Susan E. Daniels

Correlation of peripheral-blood gene expression with the extent of coronary artery stenosis.

Background—The molecular pathophysiology of coronary artery disease (CAD) includes cytokine release and a localized inflammatory response within the vessel wall. The extent to which CAD and its severity is reflected by gene expression in circulating cells is unknown. Methods and Results—From an initial coronary catheterization cohort we identified 41 patients, comprising 27 cases with angiographically significant CAD and 14 controls without coronary stenosis. Whole-genome microarray analysis performed on peripheral-blood mononuclear cells yielded 526 genes with >1.3-fold differential expression (P<0.05) between cases and controls. Real-time polymerase chain reaction on 106 genes (the 50 most significant microarray genes and 56 additional literature genes) in an independent subset of 95 patients (63 cases, 32 controls) from the same cohort yielded 14 genes (P<0.05) that independently discriminated CAD state in a multivariable analysis that included clinical and demographic factors. From an independent second catheterization cohort, 215 patients were selected for real-time polymerase chain reaction–based replication. A case:control subset of 107 patients (86 cases, 21 controls) replicated 11 of the 14 multivariably significant genes from the first cohort. An analysis of the 14 genes in the entire set of 215 patients demonstrated that gene expression was proportional to maximal coronary artery stenosis (P<0.001 by ANOVA). Conclusions—Gene expression in peripheral-blood cells reflects the presence and extent of CAD in patients undergoing angiography.

Multicenter Validation of the Diagnostic Accuracy of a Blood-Based Gene Expression Test for Assessing Obstructive Coronary Artery Disease in Nondiabetic Patients

BACKGROUND Diagnosing obstructive coronary artery disease (CAD) in at-risk patients can be challenging and typically requires both noninvasive imaging methods and coronary angiography, the gold standard. Previous studies have suggested that peripheral blood gene expression can indicate the presence of CAD. OBJECTIVE To validate a previously developed 23-gene, expression-based classification test for diagnosis of obstructive CAD in nondiabetic patients. DESIGN Multicenter prospective trial with blood samples obtained before coronary angiography. (ClinicalTrials.gov registration number: NCT00500617) SETTING: 39 centers in the United States. PATIENTS An independent validation cohort of 526 nondiabetic patients with a clinical indication for coronary angiography. MEASUREMENTS Receiver-operating characteristic (ROC) analysis of classifier score measured by real-time polymerase chain reaction, additivity to clinical factors, and reclassification of patient disease likelihood versus disease status defined by quantitative coronary angiography. Obstructive CAD was defined as 50% or greater stenosis in 1 or more major coronary arteries by quantitative coronary angiography. RESULTS The area under the ROC curve (AUC) was 0.70 ± 0.02 (P < 0.001); the test added to clinical variables (Diamond-Forrester method) (AUC, 0.72 with the test vs. 0.66 without; P = 0.003) and added somewhat to an expanded clinical model (AUC, 0.745 with the test vs. 0.732 without; P = 0.089). The test improved net reclassification over both the Diamond-Forrester method and the expanded clinical model (P < 0.001). At a score threshold that corresponded to a 20% likelihood of obstructive CAD (14.75), the sensitivity and specificity were 85% and 43% (yielding a negative predictive value of 83% and a positive predictive value of 46%), with 33% of patient scores below this threshold. LIMITATION Patients with chronic inflammatory disorders, elevated levels of leukocytes or cardiac protein markers, or diabetes were excluded. CONCLUSION A noninvasive whole-blood test based on gene expression and demographic characteristics may be useful for assessing obstructive CAD in nondiabetic patients without known CAD. PRIMARY FUNDING SOURCE CardioDx.

Development of a blood-based gene expression algorithm for assessment of obstructive coronary artery disease in non-diabetic patients.

BackgroundAlterations in gene expression in peripheral blood cells have been shown to be sensitive to the presence and extent of coronary artery disease (CAD). A non-invasive blood test that could reliably assess obstructive CAD likelihood would have diagnostic utility.ResultsMicroarray analysis of RNA samples from a 195 patient Duke CATHGEN registry case:control cohort yielded 2,438 genes with significant CAD association (p < 0.05), and identified the clinical/demographic factors with the largest effects on gene expression as age, sex, and diabetic status. RT-PCR analysis of 88 CAD classifier genes confirmed that diabetic status was the largest clinical factor affecting CAD associated gene expression changes. A second microarray cohort analysis limited to non-diabetics from the multi-center PREDICT study (198 patients; 99 case: control pairs matched for age and sex) evaluated gene expression, clinical, and cell population predictors of CAD and yielded 5,935 CAD genes (p < 0.05) with an intersection of 655 genes with the CATHGEN results. Biological pathway (gene ontology and literature) and statistical analyses (hierarchical clustering and logistic regression) were used in combination to select 113 genes for RT-PCR analysis including CAD classifiers, cell-type specific markers, and normalization genes.RT-PCR analysis of these 113 genes in a PREDICT cohort of 640 non-diabetic subject samples was used for algorithm development. Gene expression correlations identified clusters of CAD classifier genes which were reduced to meta-genes using LASSO. The final classifier for assessment of obstructive CAD was derived by Ridge Regression and contained sex-specific age functions and 6 meta-gene terms, comprising 23 genes. This algorithm showed a cross-validated estimated AUC = 0.77 (95% CI 0.73-0.81) in ROC analysis.ConclusionsWe have developed a whole blood classifier based on gene expression, age and sex for the assessment of obstructive CAD in non-diabetic patients from a combination of microarray and RT-PCR data derived from studies of patients clinically indicated for invasive angiography.Clinical trial registration informationPREDICT, Personalized Risk Evaluation and Diagnosis in the Coronary Tree, http://www.clinicaltrials.gov, NCT00500617

A Blood-Based Gene Expression Test for Obstructive Coronary Artery Disease Tested in Symptomatic Nondiabetic Patients Referred for Myocardial Perfusion Imaging The COMPASS Study

Background—Obstructive coronary artery disease diagnosis in symptomatic patients often involves noninvasive testing before invasive coronary angiography. A blood-based gene expression score (GES) was previously validated in nondiabetic patients referred for invasive coronary angiography but not in symptomatic patients referred for myocardial perfusion imaging (MPI). Methods and Results—This prospective, multicenter study obtained peripheral blood samples for GES before MPI in 537 consecutive patients. Patients with abnormal MPI usually underwent invasive coronary angiography; all others had research coronary computed tomographic angiography, with core laboratories defining coronary anatomy. A total of 431 patients completed GES, coronary imaging (invasive coronary angiography or computed tomographic angiography), and MPI. Mean age was 56±10 years (48% women). The prespecified primary end point was GES receiver-operating characteristics analysis to discriminate ≥50% stenosis (15% prevalence by core laboratory analysis). Area under the receiver-operating characteristics curve for GES was 0.79 (95% confidence interval, 0.73–0.84; P<0.001), with sensitivity, specificity, and negative predictive value of 89%, 52%, and 96%, respectively, at a prespecified threshold of ⩽15 with 46% of patients below this score. The GES outperformed clinical factors by receiver-operating characteristics and reclassification analysis and showed significant correlation with maximum percent stenosis. Six-month follow-up on 97% of patients showed that 27 of 28 patients with adverse cardiovascular events or revascularization had GES >15. Site and core-laboratory MPI had areas under the curve of 0.59 and 0.63, respectively, significantly less than GES. Conclusions—GES has high sensitivity and negative predictive value for obstructive coronary artery disease. In this population clinically referred for MPI, the GES outperformed clinical factors and MPI. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01117506.

Identification of Factors Contributing to Variability in a Blood-Based Gene Expression Test

Background Corus CAD is a clinically validated test based on age, sex, and expression levels of 23 genes in whole blood that provides a score (1–40 points) proportional to the likelihood of obstructive coronary disease. Clinical laboratory process variability was examined using whole blood controls across a 24 month period: Intra-batch variability was assessed using sample replicates; inter-batch variability examined as a function of laboratory personnel, equipment, and reagent lots. Methods/Results To assess intra-batch variability, five batches of 132 whole blood controls were processed; inter-batch variability was estimated using 895 whole blood control samples. ANOVA was used to examine inter-batch variability at 4 process steps: RNA extraction, cDNA synthesis, cDNA addition to assay plates, and qRT-PCR. Operator, machine, and reagent lots were assessed as variables for all stages if possible, for a total of 11 variables. Intra- and inter-batch variations were estimated to be 0.092 and 0.059 Cp units respectively (SD); total laboratory variation was estimated to be 0.11 Cp units (SD). In a regression model including all 11 laboratory variables, assay plate lot and cDNA kit lot contributed the most to variability (p = 0.045; 0.009 respectively). Overall, reagent lots for RNA extraction, cDNA synthesis, and qRT-PCR contributed the most to inter-batch variance (52.3%), followed by operators and machines (18.9% and 9.2% respectively), leaving 19.6% of the variance unexplained. Conclusion Intra-batch variability inherent to the PCR process contributed the most to the overall variability in the study while reagent lot showed the largest contribution to inter-batch variability.

Natural fractures in a United Kingdom shale reservoir analog, Cleveland Basin, northeast England

Faults and fractures within the well-exposed Lower Jurassic Cleveland Ironstone and Whitby Mudstone Formations may provide insights into the tectonic history of gas-prospective, Mississippian shale in northern England. Subvertical opening mode fractures occur throughout the Cleveland Basin. Bed-parallel fractures, some of which contain blocky calcite fills, occur preferentially within well-bedded, clay-rich mudstones of the Cleveland Ironstone and Whitby Mudstone Formations at Jet Wyke and Port Mulgrave. Subvertical fractures display abutting or curving-parallel relationships with under- and overlying bed-parallel fractures. Together, these observations suggest that bed-parallel fractures, at times, acted as free surfaces. Some bed-parallel fractures curve toward and branch from calcite-filled fault slip surfaces, indicating that bed-parallel fracturing and normal faulting were synchronous, occurring within a regional stress field with vertical maximum principal stress. This apparent paradox can be explained by normal compaction, followed by cementation and coupling between pore pressure and minimum horizontal stress driven by poroelastic deformation or incipient slip along critically stressed normal faults, causing elevation of horizontal stress in excess of the vertical stress within clay-rich units. Propagation of bed-parallel fractures was enhanced by dilatational strains adjacent to normal fault planes. Bed-parallel fractures have not been observed within more -rich units at the top of the Whitby Mudstone Formation at Whitby East Cliff, or within well-bedded, clay-rich shale at Saltwick Nab. This observation is consistent with the lack of normal faulting at Saltwick Nab, and the Whitby Mudstone Formation having been drained by structural and/or stratigraphical juxtaposition against permeable Middle Jurassic sandstones at both these localities.

An age- and sex-specific gene expression score is associated with revascularization and coronary artery disease: Insights from the Prospective Multicenter Imaging Study for Evaluation of Chest Pain (PROMISE) trial

Background Identifying predictors of coronary artery disease (CAD)‐related procedures and events remains a priority. Methods We measured an age‐ and sex‐specific gene expression score (ASGES) previously validated to detect obstructive CAD (oCAD) in symptomatic nondiabetic patients in the PROMISE trial. The outcomes were oCAD (≥70% stenosis in ≥1 vessel or ≥50% left main stenosis on CT angiography [CTA]) and a composite endpoint of death, myocardial infarction, revascularization, or unstable angina. Results The ASGES was determined in 2370 nondiabetic participants (47.5% male, median age 59.5 years, median follow‐up 25 months), including 1137 with CTA data. An ASGES >15 was associated with oCAD (odds ratio 2.5 [95% CI 1.6‐3.8], P < .001) and the composite endpoint (hazard ratio [HR] 2.6 [95% CI 1.8‐3.9], P < .001) in unadjusted analyses. After adjustment for Framingham risk, an ASGES >15 remained associated with the composite endpoint (P = .02); the only component that was associated was revascularization (adjusted HR 2.69 [95% CI 1.52‐4.79], P < .001). Compared to noninvasive testing, the ASGES improved prediction for the composite (increase in c‐statistic = 0.036; continuous net reclassification index = 43.2%). Patients with an ASGES ≤15 had a composite endpoint rate no different from those with negative noninvasive test results (3.2% vs. 2.6%, P = .29). Conclusions A blood‐based genomic test for detecting oCAD significantly predicts near‐term revascularization procedures, but not non‐revascularization events. Larger studies will be needed to clarify the risk with non‐revascularization events.

A VALIDATED, PERIPHERAL BLOOD GENE EXPRESSION PATTERN CORRELATES WITH CORONARY ARTERIAL PLAQUE BURDEN BY QUANTITATIVE CORONARY ANGIOGRAPHY AND CORONARY ARTERY CALCIUM SCORING

Category: 48. Genetics and Clinical Outcomes Presentation Number: 924-4 Citation: J. Am. Coll. Cardiol. 2011;57;E1157 Authors: Szilard Voros, Michael R. Elashoff, Amy J. Sehnert, Hsiao D. Lieu, James A. Wingrove, Susan E. Daniels, Steven Rosenberg, Alexandra Lansky, Robert S. Schwartz, William E. Kraus, Eric J. Topol, Piedmont Heart Institute, Atlanta, GA, CardioDx Inc, Palo Alto, CA Abstract: Background: Coronary plaque burden can be measured indirectly by quantitative coronary angiography (QCA) and by coronary artery calcium (CAC) scoring by CT. We previously validated a real-time-PCR-based 23-geneexpression test, Corus CAD, to predict obstructive CAD using QCA (≥50% stenosis). Background: Coronary plaque burden can be measured indirectly by quantitative coronary angiography (QCA) and by coronary artery calcium (CAC) scoring by CT. We previously validated a real-time-PCR-based 23-geneexpression test, Corus CAD, to predict obstructive CAD using QCA (≥50% stenosis). Methods: In the validation cohort of the PREDICT study (NCT 00500617), 526 patients had gene expression score and QCA-derived plaque burden available and in 79 patients (49 cases, 30 controls), CAC score also. Plaque burden was defined as the sum of occluded luminal volumes for all QCA lesions; occluded volume is calculated from lesion length and reference and minimal vessel luminal diameter, assuming a symmetrical hourglass shape. Linear regression analysis was used to evaluate relationships. Results: In the 526 patients, there was significant correlation between gene expression score and QCA-derived plaque burden (p=1.4x10-8, R=0.38). In the CT subset, gene expression and CAC scores (range 0-3189; median=270) were correlated with R=0.63 (p=3x10-10). ROC analysis for the prediction of non-zero CAC by gene expression score showed an AUC 0.92. Both scores were significantly associated with disease burden by QCA (R=0.57 and 0.51,

GENE EXPRESSION SCORES FOR CAD ARE STABLE OVER ONE YEAR: RESULTS FROM THE COMPASS TRIAL

Background: Peripheral whole blood cell gene expression measurements have significant promise in diagnosis and monitoring of progressive inflammatory disorders such as atherosclerosis. However, the sensitivity of gene expression to environmental and clinical factors and thus stability over time have not been evaluated, and are key to clinical utility. We previously validated a gene expression score (GES, 1-40 scale) for obstructive CAD diagnosis in non--diabetic patients in the COMPASS (NCT01117506) trial and found a threshold of 15 to have a 96% negative predictive value. We also found real--time PCR laboratory based GES variation of 0.97 score units was substantially less than population variation of 10.5. In the current study we sought to determine the frequency and magnitude of changes in this GES over 1 year.