Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics
Abstract
:1. Introduction
2. Materials and Methods
2.1. DNA and RNA Samples
2.2. Library Preparation and Sequencing of DNA Modules
2.3. Library Preparation and Sequencing of RNA Modules
2.4. Sequence Data Analysis
2.5. Analysis of Discordant Samples
2.6. Preparation of Artificial Samples
3. Results
3.1. Design of the Compact Panel
3.2. Sensitivity of Mutation Detection Using DNA as a Template
3.3. Sensitivity of Fusion Detection Using RNA as a Template
3.4. Quantification of Mutation Detection
3.5. Concordance with Conventional Diagnostic Tests
3.6. Incidence of Mutations and Fusions
4. 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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Gene | Target Mutations/Fusion Variants | |
---|---|---|
DNA module I | EGFR | Exon 19 deletion, L858R, T790M, L861Q, L861R |
BRAF | V600E | |
KRAS | G12C | |
DNA module II | EGFR | G719X, E709X, S768I, exon 20 insertion |
HER2 | Exon 20 mutations | |
MET | Exon 14 skipping | |
RNA module I | ALK | EML4, 22 variants; KIF5, 3; TFG, 1; HIP, 3; KLC1, 1 |
MET | Exon 14 skipping | |
RNA module II | ROS1 | CD74, 5 variants; SLC34A2, 7; EZR, 1; GOPC, 2; SDC4, 4; LRIG, 1; TPM3, 1; CCDC6, 1; KDELR2, 1 |
RET | KIF5B, 7 variants; CCDC6, 1; NCOA4, 1 |
Threshold of Detection DNA, % Allele Frequency; RNA, TM Score | Sensitivity Test | ||
---|---|---|---|
False Negative | False Positive | ||
DNA module | |||
EGFR exon 19 del | 0.14 | 0 | 0 |
EGFR L858R | 0.2 | 0 | 0 |
EGFR T790M | 0.48 | 0 | 0 |
BRAF V600E | 0.24 | 0 | 0 |
KRAS G12C | 0.2 | 0 | 0 |
RNA module | |||
ALK fusion | 188 | 0 | 1 |
ROS1 fusion | 32 | 0 | 1 |
RET fusion | 18 | 0 | 0 |
MET exon 14 skipping | 28 | 0 | 0 |
Compact Panel Positive | Compact Panel Negative | Positive Identity Rate | Negative Identity Rate | Reference Test | ||
---|---|---|---|---|---|---|
EGFR | Reference test positive | 73 | 0 | 100 (95.5–100) | - | Cobas EGFR Mutation Test v2 |
Reference test negative | 7 | 70 | - | 90.9 (82.2–96.3) | ||
BRAF V600E | Reference test positive | 7 | 0 | 100 (59.0–100) | - | Oncomine Dx Target Test |
Reference test negative | 0 | 70 | - | 100 (94.9–100) | ||
KRAS G12C | Reference test positive | 49 | 0 | 100 (92.7–100) | - | therascreen KRAS RGQ PCR Kit |
Reference test negative | 0 | 51 | - | 100 (93.0–100) | ||
ALK | Reference test positive | 29 | 1 | 96.7 (83.8–99.9) | - | Histofine ALK iAEP kit/Vysis ALK Break Apart FISH Probe Kit |
Reference test negative | 11 | 692 | - | 98.4 (97.2–99.2) | ||
ROS1 | Reference test positive | 9 | 0 | 100 (66.4–100) | - | OncoGuide AmoyDx ROS1 |
Reference test negative | 1 | 99 | - | 99.0 (94.6–100) | ||
MET | Reference test positive | 48 | 1 | 98.0 (89.0–99.9) | - | ArcherMET |
Reference test negative | 0 | 50 | - | 100 (92.8–100) | ||
RET | Reference test positive | 15 | 1 | 93.8 (69.8–100) | - | Oncomine Dx Target Test |
Reference test negative | 0 | 70 | - | 100 (94.9–100) |
Compact Panel Positive | Compact Panel Negative | Positive Identity Rate | Reference Test | ||
---|---|---|---|---|---|
BRAF V600E | Reference test positive | 77 | 0 | 100 (95.3–100) | Oncomine Dx Target Test |
Reference test no call | 3 | 0 | - | ||
ROS1 | Reference test positive | 99 | 1 | 99.0 (94.6–100) | OncoGuide AmoyDx ROS1 |
RET | Reference test positive | 79 | 1 | 98.8 (93.2–100) | Oncomine Dx Target Test |
Osaka International Cancer Institute | Scrum-Japan | |||
---|---|---|---|---|
Assay system | Compact panel | Oncomine comprehensive assay | ||
Total number of samples | 827 | 3919 | ||
Gene | Number of variant-positive samples | Percent frequency | Number of variant-positive samples | Percent frequency |
KRAS | 202 | 24.4 (21.5–27.5) | 382 | 9.7 (8.8–10.7) |
BRAF | 36 | 4.4 (3.1–6.0) | 97 | 2.5 (2.0–3.0) |
ALK | 41 | 5 (3.6–6.7) | 97 | 2.5 (2.0–3.0) |
ROS1 | 10 | 1.2 (0.6–2.2) | 142 | 3.6 (3.1–4.3) |
MET | 56 | 6.8 (5.2–8.7) | 98 | 2.5 (2.0–3.0) |
RET | 18 | 2.2 (1.3–3.4) | 100 | 2.6 (2.1–3.1) |
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Kato, K.; Okami, J.; Nakamura, H.; Honma, K.; Sato, Y.; Nakamura, S.; Kukita, Y.; Nakatsuka, S.-i.; Higashiyama, M. Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics. Diagnostics 2023, 13, 1476. https://doi.org/10.3390/diagnostics13081476
Kato K, Okami J, Nakamura H, Honma K, Sato Y, Nakamura S, Kukita Y, Nakatsuka S-i, Higashiyama M. Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics. Diagnostics. 2023; 13(8):1476. https://doi.org/10.3390/diagnostics13081476
Chicago/Turabian StyleKato, Kikuya, Jiro Okami, Harumi Nakamura, Keiichiro Honma, Yoshiharu Sato, Seiji Nakamura, Yoji Kukita, Shin-ichi Nakatsuka, and Masahiko Higashiyama. 2023. "Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics" Diagnostics 13, no. 8: 1476. https://doi.org/10.3390/diagnostics13081476