Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics
Abstract
1. Introduction
2. Results
2.1. Validation Cohort
2.2. ROC Curve Analysis of MSI-NGS Diagnostic Performance
2.3. Determination of MSI Score Cut-Off Value and Evaluation of Diagnostic Performance Across Different MSI Score Cut-Offs
2.4. MSI Score Distributions in MSI-H and MSI-S Tumor Samples
2.5. Usable and Unstable Microsatellite Sites in MSI-H and MSI-S Tumor Samples
2.6. Association Between MSI Score, Tumor Cell Content, and Tumor Mutational Burden in MSI-H Tumor Samples
2.7. Tumor Mutational Burden in MSI-H and MSI-S Tumor Samples
2.8. Suggested Workflow for the Detection of MSI-H Tumors Based on MSI Scores and Tumor Mutational Burden (TMB)
3. Discussion
4. Materials and Methods
4.1. Patients and Tumor Samples
4.2. DNA Isolation
4.3. MSI Detection Using PCR (MSI-PCR)
4.4. MSI Detection Using Next Generation Sequencing (MSI-NGS) and MSI Score Determination
4.5. Tumor Mutational Burden (TMB)
4.6. Immunohistochemical (IHC) Analysis
4.7. Methylation-Specific MLPA (MS-MLPA)
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
MSI | Microsatellite instability |
MSI-H | Microsatellite instability high |
MSI-S | Microsatellite stable |
MS loci | Microsatellite loci |
IHC | Immunohistochemistry |
PCR | Polymerase chain reaction |
NGS | Next-generation sequencing |
MMR | Mismatch repair |
TMB | Tumor mutational burden |
ROC | Receiver operating characteristic curve |
AUC | Area under the curve |
LR | Likelihood ratio |
PPV | Positive predictive value |
NPV | Negative predictive value |
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Tumor Type | Lynch Syndrome-Associated Cancer | Initial Tumor Samples Group (n) | Validation Cohort (n) |
---|---|---|---|
Colorectal cancer | yes | 209 | 201 |
Prostate cancer | yes | 64 | 58 |
Gastric cancer | yes | 16 | 16 |
Biliary tract cancers | yes | 12 | 11 |
Pancreatic cancer | yes | 13 | 12 |
Endometrial cancer | yes | 4 | 3 |
Urothelial cancer | yes | 3 | 3 |
Esophageal cancer | no | 3 | 3 |
Origo ignota | no | 2 | 2 |
Malignant melanoma | no | 1 | 1 |
Bone cancer | no | 1 | 1 |
Penile cancer | no | 1 | 1 |
Adrenal gland tumor | no | 1 | 1 |
Breast cancer | no | 1 | 1 |
All tumor samples | 331 | 314 |
Analytical Parameter | MSI Score Cut-Off Value (%) | |||
---|---|---|---|---|
≥5.7 | ≥8.7 | ≥9.7 | ≥13.8 | |
Specificity % (95% CI) | 91.9 (88.2–94.6) | 98.6 (96.5–99.5) | 99.7 (98.1–100.0) | 100.0 (98.7–100.0) |
Sensitivity % (95% CI) | 78.5 (60.4–90.0) | 75.0 (56.6–87.3) | 71.4 (52.9–84.8) | 50.0 (32.6–67.4) |
Positive predictive value % | 48.9 | 84.0 | 95.2 | 100.0 |
Negative predictive value % | 97.8 | 97.6 | 97.2 | 95.3 |
Accuracy | 90.8 | 96.5 | 97.2 | 95.5 |
TPR + TNR | 170.5 | 173.6 | 171.1 | 150.0 |
Likelihood Ratio | 10 | 53 | 204 | / |
MSI Score (%) | MSI-H (n = 28) | MSI-S (n = 286) |
---|---|---|
Mean ± SD | 14.28 ± 8.51 | 2.90 ± 1.97 |
Minimum | 1.12 | 0 |
Maximum | 34.78 | 13.41 |
Range | 33.66 | 13.41 |
Lower 95% CI of mean | 10.99 | 2.67 |
Upper 95% CI of mean | 17.58 | 3.13 |
MSI Status | MSI-S | MSI-H | All Samples |
---|---|---|---|
No. of usable MSI sites (mean ± SD) | 88.92 ± 5.82 | 91.04 ± 8.67 | 89.11 ± 6.15 |
No. of unstable MSI sites (mean ± SD) | 2.56 ± 1.70 | 13.18 ± 8.57 |
MSI Score Cut-Off Value in MSI-H Tumor Samples | MSI Score Cut-Off Value ≥8.7% (n = 21) | MSI Score Cut-Off Value <8.7% (n = 7) |
---|---|---|
MSI score (%) (mean ± SD) | 17.67 ± 6.80 | 4.13 ± 1.68 |
% of tumor cells in tumor sample (mean ± SD) | 73.53 ± 33.84 | 56.43 ± 24.31 |
TMB (mean ± SD) | 38.15 ± 17.91 | 27.14 ± 13.47 |
TMB | MSI-H (n = 28) | MSI-S * (n = 269) |
---|---|---|
Mean ± SD | 33.77 ± 18.75 | 6.90 ± 4.00 |
Minimum | 4 | 0 |
Maximum | 82 | 22 |
Range | 78 | 22 |
Lower 95% CI of mean | 25.46 | 6.4 |
Upper 95% CI of mean | 42.08 | 7.4 |
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Škerl, P.; Vogrič, V.; Stegel, V.; Šetrajčič Dragoš, V.; Blatnik, O.; Klančar, G.; Novaković, S. Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics. Int. J. Mol. Sci. 2025, 26, 7138. https://doi.org/10.3390/ijms26157138
Škerl P, Vogrič V, Stegel V, Šetrajčič Dragoš V, Blatnik O, Klančar G, Novaković S. Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics. International Journal of Molecular Sciences. 2025; 26(15):7138. https://doi.org/10.3390/ijms26157138
Chicago/Turabian StyleŠkerl, Petra, Vesna Vogrič, Vida Stegel, Vita Šetrajčič Dragoš, Olga Blatnik, Gašper Klančar, and Srdjan Novaković. 2025. "Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics" International Journal of Molecular Sciences 26, no. 15: 7138. https://doi.org/10.3390/ijms26157138
APA StyleŠkerl, P., Vogrič, V., Stegel, V., Šetrajčič Dragoš, V., Blatnik, O., Klančar, G., & Novaković, S. (2025). Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics. International Journal of Molecular Sciences, 26(15), 7138. https://doi.org/10.3390/ijms26157138