Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett’s Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Sample Collection
2.3. DNA Extraction from EsoCheck Balloon Samples
2.4. EsoGuard Assay
2.5. Bioinformatics Analysis
2.6. Sanger Sequencing
2.7. Statistical Methods
3. Results
3.1. Accuracy of Methylation Calling
3.2. Analytical Sensitivity, Specificity, and Accuracy
3.3. Bioinformatic Pipeline Accuracy
3.4. Accuracy for Sequencing Platform
4. Intra-Assay and Inter-Assay Precision
4.1. EsoGuard® Assay Precision
4.2. Assay Linearity and Limit of Detection (LOD)
4.3. Assay Input Range
4.4. Reference Range
4.5. Reportable Range:
4.6. Limit of Blank (LOB)
4.7. Sample Stability in Preservative Media
4.8. Interference Testing
4.9. Percentage of Samples near Cutoff
5. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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EG/EC Testing Performed in Academic Lab | EG/EC Testing Performed in Commercial Lab | |||
---|---|---|---|---|
Study Name | STM (Pivotal) study [16] | BETRNet Study [18] | Cleveland VA Study [17] | Lucid BE1 Study [19] |
Study Design | Case–control study | Case–control study | Single-arm study in intended use population | Single-arm study in intended use population |
Evaluable Subjects (n) | Total: 86 (36 controls and 50 cases) | Total: 243 (155 controls and 88 cases) | Total: 111 | Total: 93 |
Disease Distribution | 42 BE and 8 EAC cases | 70 BE and 18 EAC cases | 12 BE and 2 EAC cases | 8 BE cases |
BE and EAC Sensitivity (95%CI) | 88.0% (N/A) | 85% (78–93) | 92.9% (66.1–99.8) | 87.5% (47.4–99.7) |
Specificity (95%CI) | 91.7% (N/A) | 85% (79–90) | 72.2% (62.1–80.8) | 81.2% (71.2–88.8) |
PPV (95%CI) | N/A (N/A) | 38.6% (N/A) * | 32.5% (18.6–49.1) | 30.4% (13.2–52.9) |
NPV (95%CI) | N/A (N/A) | 98.1% (N/A) * | 98.6% (92.4–100) | 98.6% (92.3–99.96) |
(A) | ||||||||
---|---|---|---|---|---|---|---|---|
Methylation Site # | H-1975 (0% Methylation) | SK-GT-4 (100% Methylation) | ||||||
Rep1 | Rep2 | Rep3 | %CV | Rep1 | Rep2 | Rep3 | %CV | |
%VIM-Meth at POS (17271467)-CpG1 | 0.2% | 0.2% | 0.3% | 24.7% | 96.7% | 96.8% | 97.2% | 0.3% |
%VIM-Meth at POS (17271470)-CpG2 | 0.5% | 0.3% | 0.3% | 31.5% | 96.2% | 96.4% | 97.2% | 0.5% |
%VIM-Meth at POS (17271475)-CpG3 | 0.5% | 0.9% | 0.4% | 44.1% | 97.3% | 96.4% | 96.7% | 0.5% |
%VIM-Meth at POS (17271482)-CpG4 | 0.2% | 0.4% | 0.4% | 34.6% | 97.3% | 97.4% | 96.8% | 0.3% |
%VIM-Meth at POS (17271484)-CpG5 | 0.3% | 0.5% | 0.3% | 31.5% | 95.6% | 97.3% | 97.0% | 0.9% |
%VIM-Meth at POS (17271489)-CpG6 | 0.2% | 0.3% | 0.3% | 21.7% | 97.0% | 95.9% | 96.6% | 0.6% |
%VIM-Meth at POS (17271493)-CpG7 | 0.2% | 0.3% | 0.3% | 21.7% | 96.5% | 96.7% | 97.3% | 0.4% |
%VIM-Meth at POS (17271504)-CpG8 | 0.5% | 0.5% | 0.4% | 12.4% | 97.0% | 96.8% | 97.2% | 0.2% |
%VIM-Meth at POS (17271512)-CpG9 | 0.3% | 0.5% | 0.4% | 25.0% | 97.2% | 96.1% | 97.2% | 0.7% |
%VIM-Meth at POS (17271520)-CpG10 | 0.3% | 0.5% | 0.4% | 25.0% | 97.2% | 97.0% | 97.2% | 0.1% |
(B) | ||||||||
Methylation Site # | H-1975 (0% Methylation) | SK-GT-4 (100% Methylation) | ||||||
Rep1 | Rep2 | Rep3 | %CV | Rep1 | Rep2 | Rep3 | %CV | |
%CCNA1-Meth at POS (37005878)-CpG1 | 0.5% | 0.7% | 0.8% | 22.9% | 96.3% | 96.1% | 95.6% | 0.4% |
%CCNA1-Meth at POS (37005881)-CpG2 | 0.5% | 0.8% | 0.5% | 28.9% | 96.5% | 98.1% | 97.1% | 0.8% |
%CCNA1-Meth at POS (37005883)-CpG3 | 0.8% | 0.7% | 0.7% | 7.9% | 95.9% | 95.9% | 95.5% | 0.2% |
%CCNA1-Meth at POS (37005892)-CpG4 | 0.3% | 0.4% | 0.5% | 25.0% | 96.6% | 95.2% | 95.9% | 0.7% |
%CCNA1-Meth at POS (37005899)-CpG5 | 0.9% | 0.7% | 0.4% | 37.7% | 97.3% | 96.1% | 97.0% | 0.6% |
%CCNA1-Meth at POS (37005909)-CpG6 | 1.1% | 0.4% | 0.6% | 51.5% | 96.7% | 95.4% | 95.8% | 0.7% |
%CCNA1-Meth at POS (37005924)-CpG7 | 0.5% | 0.5% | 0.7% | 20.4% | 90.2% | 91.4% | 87.0% | 2.5% |
%CCNA1-Meth at POS (37005929)-CpG8 | 0.5% | 0.9% | 1.2% | 40.5% | 92.0% | 92.2% | 91.0% | 0.7% |
%CCNA1-Meth at POS (37005934)-CpG9 | 0.8% | 0.6% | 0.6% | 17.3% | 95.8% | 94.3% | 95.1% | 0.8% |
%CCNA1-Meth at POS (37005939)-CpG10 | 0.3% | 0.8% | 0.8% | 45.6% | 95.9% | 95.5% | 95.0% | 0.5% |
%CCNA1-Meth at POS (37005941)-CpG11 | 0.3% | 0.8% | 0.6% | 44.4% | 97.0% | 96.9% | 97.3% | 0.2% |
%CCNA1-Meth at POS (37005947)-CpG12 | 0.3% | 0.2% | 0.4% | 33.3% | 92.1% | 92.4% | 93.1% | 0.6% |
%CCNA1-Meth at POS (37005955)-CpG13 | 0.5% | 0.4% | 0.7% | 28.6% | 93.6% | 91.8% | 93.9% | 1.2% |
%CCNA1-Meth at POS (37005966)-CpG14 | 1.5% | 0.9% | 0.9% | 31.5% | 96.2% | 95.5% | 95.2% | 0.5% |
%CCNA1-Meth at POS (37005969)-CpG15 | 0.9% | 0.8% | 1.5% | 35.5% | 96.2% | 94.9% | 95.0% | 0.8% |
%CCNA1-Meth at POS (37005978)-CpG16 | 1.7% | 0.9% | 1.0% | 36.3% | 97.4% | 96.5% | 97.4% | 0.5% |
%CCNA1-Meth at POS (37005986)-CpG17 | 0.8% | 1.1% | 1.0% | 15.8% | 95.0% | 94.8% | 95.5% | 0.4% |
%CCNA1-Meth at POS (37005995)-CpG18 | 0.7% | 1.5% | 0.5% | 58.8% | 96.6% | 96.0% | 95.7% | 0.5% |
%CCNA1-Meth at POS (37005997)-CpG19 | 0.6% | 0.8% | 0.5% | 24.1% | 97.6% | 96.7% | 97.2% | 0.5% |
%CCNA1-Meth at POS (37006000)-CpG20 | 0.7% | 0.4% | 1.0% | 42.86% | 97.5% | 97.0% | 97.2% | 0.26% |
%CCNA1-Meth at POS (37006008)-CpG21 | 0.6% | 0.2% | 0.5% | 48.0% | 96.8% | 97.3% | 96.0% | 0.7% |
(C) | ||||||||
VIM | CCNA1 | |||||||
Sanger Sequencing | Average EsoGuard Assay Computed Methylation % Across All 10 CpG Sites | Sanger Sequencing | Average EsoGuard Assay Computed Methylation % Across All 21 CpG Sites | |||||
H-1975 | 0% | 0% | 0% | 0% | ||||
SK-TG-4 | 100% | 99.5% | 100% | 99.4% |
(A) | |||
---|---|---|---|
Analytical Accuracy (Singleplex)—EsoCheck Balloon Samples | EsoGuard® Assay Performed at Reference Lab (CWRU) | ||
Positive | Negative | ||
EsoGuard assay performed at test lab (LucidDx Labs) | Positive | 32 | 3 |
Negative | 1 | 13 | |
(B) | |||
Analytical Accuracy (Singleplex)—Contrived Samples | EsoGuard® Assay Performed at Reference Lab (CWRU) | ||
Positive | Negative | ||
EsoGuard assay performed at test lab (LucidDx Labs) | Positive | 33 | 0 |
Negative | 0 | 7 | |
(C) | |||
EsoCheck Balloon Samples (n = 49) | Contrived Samples (n = 40) | Overall (n = 89) | |
Analytical Sensitivity | 96.9% | 100% | 98.5% |
Analytical Specificity | 81.3% | 100% | 87.0% |
Analytical Accuracy | 91.8% | 100% | 95.5% |
Analytical Accuracy (Multiplex)—EsoCheck Balloon Samples | EsoGuard Assay (Singleplex) | Analytical Accuracy | ||
---|---|---|---|---|
Positive | Negative | |||
EsoGuard Assay (Multiplex) | Positive | 24 | 0 | 96.1% |
Negative | 3 | 50 | ||
Analytical Sensitivity and Specificity | 88.9% | 100% |
Intra-Assay | Genes | Average (Methylation%) | Standard Deviation | %CV | EG Assay Concordance |
---|---|---|---|---|---|
Contrived—10% (Medium positive) | VIM | 29.2 | 0.4 | 1% | C (3/3) |
CCNA1 | 52.6 | 4.8 | 9% | ||
Contrived—1% (Low-positive) | VIM | 7.9 | 2.5 | 32% | C (3/3) |
CCNA1 | 12.4 | 5.3 | 42% | ||
Contrived—0.5% (Low-positive) | VIM | 3.2 | 1.1 | 34% | C (4/4) |
CCNA1 | 5.2 | 3.5 | 67% | ||
Contrived—0.0% * (Negative) | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Positive-1 | VIM | 30.6 | 1.0 | 3% | C (3/3) |
CCNA1 | 28.9 | 3.2 | 11% | ||
EsoCheck Balloon Positive-2 | VIM | 27.5 | 1.3 | 5% | C (3/3) |
CCNA1 | 28.7 | 7.7 | 27% | ||
EsoCheck Balloon Positive-3 | VIM | 3.0 | 0.8 | 28% | C (3/3) |
CCNA1 | 1.1 | 0.4 | 39% | ||
EsoCheck Balloon Negative-1 * | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Negative-2 * | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Negative-3 * | VIM | 0.0 | 0.1 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% |
Inter-Assay | Genes | Average (Methylation%) | Standard Deviation | %CV | Assay Concordance |
---|---|---|---|---|---|
Contrived—10% (Medium positive) | VIM | 36.9 | 9.8 | 27% | C (3/3) |
CCNA1 | 50.5 | 3.1 | 6% | ||
Contrived—1% (Low positive) | VIM | 8.8 | 1.1 | 13% | C (3/3) |
CCNA1 | 11.3 | 1.7 | 16% | ||
Contrived—0.5% (Low positive) | VIM | 4.8 | 1.6 | 33% | C (3/3) |
CCNA1 | 7.8 | 3.0 | 38% | ||
Contrived—0.0% * (Negative) | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Positive-1 | VIM | 30.9 | 1.5 | 5% | C (3/3) |
CCNA1 | 23.9 | 2.3 | 9% | ||
EsoCheck Balloon Positive-2 | VIM | 22.9 | 3.0 | 13% | C (3/3) |
CCNA1 | 19.7 | 0.4 | 2% | ||
EsoCheck Balloon Positive-3 | VIM | 2.4 | 0.5 | 21% | C (3/3) |
CCNA1 | 1.5 | 0.6 | 45% | ||
EsoCheck Balloon Negative-1 * | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Negative-2 * | VIM | 0.0 | 0.0 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% | ||
EsoCheck Balloon Negative-3 * | VIM | 0.1 | 0.1 | 0% | C (3/3) |
CCNA1 | 0.0 | 0.0 | 0% |
n | Average VIM | Average CCNA1 | # of VIM Positive | # CCNA1 Positive | EsoGuard® Positive | |
---|---|---|---|---|---|---|
1P-Spike-in | 24 | 9.5 | 10.8 | 24 | 24 | 24 (100%) |
0.5P-Spike-in | 27 | 4.8 | 7.4 | 26 | 20 | 27 (100%) |
0.25P-Spike-in | 24 | 3.0 | 5.6 | 21 | 21 | 24 (100%) |
0.13P-Spike-in | 20 | 1.6 | 2.2 | 13 | 12 | 18 (90%) |
EGD Result | ||
---|---|---|
Positive | Negative | |
EsoGuard Assay Positive | 0 | 13 |
EsoGuard Assay Negative | 0 | 69 |
Specificity | 84.1% |
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Ghosal, A.; Verma, S.; Le, I.T.; Lee, V.T.; deGuzman, B.J.; Aklog, L. Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett’s Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device. Diagnostics 2024, 14, 1784. https://doi.org/10.3390/diagnostics14161784
Ghosal A, Verma S, Le IT, Lee VT, deGuzman BJ, Aklog L. Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett’s Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device. Diagnostics. 2024; 14(16):1784. https://doi.org/10.3390/diagnostics14161784
Chicago/Turabian StyleGhosal, Abhisek, Suman Verma, Ivy T. Le, Victoria T. Lee, Brian J. deGuzman, and Lishan Aklog. 2024. "Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett’s Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device" Diagnostics 14, no. 16: 1784. https://doi.org/10.3390/diagnostics14161784