Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of Pre-Characterized Reference Materials
2.2. Preparation of Clinical Samples
2.3. Library Preparation, Sequencing, and Variant Calling
3. Results
3.1. Summary of the Run
3.2. Analytical Sensitivity
3.3. Analytical Specificity
3.4. Precision
3.5. Comparison Between Cobas-EGFR and Pan-Cancer Cell-Free Assay
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
- Stroun, M.; Lyautey, J.; Lederrey, C.; Olson-Sand, A.; Anker, P. About the possible origin and mechanism of circulating DNA apoptosis and active DNA release. Clin. Chim. Acta 2001, 313, 139–142. [Google Scholar] [CrossRef]
- Mouliere, F.; Chandrananda, D.; Piskorz, A.M.; Moore, E.K.; Morris, J.; Ahlborn, L.B.; Mair, R.; Goranova, T.; Marass, F.; Heider, K.; et al. Enhanced detection of circulating tumor DNA by fragment size analysis. Sci. Transl. Med. 2018, 10, eaat4921. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schwarzenbach, H.; Hoon, D.S.B.; Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 2011, 11, 426–437. [Google Scholar] [CrossRef]
- Thress, K.S.; Brant, R.; Carr, T.H.; Dearden, S.; Jenkins, S.; Brown, H.; Hammett, T.; Cantarini, M.; Barrett, J.C. EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 2015, 90, 509–515. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Arriola, E.; Paredes-Lario, A.; García-Gomez, R.; Diz-Tain, P.; Constenla, M.; García-Girón, C.; Márquez, G.; Reck, M.; López-Vivanco, G. Comparison of plasma ctDNA and tissue/cytology-based techniques for the detection of EGFR mutation status in advanced NSCLC: Spanish data subset from ASSESS. Clin. Transl. Oncol. 2018, 20, 1261–1267. [Google Scholar] [CrossRef] [Green Version]
- Diaz, L.A.J.; Bardelli, A. Liquid biopsies: Genotyping circulating tumor DNA. J. Clin. Oncol. 2014, 32, 579–586. [Google Scholar] [CrossRef]
- Merker, J.D.; Oxnard, G.R.; Compton, C.; Diehn, M.; Hurley, P.; Lazar, A.J.; Lindeman, N.; Lockwood, C.M.; Rai, A.J.; Schilsky, R.L.; et al. Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review. J. Clin. Oncol. 2018, 36, 1631–1641. [Google Scholar] [CrossRef] [PubMed]
- Barbany, G.; Arthur, C.; Lieden, A.; Nordenskjold, M.; Rosenquist, R.; Tesi, B.; Wallander, K.; Tham, E. Cell-free tumour DNA testing for early detection of cancer—A potential future tool. J. Intern. Med. 2019, 286, 118–136. [Google Scholar] [CrossRef] [Green Version]
- U.S. Food and Drug Administration: Medical Devices: Cobas EGFR Mutation Test v2-P150047. Available online: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpma/pma.cfm?id=P150047 (accessed on 6 June 2016).
- Li, H.; Jing, C.; Wu, J.; Ni, J.; Sha, H.; Xu, X.; Du, Y.; Lou, R.; Dong, S.; Feng, J. Circulating tumor DNA detection: A potential tool for colorectal cancer management (Review). Oncol. Lett. 2018, 17, 1409–1416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Salk, J.J.; Schmitt, M.W.; Loeb, L.A. Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations. Nat. Rev. Genet. 2018, 19, 269–285. [Google Scholar] [CrossRef] [PubMed]
- Kinde, I.; Wu, J.; Papadopoulos, N.; Kinzler, K.W.; Vogelstein, B. Detection and quantification of rare mutations with massively parallel sequencing. Proc. Natl. Acad. Sci. USA 2011, 108, 9530–9535. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Aravanis, A.M.; Lee, M.; Klausner, R.D. Next-Generation Sequencing of Circulating Tumor DNA for Early Cancer Detection. Cell 2017, 168, 571–574. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Heitzer, E.; Haque, I.S.; Roberts, C.E.S.; Speicher, M.R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet. 2019, 20, 71–88. [Google Scholar] [CrossRef]
- Phallen, J.; Sausen, M.; Adleff, V.; Leal, A.; Hruban, C.; White, J.; Anagnostou, V.; Fiksel, J.; Cristiano, S.; Papp, E.; et al. Direct detection of early-stage cancers using circulating tumor DNA. Sci. Transl. Med. 2017, 9, eaan2415. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pairawan, S.; Hess, K.R.; Janku, F.; Sanchez, N.S.; Shaw, K.R.M.; Eng, C.; Damodaran, S.; Javle, M.; Kaseb, A.O.; Hong, D.S.; et al. Cell-free Circulating Tumor DNA Variant Allele Frequency Associates with Survival in Metastatic Cancer. Clin. Cancer Res. 2019, 26, 1924–1931. [Google Scholar] [CrossRef] [PubMed]
- Lih, C.J.; Harrington, R.D.; Sims, D.J.; Harper, K.N.; Bouk, C.H.; Datta, V.; Yau, J.; Singh, R.R.; Routbort, M.J.; Luthra, R.; et al. Analytical validation of the next-generation sequencing assay for a nationwide signal-finding clinical trial: Molecular analysis for Therapy Choice Clinical Trial. J. Mol. Diagn 2017, 19, 313–327. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gargis, A.S.; Kalman, L.; Berry, M.W.; Bick, D.P.; Dimmock, D.P.; Hambuch, T.; Lu, F.; Lyon, E.; Voelkerding, K.V.; Zehnbauer, B.A.; et al. Assuring the quality of next-generation sequencing in clinical laboratory practice. Nat. Biotechnol. 2012, 30, 1033–1036. [Google Scholar] [CrossRef]
- Shi, Y.; Au, J.S.-K.; Thongprasert, S.; Srinivasan, S.; Tsai, C.-M.; Khoa, M.T.; Heeroma, K.; Itoh, Y.; Cornelio, G.; Yang, P.-C. A Prospective, Molecular Epidemiology Study of EGFR Mutations in Asian Patients with Advanced Non–Small-Cell Lung Cancer of Adenocarcinoma Histology (PIONEER). J. Thorac. Oncol. 2014, 9, 154–162. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kim, Y.; Shin, S.; Lee, K.-A. A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories. BioMed Res. Int. 2018, 2018, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- De Luca, G.; Lastraioli, S.; Conte, R.; Mora, M.; Genova, C.; Rossi, G.; Tagliamento, M.; Coco, S.; Bello, M.G.D.; Zupo, S.; et al. Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice. Appl. Sci. 2020, 10, 2895. [Google Scholar] [CrossRef]
- Williamson, J.B.; Solano, L.; Yuki, A.; Burkhart, V.D.; Chitwood, J.; Cao, R.; Schageman, J.; Saunders, H.; Cheng, A.; Ha, T.; et al. Analytical validation of the Oncomine Pan-Cancer Cell-Free Assay in a CLIA- and CAP-regulated laboratory for detection of solid tumor-derived variants in blood plasma. J. Clin. Oncol. 2019, 37, e14614. [Google Scholar] [CrossRef]
- Moss, E.L.; Gorsia, D.N.; Collins, A.; Sandhu, P.; Foreman, N.; Gore, A.; Wood, J.; Kent, C.; Silcock, L.; Guttery, D.S. Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression. Cancers 2020, 12, 2231. [Google Scholar] [CrossRef] [PubMed]
- Shah, M.; Takayasu, T.; Moghadamtousi, S.Z.; Arevalo, O.; Chen, M.; Lan, C.; Duose, D.; Hu, P.; Zhu, J.-J.; Roy-Chowdhuri, S.; et al. Evaluation of the Oncomine Pan-Cancer Cell-Free Assay for Analyzing Circulating Tumor DNA in the Cerebrospinal Fluid in Patients with Central Nervous System Malignancies. J. Mol. Diagn. 2021, 23, 171–180. [Google Scholar] [CrossRef] [PubMed]
- Chan, R.-H.; Lin, P.-C.; Chen, S.-H.; Lin, S.-C.; Chen, P.-C.; Lin, B.-W.; Shen, M.-R.; Yeh, Y.-M. Clinical Utility of a Cell-Free DNA Assay in Patients With Colorectal Cancer. Front. Oncol. 2021, 11, 589673. [Google Scholar] [CrossRef]
Gene | Variant | Variant Allele Frequency (%) According to Input (ng) | |||||||
---|---|---|---|---|---|---|---|---|---|
0.1% | 0.5% | 1% | |||||||
5 ng | 10 ng | 20 ng | 20 ng | 20 ng | |||||
EGFR | p.L858R | 0.7 | 0.94 | 0.62 | |||||
p.E746_A750del | 0.26 | 0.25 | 0.55 | 0.59 | 1.82 | 1.44 | |||
p.T790M | 0.2 | 0.44 | 0.52 | 1.08 | 1.31 | ||||
p.A767_V769dup | 0.16 | 0.08 | 0.32 | 0.32 | 1.07 | 0.67 | |||
KRAS | p.G12D | 0.53 | 0.2 | 0.37 | 0.68 | 1.22 | 1.25 | ||
NRAS | p.Q61K | 0.18 | 1.27 | 0.86 | 1.99 | 1.36 | |||
p.A59T | 0.72 | 0.55 | 1.35 | 2.41 | |||||
PIK3CA | p.E545K | 0.16 | 0.32 | 0.41 | 0.97 | 1.34 | 1.44 | ||
Molecular coverage | 688× | 1139× | 2091× | 2115× | 2017× | ||||
Analytical sensitivity | 0% (0/8) | 25% (2/8) | 50% (8/16) | 94% (15/16) | 100% (16/16) |
Variant (Fusion) | VAF 1% | VAF 5% | ||||
Result | Mol. Cov. for Fusion | Read Cov. for Fusion | Result | Mol. Cov. for Fusion | Read Cov. for Fusion | |
SLC34A2–ROS1 | Detected | 31 | 1473 | Detected | 127 | 4850 |
CCDC6–RET | Detected | 12 | 510 | Detected | 32 | 1357 |
Variant (CNV) | 2.5 Copies (CN Ratio of 1.25) | 4.5 Copies (CN Ratio of 2.25) | ||||
Result | Observed CN Ratio | MAPD | Result | Observed CN Ratio | MAPD | |
MET amplification | Detected | 1.44 | 0.233 | Detected | 1.8 | 0.24 |
Gene | Variants | Expected VAF (%) | Mean and SD of Observed VAF (%) | CV (%) | Detection Rate |
---|---|---|---|---|---|
EGFR | p.L858R | 1.0 | 0.99 ± 0.23 | 23 | 87.5% (7/8) |
p.E746_A750del | 1.38 ± 0.37 | 27 | 100% (8/8) | ||
p.T790M | 1.00 ± 0.37 | 37 | 100% (8/8) | ||
p.A767_V769dup | 0.94 ± 0.29 | 31 | 100% (8/8) | ||
KRAS | p.G12D | 1.3 | 1.29 ± 0.32 | 25 | 100% (8/8) |
NRAS | p.Q61K | 1.43 ± 0.23 | 16 | 100% (8/8) | |
p.A59T | 1.66 ± 0.44 | 27 | 100% (8/8) | ||
PIK3CA | p.E545K | 0.95 ± 0.58 | 61 | 100% (8/8) |
Variant | 0.1% | 0.5% | 1% | |||
---|---|---|---|---|---|---|
Pan-Cancer | Cobas | Pan-Cancer | Cobas | Pan-Cancer | Cobas | |
VAF (%) | SQI | VAF (%) | SQI | VAF (%) | SQI | |
p.E746_A750del | 0.26 | 11.36 | 0.59 | 12.92 | 1.82 | 14.11 |
p.A767_V769dup | ND | ND | 0.32 | 2.72 | 1.07 | 3.1 |
p.T790M | 0.2 | ND | 0.52 | 8.75 | 1.08 | 9.42 |
p.L858R | ND | ND | 0.7 | 8.2 | 0.94 | 8.5 |
Library No. * | Variant | Cobas-EGFR Assay | Pan-Cancer Assay | ||
---|---|---|---|---|---|
SQI Value | Molecular Depth | Molecular Count | Observed VAF (%) | ||
1 | p.E746_A750del | 12.04 | 3144 | 3 | 0.1 |
p.E746_A750del | 11.18 | ||||
2 | p.L858R | 6.66 | 2110 | 210 | 9.95 |
p.T790M | 11.61 | 886 | 83 | 9.37 | |
3 | p.L858R | 7.01 | 1540 | 32 | 2.08 |
p.L858R | 7.87 | ||||
4 | p.L858R | 5.01 | 1770 | 5 | 0.28 |
p.L858R | 5.99 |
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So, M.-K.; Park, J.-H.; Kim, J.-W.; Jang, J.-H. Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations. Diagnostics 2021, 11, 1022. https://doi.org/10.3390/diagnostics11061022
So M-K, Park J-H, Kim J-W, Jang J-H. Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations. Diagnostics. 2021; 11(6):1022. https://doi.org/10.3390/diagnostics11061022
Chicago/Turabian StyleSo, Min-Kyung, Jong-Ho Park, Jong-Won Kim, and Ja-Hyun Jang. 2021. "Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations" Diagnostics 11, no. 6: 1022. https://doi.org/10.3390/diagnostics11061022