNE3.TLR4
0.000644
0.000296
6
6
Yes


CASP5.IL18RAP.TNFAIP6.TNFRSF10C.KCNE3.TLR4
−0.0012
−0.00038
6
6
Yes


CASP5.IL8RB.TNFRSF10C.KCNE3.TLR4
−0.01475
−0.01514
5
6
Yes


IL18RAP.IL8RB.TNFRSF10C.KCNE3.TLR4
−0.00966
−0.00894
5
6
Yes


CASP5.IL18RAP.IL8RB.TNFRSF10C.KCNE3
−0.00788
−0.008
5
6
Yes


CASP5.IL18RAP.IL8RB.TNFRSF10C.TLR4
−0.00707
−0.00668
5
6
Yes


CASP5.IL18RAP.IL8RB.KCNE3.TLR4
−0.00706
−0.00693
5
6
Yes


CASP5.IL18RAP.TNFRSF10C.KCNE3.TLR4
−0.00785
−0.00776
5
6
Yes


TNFAIP6.IL8RB.TNFRSF10C.KCNE3.TLR4
−0.00696
−0.00669
5
6
Yes


CASP5.TNFAIP6.IL8RB.TNFRSF10C.KCNE3
−0.00842
−0.0081
5
6
Yes


CASP5.TNFAIP6.IL8RB.TNFRSF10C.TLR4
−0.00771
−0.00776
5
6
Yes


CASP5.TNFAIP6.IL8RB.KCNE3.TLR4
−0.00722
−0.00741
5
6
Yes


CASP5.TNFAIP6.TNFRSF10C.KCNE3.TLR4
−0.00815
−0.00809
5
6
Yes


IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.KCNE3
0.001066
0.00151
5
6
Yes


IL18RAP.TNFAIP6.IL8RB.TNFRSF10C.TLR4
0.001795
0.003252
5
6
Yes


IL18RAP.TNFAIP6.IL8RB.KCNE3.TLR4
0.002745
0.003822
5
6
Yes


IL18RAP.TNFAIP6.TNFRSF10C.KCNE3.TLR4
0.001626
0.003305
5
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.TNFRSF10C
−0.00061
−0.00029
5
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.KCNE3
−0.00103
1.06E−05
5
6
Yes


CASP5.IL18RAP.TNFAIP6.TNFRSF10C.KCNE3
−0.00291
−0.00322
5
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB.TLR4
0.002492
0.002714
5
6
Yes


CASP5.IL18RAP.TNFAIP6.TNFRSF10C.TLR4
−0.0005
−0.00052
5
6
Yes


CASP5.IL18RAP.TNFAIP6.KCNE3.TLR4
−0.00096
−0.0004
5
6
Yes


CASP5.IL8RB.TNFRSF10C.KCNE3
−0.01477
−0.01564
4
6
Yes


CASP5.IL8RB.TNFRSF10C.TLR4
−0.01456
−0.01506
4
6
Yes


CASP5.IL8RB.KCNE3.TLR4
−0.01438
−0.01466
4
6
Yes


CASP5.TNFRSF10C.KCNE3.TLR4
−0.01449
−0.01482
4
6
Yes


IL18RAP.IL8RB.TNFRSF10C.KCNE3
−0.00979
−0.00959
4
6
Yes


IL18RAP.IL8RB.TNFRSF10C.TLR4
−0.00982
−0.00881
4
6
Yes


IL18RAP.IL8RB.KCNE3.TLR4
−0.00926
−0.00856
4
6
Yes


IL18RAP.TNFRSF10C.KCNE3.TLR4
−0.00952
−0.00899
4
6
Yes


CASP5.IL18RAP.IL8RB.TNFRSF10C
−0.00777
−0.00745
4
6
Yes


CASP5.IL18RAP.IL8RB.KCNE3
−0.00763
−0.00746
4
6
Yes


CASP5.IL18RAP.TNFRSF10C.KCNE3
−0.00871
−0.00871
4
6
Yes


CASP5.IL18RAP.IL8RB.TLR4
−0.00598
−0.00571
4
6
Yes


CASP5.IL18RAP.TNFRSF10C.TLR4
−0.00733
−0.00724
4
6
Yes


CASP5.IL18RAP.KCNE3.TLR4
−0.00755
−0.00775
4
6
Yes


TNFAIP6.IL8RB.TNFRSF10C.KCNE3
−0.00715
−0.00729
4
6
Yes


TNFAIP6.IL8RB.TNFRSF10C.TLR4
−0.00744
−0.00638
4
6
Yes


TNFAIP6.IL8RB.KCNE3.TLR4
−0.00641
−0.00627
4
6
Yes


TNFAIP6.TNFRSF10C.KCNE3.TLR4
−0.00668
−0.00641
4
6
Yes


CASP5.TNFAIP6.IL8RB.TNFRSF10C
−0.00867
−0.0087
4
6
Yes


CASP5.TNFAIP6.IL8RB.KCNE3
−0.00781
−0.00814
4
6
Yes


CASP5.TNFAIP6.TNFRSF10C.KCNE3
−0.00926
−0.00908
4
6
Yes


CASP5.TNFAIP6.IL8RB.TLR4
−0.0068
−0.00666
4
6
Yes


CASP5.TNFAIP6.TNFRSF10C.TLR4
−0.00852
−0.00834
4
6
Yes


CASP5.TNFAIP6.KCNE3.TLR4
−0.00793
−0.00791
4
6
Yes


IL18RAP.TNFAIP6.IL8RB.TNFRSF10C
−6.34E−05
0.000813
4
6
Yes


IL18RAP.TNFAIP6.IL8RB.KCNE3
0.001542
0.002175
4
6
Yes


IL18RAP.TNFAIP6.TNFRSF10C.KCNE3
0.000285
0.00057
4
6
Yes


IL18RAP.TNFAIP6.IL8RB.TLR4
0.003347
0.004878
4
6
Yes


IL18RAP.TNFAIP6.TNFRSF10C.TLR4
0.001594
0.002502
4
6
Yes


IL18RAP.TNFAIP6.KCNE3.TLR4
0.001795
0.002566
4
6
Yes


CASP5.IL18RAP.TNFAIP6.IL8RB
−0.00015
0.001193
4
6
Yes


CASP5.IL18RAP.TNFAIP6.TNFRSF10C
−0.00385
−0.00361
4
6
Yes


CASP5.IL18RAP.TNFAIP6.KCNE3
−0.00364
−0.0031
4
6
Yes


CASP5.IL18RAP.TNFAIP6.TLR4
−0.00058
−0.00041
4
6
Yes


CASP5.IL8RB.TNFRSF10C
−0.01523
−0.01583
3
6
Yes


CASP5.IL8RB.KCNE3
−0.01468
−0.01507
3
6
Yes


CASP5.TNFRSF10C.KCNE3
−0.01494
−0.01568
3
6
Yes


CASP5.IL8RB.TLR4
−0.01426
−0.01487
3
6
Yes


CASP5.TNFRSF10C.TLR4
−0.01482
−0.0152
3
6
Yes


CASP5.KCNE3.TLR4
−0.01459
−0.01474
3
6
Yes


IL18RAP.IL8RB.TNFRSF10C
−0.01037
−0.00964
3
6
Yes


IL18RAP.IL8RB.KCNE3
−0.01
−0.00946
3
6
Yes


IL18RAP.TNFRSF10C.KCNE3
−0.01029
−0.00963
3
6
Yes


IL18RAP.IL8RB.TLR4
−0.00926
−0.00805
3
6
Yes


IL18RAP.TNFRSF10C.TLR4
−0.00991
−0.0089
3
6
Yes


IL18RAP.KCNE3.TLR4
−0.0091
−0.00833
3
6
Yes


CASP5.IL18RAP.IL8RB
−0.00718
−0.00648
3
6
Yes


CASP5.IL18RAP.TNFRSF10C
−0.00915
−0.00916
3
6
Yes


CASP5.IL18RAP.KCNE3
−0.00891
−0.00872
3
6
Yes


CASP5.IL18RAP.TLR4
−0.00688
−0.00775
3
6
Yes


TNFAIP6.IL8RB.TNFRSF10C
−0.00786
−0.00752
3
6
Yes


TNFAIP6.IL8RB.KCNE3
−0.00686
−0.00644
3
6
Yes


TNFAIP6.TNFRSF10C.KCNE3
−0.00771
−0.00749
3
6
Yes


TNFAIP6.IL8RB.TLR4
−0.00668
−0.0056
3
6
Yes


TNFAIP6.TNFRSF10C.TLR4
−0.00765
−0.0068
3
6
Yes


TNFAIP6.KCNE3.TLR4
−0.00623
−0.00608
3
6
Yes


CASP5.TNFAIP6.IL8RB
−0.0079
−0.00708
3
6
Yes


CASP5.TNFAIP6.TNFRSF10C
−0.00996
−0.00988
3
6
Yes


CASP5.TNFAIP6.KCNE3
−0.00935
−0.00929
3
6
Yes


CASP5.TNFAIP6.TLR4
−0.00745
−0.00787
3
6
Yes


IL18RAP.TNFAIP6.IL8RB
0.000549
0.00189
3
6
Yes


IL18RAP.TNFAIP6.TNFRSF10C
−0.00174
−0.00122
3
6
Yes


IL18RAP.TNFAIP6.KCNE3
−0.00038
0.00037
3
6
Yes


IL18RAP.TNFAIP6.TLR4
0.001552
0.002249
3
6
Yes


CASP5.IL8RB
−0.01498
−0.01537
2
6
Yes


CASP5.TNFRSF10C
−0.01527
−0.01602
2
6
Yes


CASP5.KCNE3
−0.01471
−0.0152
2
6
Yes


CASP5.TLR4
−0.01426
−0.01449
2
6
Yes


IL18RAP.IL8RB
−0.00983
−0.00922
2
6
Yes


IL18RAP.TNFRSF10C
−0.01126
−0.01029
2
6
Yes


IL18RAP.KCNE3
−0.0097
−0.01001
2
6
Yes


IL18RAP.TLR4
−0.00878
−0.00829
2
6
Yes


TNFAIP6.IL8RB
−0.00724
−0.00667
2
6
Yes


TNFAIP6.TNFRSF10C
−0.00924
−0.00868
2
6
Yes


TNFAIP6.KCNE3
−0.00752
−0.00694
2
6
Yes


TNFAIP6.TLR4
−0.00663
−0.00632
2
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01883
−0.01929
7
6
Yes


H3F3B.TXN.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01979
−0.02006
6
6
Yes


H3F3B.PLAUR.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01852
−0.01892
6
6
Yes


TXN.PLAUR.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01845
−0.01856
6
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.PTAFR.LAMP2
−0.01891
−0.01913
6
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.PTAFR.TYROBP
−0.01909
−0.01945
6
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.LAMP2.TYROBP
−0.01989
−0.02008
6
6
Yes


H3F3B.TXN.PLAUR.PTAFR.LAMP2.TYROBP
−0.01823
−0.01798
6
6
Yes


H3F3B.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01985
−0.01997
5
6
Yes


TXN.BCL2A1.PTAFR.LAMP2.TYROBP
−0.01978
−0.0199
5
6
Yes


H3F3B.TXN.BCL2A1.PTAFR.LAMP2
−0.0195
−0.01968
5
6
Yes


H3F3B.TXN.BCL2A1.PTAFR.TYROBP
−0.02001
−0.02022
5
6
Yes


H3F3B.TXN.BCL2A1.LAMP2.TYROBP
−0.02077
−0.02088
5
6
No


H3F3B.TXN.PTAFR.LAMP2.TYROBP
−0.01929
−0.01949
5
6
Yes


PLAUR.BCL2A1.PTAFR.LAMP2.TYROBP
−0.0192
−0.0192
5
6
Yes


H3F3B.PLAUR.BCL2A1.PTAFR.LAMP2
−0.01879
−0.01932
5
6
Yes


H3F3B.PLAUR.BCL2A1.PTAFR.TYROBP
−0.01904
−0.0194
5
6
Yes


H3F3B.PLAUR.BCL2A1.LAMP2.TYROBP
−0.01934
−0.01953
5
6
Yes


H3F3B.PLAUR.PTAFR.LAMP2.TYROBP
−0.01801
−0.01798
5
6
Yes


TXN.PLAUR.BCL2A1.PTAFR.LAMP2
−0.01863
−0.01884
5
6
Yes


TXN.PLAUR.BCL2A1.PTAFR.TYROBP
−0.01971
−0.01955
5
6
Yes


TXN.PLAUR.BCL2A1.LAMP2.TYROBP
−0.01923
−0.01932
5
6
Yes


TXN.PLAUR.PTAFR.LAMP2.TYROBP
−0.01834
−0.01792
5
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.PTAFR
−0.01945
−0.01958
5
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.LAMP2
−0.01999
−0.02012
5
6
Yes


H3F3B.TXN.PLAUR.PTAFR.LAMP2
−0.01816
−0.01784
5
6
Yes


H3F3B.TXN.PLAUR.BCL2A1.TYROBP
−0.02038
−0.02064
5
6
Yes


H3F3B.TXN.PLAUR.PTAFR.TYROBP
−0.01949
−0.0195
5
6
Yes


H3F3B.TXN.PLAUR.LAMP2.TYROBP
−0.01904
−0.01909
5
6
Yes


H3F3B.BCL2A1.PTAFR.LAMP2
−0.01974
−0.02007
4
6
Yes


H3F3B.BCL2A1.PTAFR.TYROBP
−0.01984
−0.02014
4
6
Yes


H3F3B.BCL2A1.LAMP2.TYROBP
−0.01983
−0.02014
4
6
Yes


H3F3B.PTAFR.LAMP2.TYROBP
−0.01896
−0.01932
4
6
Yes


TXN.BCL2A1.PTAFR.LAMP2
−0.01948
−0.01943
4
6
Yes


TXN.BCL2A1.PTAFR.TYROBP
−0.02009
−0.02005
4
6
Yes


TXN.BCL2A1.LAMP2.TYROBP
−0.02014
−0.02012
4
6
Yes


TXN.PTAFR.LAMP2.TYROBP
−0.01931
−0.01992
4
6
Yes


H3F3B.TXN.BCL2A1.PTAFR
−0.02017
−0.02014
4
6
Yes


H3F3B.TXN.BCL2A1.LAMP2
−0.02053
−0.02056
4
6
No


H3F3B.TXN.PTAFR.LAMP2
−0.01819
−0.01867
4
6
Yes


H3F3B.TXN.BCL2A1.TYROBP
−0.02078
−0.02122
4
6
No


H3F3B.TXN.PTAFR.TYROBP
−0.02018
−0.02033
4
6
Yes


H3F3B.TXN.LAMP2.TYROBP
−0.01999
−0.02022
4
6
Yes


PLAUR.BCL2A1.PTAFR.LAMP2
−0.0192
−0.01915
4
6
Yes


PLAUR.BCL2A1.PTAFR.TYROBP
−0.01966
−0.02001
4
6
Yes


PLAUR.BCL2A1.LAMP2.TYROBP
−0.01947
−0.01983
4
6
Yes


PLAUR.PTAFR.LAMP2.TYROBP
−0.01847
−0.01839
4
6
Yes


H3F3B.PLAUR.BCL2A1.PTAFR
−0.01925
−0.01924
4
6
Yes


H3F3B.PLAUR.BCL2A1.LAMP2
−0.01962
−0.01999
4
6
Yes


H3F3B.PLAUR.PTAFR.LAMP2
−0.01779
−0.01779
4
6
Yes


H3F3B.PLAUR.BCL2A1.TYROBP
−0.01978
−0.02003
4
6
Yes


H3F3B.PLAUR.PTAFR.TYROBP
−0.01903
−0.01932
4
6
Yes


H3F3B.PLAUR.LAMP2.TYROBP
−0.01835
−0.01842
4
6
Yes


TXN.PLAUR.BCL2A1.PTAFR
−0.01895
−0.0189
4
6
Yes


TXN.PLAUR.BCL2A1.LAMP2
−0.01947
−0.01941
4
6
Yes


TXN.PLAUR.PTAFR.LAMP2
−0.01748
−0.01746
4
6
Yes


TXN.PLAUR.BCL2A1.TYROBP
−0.02086
−0.02136
4
6
No


TXN.PLAUR.PTAFR.TYROBP
−0.01936
−0.01917
4
6
Yes


TXN.PLAUR.LAMP2.TYROBP
−0.01876
−0.01874
4
6
Yes


H3F3B.TXN.PLAUR.BCL2A1
−0.02057
−0.02098
4
6
No


H3F3B.TXN.PLAUR.PTAFR
−0.0182
−0.01799
4
6
Yes


H3F3B.TXN.PLAUR.LAMP2
−0.0185
−0.0186
4
6
Yes


H3F3B.TXN.PLAUR.TYROBP
−0.02026
−0.02086
4
6
Yes


H3F3B.BCL2A1.PTAFR
−0.0199
−0.02011
3
6
Yes


H3F3B.BCL2A1.LAMP2
−0.02034
−0.02054
3
6
Yes


H3F3B.PTAFR.LAMP2
−0.01899
−0.01887
3
6
Yes


H3F3B.BCL2A1.TYROBP
−0.02078
−0.02105
3
6
No


H3F3B.PTAFR.TYROBP
−0.02001
−0.02029
3
6
Yes


H3F3B.LAMP2.TYROBP
−0.01974
−0.02007
3
6
Yes


TXN.BCL2A1.PTAFR
−0.01986
−0.01992
3
6
Yes


TXN.BCL2A1.LAMP2
−0.01978
−0.02016
3
6
Yes


TXN.PTAFR.LAMP2
−0.01891
−0.01887
3
6
Yes


TXN.BCL2A1.TYROBP
−0.02107
−0.02132
3
6
No


TXN.PTAFR.TYROBP
−0.02
−0.02024
3
6
Yes


TXN.LAMP2.TYROBP
−0.01962
−0.02003
3
6
Yes


H3F3B.TXN.BCL2A1
−0.0211
−0.02123
3
6
No


H3F3B.TXN.PTAFR
−0.01906
−0.01911
3
6
Yes


H3F3B.TXN.LAMP2
−0.0193
−0.01958
3
6
Yes


H3F3B.TXN.TYROBP
−0.0212
−0.02144
3
6
No


PLAUR.BCL2A1.PTAFR
−0.01948
−0.01956
3
6
Yes


PLAUR.BCL2A1.LAMP2
−0.01952
−0.0198
3
6
Yes


PLAUR.PTAFR.LAMP2
−0.01797
−0.01784
3
6
Yes


PLAUR.BCL2A1.TYROBP
−0.02012
−0.02041
3
6
Yes


PLAUR.PTAFR.TYROBP
−0.01907
−0.01933
3
6
Yes


PLAUR.LAMP2.TYROBP
−0.01889
−0.01879
3
6
Yes


H3F3B.PLAUR.BCL2A1
−0.02017
−0.02029
3
6
Yes


H3F3B.PLAUR.PTAFR
−0.01819
−0.01819
3
6
Yes


H3F3B.PLAUR.LAMP2
−0.01851
−0.01851
3
6
Yes


H3F3B.PLAUR.TYROBP
−0.01981
−0.01982
3
6
Yes


TXN.PLAUR.BCL2A1
−0.02091
−0.02139
3
6
No


TXN.PLAUR.PTAFR
−0.01811
−0.01817
3
6
Yes


TXN.PLAUR.LAMP2
−0.01777
−0.01807
3
6
Yes


TXN.PLAUR.TYROBP
−0.02058
−0.02118
3
6
No


H3F3B.BCL2A1
−0.02045
−0.02078
2
6
No


H3F3B.PTAFR
−0.01928
−0.0197
2
6
Yes


H3F3B.LAMP2
−0.01952
−0.01982
2
6
Yes


H3F3B.TYROBP
−0.02049
−0.02082
2
6
No


TXN.BCL2A1
−0.02134
−0.02159
2
6
No


TXN.PTAFR
−0.01891
−0.01933
2
6
Yes


TXN.LAMP2
−0.01943
−0.0199
2
6
Yes


TXN.TYROBP
−0.02144
−0.02172
2
6
No


PLAUR.BCL2A1
−0.0202
−0.02062
2
6
Yes


PLAUR.PTAFR
−0.01811
−0.01813
2
6
Yes


PLAUR.LAMP2
−0.01833
−0.01797
2
6
Yes


PLAUR.TYROBP
−0.01995
−0.02038
2
6
Yes


CASP5.IL8RB1
−0.01498
−0.01537
2
6
Yes


CASP5.TNFRSF10C1
−0.01527
−0.01602
2
6
Yes


CASP5.KCNE31
−0.01471
−0.0152
2
6
Yes


CASP5.TLR41
−0.01426
−0.01449
2
6
Yes


CASP5.BCL2A1
−0.01551
−0.016
2
6
Yes


CASP5.PTAFR
−0.01515
−0.01538
2
6
Yes


CASP5.LAMP2
−0.0153
−0.01552
2
6
Yes


CASP5.TYROBP
−0.01642
−0.01626
2
6
Yes


IL18RAP.IL8RB1
−0.00983
−0.00922
2
6
Yes


IL18RAP.TNFRSF10C1
−0.01126
−0.01029
2
6
Yes


IL18RAP.KCNE31
−0.0097
−0.01001
2
6
Yes


IL18RAP.TLR41
−0.00878
−0.00829
2
6
Yes


IL18RAP.BCL2A1
−0.01217
−0.01143
2
6
Yes


IL18RAP.PTAFR
−0.01153
−0.01101
2
6
Yes


IL18RAP.LAMP2
−0.01012
−0.00998
2
6
Yes


IL18RAP.TYROBP
−0.01198
−0.01196
2
6
Yes


TNFAIP6.IL8RB1
−0.00724
−0.00667
2
6
Yes


TNFAIP6.TNFRSF10C1
−0.00924
−0.00868
2
6
Yes


TNFAIP6.KCNE31
−0.00752
−0.00694
2
6
Yes


TNFAIP6.TLR41
−0.00663
−0.00632
2
6
Yes


TNFAIP6.BCL2A1
−0.0102
−0.0097
2
6
Yes


TNFAIP6.PTAFR
−0.00952
−0.00906
2
6
Yes


TNFAIP6.LAMP2
−0.00845
−0.00774
2
6
Yes


TNFAIP6.TYROBP
−0.01093
−0.0105
2
6
Yes


H3F3B.IL8RB
−0.0199
−0.02009
2
6
Yes


H3F3B.TNFRSF10C
−0.01956
−0.01973
2
6
Yes


H3F3B.KCNE3
−0.01699
−0.0169
2
6
Yes


H3F3B.TLR4
−0.01707
−0.01722
2
6
Yes


H3F3B.BCL2A1
−0.02045
−0.02078
2
6
No


H3F3B.PTAFR
−0.01928
−0.0197
2
6
Yes


H3F3B.LAMP2
−0.01952
−0.01982
2
6
Yes


H3F3B.TYROBP
−0.02049
−0.02082
2
6
No


TXN.IL8RB
−0.02074
−0.02073
2
6
No


TXN.TNFRSF10C
−0.02084
−0.02143
2
6
No


TXN.KCNE3
−0.01775
−0.01731
2
6
Yes


TXN.TLR4
−0.01782
−0.01819
2
6
Yes


TXN.BCL2A1
−0.02134
−0.02159
2
6
No


TXN.PTAFR
−0.01891
−0.01933
2
6
Yes


TXN.LAMP2
−0.01943
−0.0199
2
6
Yes


TXN.TYROBP
−0.02144
−0.02172
2
6
No


PLAUR.IL8RB
−0.01926
−0.0191
2
6
Yes


PLAUR.TNFRSF10C
−0.01955
−0.01981
2
6
Yes


PLAUR.KCNE3
−0.01482
−0.01437
2
6
Yes


PLAUR.TLR4
−0.0171
−0.01682
2
6
Yes


PLAUR.BCL2A1
−0.0202
−0.02062
2
6
Yes


PLAUR.PTAFR
−0.01811
−0.01813
2
6
Yes


PLAUR.LAMP2
−0.01833
−0.01797
2
6
Yes


PLAUR.TYROBP
−0.01995
−0.02038
2
6
Yes





















TABLE 23






Delta AUC
Delta AUC






Ridge
Logistic


Markers
Regression
Regression
Markers
Term
Predictive




















CD3D.TMC8.SLAMF7.KLRC4
0
0
4
7
Yes


CD3D.SLAMF7.KLRC4
−6.34E−05
1.06E−05
3
7
Yes


TMC8.SLAMF7.KLRC4
−0.00094
−0.00103
3
7
Yes


CD3D.TMC8.SLAMF7
0.003432
0.004065
3
7
Yes


CD3D.TMC8.KLRC4
−0.00269
−0.00335
3
7
Yes


CD3D.SLAMF7
0.00226
0.002766
2
7
Yes


CD3D.KLRC4
−0.0027
−0.00332
2
7
Yes


TMC8.SLAMF7
0.001594
0.001827
2
7
Yes


TMC8.KLRC4
−0.00351
−0.00365
2
7
Yes


LCK.CCT2.CX3CR1.CD8A
−0.01192
−0.01099
4
7
No


LCK.CX3CR1.CD8A
−0.01192
−0.01143
3
7
No


CCT2.CX3CR1.CD8A
−0.01192
−0.01216
3
7
No


LCK.CCT2.CX3CR1
−0.00644
−0.00646
3
7
Yes


LCK.CCT2.CD8A
−0.01323
−0.01304
3
7
No


LCK.CX3CR1
−0.00687
−0.00729
2
7
Yes


LCK.CD8A
−0.01382
−0.01289
2
7
No


CCT2.CX3CR1
−0.00646
−0.0061
2
7
Yes


CCT2.CD8A
−0.01287
−0.01253
2
7
No


CD3D.SLAMF7
0.00226
0.002766
2
7
Yes


CD3D.KLRC4
−0.0027
−0.00332
2
7
Yes


CD3D.CX3CR1
−0.00589
−0.00615
2
7
Yes


CD3D.CD8A
−0.01374
−0.01231
2
7
No


TMC8.SLAMF7
0.001594
0.001827
2
7
Yes


TMC8.KLRC4
−0.00351
−0.00365
2
7
Yes


TMC8.CX3CR1
−0.00572
−0.00602
2
7
Yes


TMC8.CD8A
−0.01199
−0.01121
2
7
No


LCK.SLAMF7
0.000116
0.000285
2
7
Yes


LCK.KLRC4
−0.00436
−0.0045
2
7
Yes


LCK.CX3CR1
−0.00687
−0.00729
2
7
Yes


LCK.CD8A
−0.01382
−0.01289
2
7
No


CCT2.SLAMF7
0.001795
0.002154
2
7
Yes


CCT2.KLRC4
−0.00403
−0.00408
2
7
Yes


CCT2.CX3CR1
−0.00646
−0.0061
2
7
Yes


CCT2.CD8A
−0.01287
−0.01253
2
7
No
















TABLE 24







Clinical and Demographic Characteristics of the Final Development and Validation Patient Sets1










Development
Validation














Obstructive
No Obstructive

Obstructive
No Obstructive




CAD2
CAD

CAD
CAD


Characteristic
(N = 230)
(N = 410)
P-value
(N = 192)
(N = 334)
P-value




















Age, mean (SD), y
63.7
(11.1)
57.2
(11.8)
<0.001
64.7
(9.8)
57.7
(11.7)
<0.001


Men, No. (%)
180
(78.3%)
193
(47.1%)
<0.001
134
(69.8%)
165
(49.4%)
<0.001


Chest pain type




<0.001




<0.001


Typical
61
(26.5%)
66
(16.1%)

42
(21.9%)
41
(12.3%)


Atypical
28
(12.2%)
56
(13.7%)

42
(21.9%)
49
(14.7%)


Non-cardiac
47
(20.4%)
137
(33.4%)

50
(26.0%)
134
(40.1%)


None
91
(39.6%)
143
(34.9%)

58
(30.2%)
109
(32.6%)


Blood pressure, mean (SD), mmHg


Systolic
138
(17.7)
133
(18.3)
<0.001
140
(17.7)
132
(18.1)
<0.001


Diastolic
79.7
(11.0)
79.6
(11.7)
0.94
79.2
(11.3)
77.5
(10.9)
0.09


Hypertension
163
(70.9%)
237
(57.8%)
  0.002
142
(74.0%)
203
(60.8%)
  0.001


Dyslipidemia
170
(73.9%)
225
(54.9%)
<0.001
133
(69.3%)
208
(62.3%)
0.11


Curent smoking
53
(23.2%)
99
(24.3%)
0.75
38
(19.8%)
68
(20.4%)
0.70


BMI, mean (SD), kg/m2
30.5
(6.0)
31.0
(7.5)
0.35
29.8
(5.5)
31.3
(7.0)
0.01


Ethnicity, White not Hispanic
210
(91.3%)
347
(84.6%)
  0.016
181
(94.3%)
293
(87.7%)
0.02


Clinical syndrome


Stable angina
123
(53.5%)
214
(52.2%)
0.78
107
(55.7%)
176
(52.7%)
0.46


Unstable angina
35
(15.2%)
81
(19.8%)
0.15
31
(16.1%)
58
(17.4%)
0.74


Asymptomatic, high risk
72
(31.3%)
113
(27.6%)
0.32
53
(27.6%)
100
(29.9%)
0.60


Medications


Aspirin and salicylates
153
(66.5%)
232
(56.6%)
0.03
139
(72.4%)
205
(61.4%)
0.01


Statins
109
(47.4%)
142
(34.6%)
  0.003
93
(48.4%)
127
(38.0%)
0.02


Beta blockers
82
(35.7%)
133
(32.4%)
0.52
85
(44.3%)
124
(37.1%)
0.11


ACE inhibitors
57
(24.8%)
67
(16.3%)
0.01
47
(24.5%)
64
(19.2%)
0.16


Angiotensin receptor
29
(12.6%)
39
(9.5%)
0.26
18
(9.4%)
34
(10.2%)
0.76


blockers


Calcium channel blockers
33
(14.3%)
46
(11.2%)
0.29
26
(13.5%)
34
(10.2%)
0.25


Antiplatelet agents
27
(11.7%)
21
(5.1%)
  0.003
16
(8.3%)
17
(5.1%)
0.14


Steroids, not systemic
23
(10.0%)
33
(8.0%)
0.45
19
(9.9%)
38
(11.4%)
0.59


NSAIDS
47
(20.4%)
78
(19.0%)
0.76
30
(15.6%)
58
(17.4%)
0.60





1Characteristics of the 640 subjects in the Algorithm Development and 526 subjects in the Validation sets. P values were calculated by t-tests for continuous variables and using chi-square tests for discrete variables. Significant p values in both sets are bolded and underlined and are bolded if significant in single sets.


2Obstructive CAD is defined as >50% luminal stenosis in ≧1 major vessel by QCA.













TABLE 25A







Reclassification analysis of Gene Expression Algorithm


with Diamond-Forrester Clinical Model











With Gene Expression





Algorithm

Reclassified %















Low
Int.
High
Total
Lower
Higher
Total


















D-F Low Risk









Patients included
118
96
38
252
0.0
15.1
15.1


Disease pts
16
19
22
57
0.0
38.6
38.6


Non disease pts
102
77
16
195
0.0
8.2
8.2


Observed risk
14%
20%
58%
23%
—
—
—


D-F Int Risk


Patients included
28
21
47
96
29.2
49.0
78.1


Disease pts
7
11
26
44
15.9
59.1
75.0


Non disease pts
21
10
21
52
40.4
40.4
80.8


Observed risk
25%
52%
55%
46%
—
—
—


D-F High Risk


Patients included
28
60
89
177
15.8
0.0
15.8


Disease pts
6
29
56
91
6.6
0.0
6.6


Non disease pts
22
31
33
86
38.4
0.0
38.4


Observed risk
21%
48%
63%
51%
—
—
—


Total Patients
174
77
174
525


included


Disease pts
29
59
104
192


Non disease pts
145
118
70
333


Observed risk
17%
33%
60%
37%





Risk categories:: Low = 0-<20%, Intermediate = ≧20-50%, High = ≧50%.


Classification improved in 18.2% of disease patients and improved in 1.8% of non disease patients for a net reclassification improvement of 20.0% (p < .001)













TABLE 25B







Reclassification analysis of Gene Expression


Algorithm with MPI Results











With Gene Expression





Algorithm

Reclassified %















Low
Int.
High
Total
Lower
Higher
Total


















MPI Negative









Patients included
41
31
15
87
0.0
17.4
17.4


Disease pts
7
8
7
22
0.0
31.8
31.8


Non disease pts
34
23
8
65
0.0
12.3
12.3


Observed risk
17%
26%
47%
25%
—
—
—


MPI Positive


Patients included
57
78
88
223
25.6
0.0
25.6


Disease pts
6
21
49
76
7.9
0.0
7.9


Non disease pts
51
57
39
147
34.7
0.0
34.7


Observed risk
11%
27%
56%
34%
—
—
—


Total
98
109
103
310


Patients included


Disease pts
13
29
56
98


Non disease pts
85
80
47
212


Observed risk
13%
27%
54%
32%





Risk categories:: Low = 0-<20%, Intermediate = ≧20-50%, High = ≧50%.


Classification improved in 1.0% of disease patients and improved in 20.3% of non disease patients for a net reclassification improvement of 21.3% (p < .001)
















SEQUENCE LISTING


Primers and Probes











Assay ID
Symbol
Forward Primer
Reverse Primer
Probe





CDXR0728-SP1
AF289562
ACAGGAGGGAGGGAT
GCCAATCACCTGCCTAAT
TCAGGCAGCCCC




GCA
GC
CCAGAG




(SEQ. ID NO. 1)
(SEQ. ID NO. 2)
(SEQ. ID NO. 3)


 


CDXR0868-SP1
AQP9
ACCTGAGTCCCAGACT
CCACTACAGGAATCCAC
CTTCAGAGCTGG




TTTCACT
CAGAAG
AAACAA




(SEQ. ID NO. 4)
(SEQ. ID NO. 5)
(SEQ. ID NO. 6)


 


CDXR0830-SP1
CASP5
CGAGCAACCTTGACAA
GGTAAATGTGCTCTTTGA
CCTGTGGTTTCAT




GAGATTTC
TGTTGACA
TTTC




(SEQ. ID NO. 7)
(SEQ. ID NO. 8)
(SEQ. ID NO. 9)


 


CDXR0884-SP2
CD79B
CAGACGCTGCTGATCA
TCGTAGGTGTGATCTTCC
CCTTGCTGTCAT




TCCT
TCCAT
CCTTGTC




(SEQ. ID NO. 10)
(SEQ. ID NO. 11)
(SEQ. ID NO. 12)


 


CDXR0863-SP1
CLEC4E
GGACGGCACACCTTTG
CCTCCAGGGTAGCTATG
CCCAGAAGCTCA




ACA
TTGTTG
GAGACT




(SEQ. ID NO. 13)
(SEQ. ID NO. 14)
(SEQ. ID NO. 15)


 


CDXR0080-SP1
IL18RAP
AGCCTGTGTTTGCTTG
TCTTCTGCTTCTCTTAAT
TCTTCTGCATACA




AAAGAGAT
AATGCTCACAA
CTCCTCC




(SEQ. ID NO. 16)
(SEQ. ID NO. 17)
(SEQ. ID NO. 18)


 


CDXR0832-SP1
IL8RB
CCCCATTGTGGTCACA
CCAGGGCAAGCTTTCTA
ACGTTCTTACTAG




GGAA
AACCAT
TTTCCC




(SEQ. ID NO. 19)
(SEQ. ID NO. 20)
(SEQ. ID NO. 21)


 


CDXR0888-SP0
KCNE3
TCTCTAAGGCTCTATC
GCTGGAACCATATATGAA
CCTACAAACACA




AGTTCTGACAT
ACTACGATACT
GTGATTACA




(SEQ. ID NO. 22)
(SEQ. ID NO. 23)
(SEQ. ID NO. 24)


 


CDXR0861-SP1
KLRC4
TGTATTGGAGTACTGG
CTGTTGGAATATGTAATC
CAATGACGTGCT




AGCAGAACA
CACTCCTCA
TTCTG




(SEQ. ID NO. 25)
(SEQ. ID NO. 26)
(SEQ. ID NO. 27)


 


CDXR0826-SP1
NCF4
CTCCCAGAAGCGCCTC
GGGACACCGTCAGCTCA
CACGCAGAAGGA




TT
TG
CAACT




(SEQ. ID NO. 28)
(SEQ. ID NO. 29)
(SEQ. ID NO. 30)


 


CDXR0056P1-
S100A12
TCTCTAAGGGTGAGCT
CCAGGCCTTGGAATATTT
CAAACACCATCAA


SP1

GAAGCA
CATCAATG
GAATAT




(SEQ. ID NO. 31)
(SEQ. ID NO. 32)
(SEQ. ID NO. 33)


 


CDXR0069P1-
S100A8
GAAGAAATTGCTAGAG
GCACCATCAGTGTTGATA
CACCCTTTTTCCT


SP1

ACCGAGTGT
TCCAACT
GATATACT




(SEQ. ID NO. 34)
(SEQ. ID NO. 35)
(SEQ. ID NO. 36)


 


CDXR0663-SP1
SLAMF7
AGCAAATACGGTTTAC
GGCATCGTGAGCAGTGA
TTTTCCATCTTTT




TCCACTGT
GT
TCGGTATTTC




(SEQ. ID NO. 37)
(SEQ. ID NO. 38)
(SEQ. ID NO. 39)


 


CDXR0840-SP1
SPIB
GAGGCCCTCGTGGCT
TGGTACAGGCGCAGCTT
CTTGCGAGTCCC




(SEQ. ID NO. 40)
(SEQ. ID NO. 41)
TGCCTC






(SEQ. ID NO. 42)


 


CDXR0672-SP1
TFCP2
ACAGAACTTTCAGGAA
CCGCACTCCTACTTCAGT
ACAATGAAAGCA




GAAGCATGT
ATGAT
GAAACC




(SEQ. ID NO. 43)
(SEQ. ID NO. 44)
(SEQ. ID NO. 45)


 


CDXR0891-SP0
TLR4
GGGAAGAGTGGATGTT
GGATGAACATTCTTTTCT
ATGTGTCTGGAA




ATCATTGAGAA
GGGAACCT
TTAATG




(SEQ. ID NO. 46)
(SEQ. ID NO. 47)
(SEQ. ID NO. 48)


 


CDXR0876-SP1
TMC8
CACAGGCTCCGGAAG
CGCGACAGGTCCTCCAC
CTGGTGTGGCAG




CA
(SEQ. ID NO. 50)
GTTC




(SEQ. ID NO. 49)

(SEQ. ID NO. 51)


 


CDXR0857-SP1
TNFAIP6
GGAGATGAGCTTCCAG
AGCTGTCACTGAAGCAT
CATCAGTACAGG




ATGACAT
CACTTAG
AAATGTC




(SEQ. ID NO. 52)
(SEQ. ID NO. 53)
(SEQ. ID NO. 54)


 


CDXR0844-SP1
TNFRSF10C
GGAATGAAAACTCCCC
CAGGACGTACAATTACTG
CTAGGGCACCTG




AGAGATGTG
ACTTGGA
CTACAC




(SEQ. ID NO. 55)
(SEQ. ID NO. 56)
(SEQ. ID NO. 57)


 


CDXR0121-SP1
AF161365
GCCTTGGAACACACCT
CAGGACACACTTCCGAT
CCCCAGGAGTTG




TCGT
GGATTTA
CTG




(SEQ. ID NO. 58)
(SEQ. ID NO. 59)
(SEQ. ID NO. 60)


 


CDXR0703-SP1
HNRPF
CCAGAAGTGTCTCCCA
GGTGATCTTGGGTGTGG
TTTGTGGCTTAAA




CTGAAG
CTTT
AACAACC




(SEQ. ID NO. 61)
(SEQ. ID NO. 62)
(SEQ. ID NO. 63)


 


A23P208358-188
RPL28
CGGACCACCATCAACA
TTCTTGCGGATCATGTGT
CTCGCGCCACGC




AGAATG
CTGA
TCA




(SEQ. ID NO. 64)
(SEQ. ID NO. 65)
(SEQ. ID NO. 66)

## Claims

1. A method for determining coronary artery disease risk in a subject comprising:
(i) performing a reverse transcriptase polymerase chain reaction (RT-PCR) assay on a sample from the subject by using a plurality of distinct primer and probe sets that specifically hybridize to mRNA corresponding to each of the following genes consisting of: S100A12, CLEC4E, S100A8, CASP5, IL18RAP, TNFAIP6, AQP9, NCF4, CD3D, TMC8, CD79B, SPIB, HNRPF, TFCP2, RPL28, AF161365, AF289562, SLAMF7, KLRC4, IL8RB, TNFRSF 10C, KCNE3, and TLR4; (ii) generating, based on said assay, data representing mRNA expression levels corresponding to each of said genes; (iii) obtaining data representing age of the subject and gender of the subject; and (iv) generating, by a computer processor, a score indicative of coronary artery disease (CAD) risk by mathematically combining the data representing the mRNA expression levels, and the data representing the age and gender of the subject, wherein a higher score relative to a control subject having <50% stenosis in all major vessels indicates an increased likelihood that the subject has CAD.

2. The method of claim 1, further comprising generating a Final Score by applying the equation: Final Score=score*40/4.52, wherein a score that is <−2.95 is rounded up to −2.95 before applying the equation; wherein a score that is >1.57 is rounded down to 1.57 before applying the equation; wherein a Final Score that is >40 is rounded down to 40, and wherein a Final Score that is <1 is rounded up to 1.

3. The method of claim 1, further comprising classifying the sample according to the score.

4. The method of claim 1, further comprising rating CAD risk using the score.

5. The method of claim 1, wherein the sample comprises RNA extracted from peripheral blood cells.

6. The method of claim 1, wherein the CAD is obstructive CAD.

7. The method of claim 1, wherein the subject has stable chest pain, the subject has typical angina or atypical angina or an anginal equivalent, the subject has no previous diagnosis of myocardial infarction (MI), the subject has not had a revascularization procedure, the subject does not have diabetes, the subject does not have a systemic autoimmune or infectious condition, and/or the subject is not currently taking a steroid, an immunosuppressive agent, or a chemotherapeutic agent.