Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients and Plasma Sample Collection
2.2. Extraction of cfDNA and Assessment of Its Quantity and Quality
2.3. cfDNA Sequencing with the AVENIO ctDNA Expanded Kit
2.4. NGS Data Analysis
2.5. Inter-Run Assay Reproducibility of cfDNA NGS Analysis
2.6. Tissue Genetic Analysis with the AVENIO Tumor Tissue Expanded Kit
2.7. Statistical Analysis and Data Analysis
3. Results
3.1. Panel Overview
3.2. Technical Assessment of cfDNA NGS Analysis
3.2.1. Assessment of Sensitivity and Limit of Detection (LOD)
3.2.2. Inter-Run Assay Reproducibility of cfDNA NGS Analysis
3.2.3. Comparison of Plasma versus Tissue NGS Analysis
3.3. Study Population
3.4. Data Collection and Interpretation
3.4.1. Assessment of ctDNA Quantity and Quality
3.4.2. Sequencing Performance
3.4.3. Gene Variant Detection
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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0.1% Multiplex I cfDNA Reference Standard (Part No.: HD779) | ||||||
---|---|---|---|---|---|---|
Gene | Variant | Expected VAF (%) | AVENIO_1 Replicate | AVENIO_2 Replicate | ||
Detected | VAF | Detected | VAF | |||
EGFR | p.L858R | 0.10% | NO | YES | 0.09% | |
EGFR | p.ΔE746-A750 | 0.10% | YES | 0.11% | NO | |
EGFR | p.T790M | 0.10% | NO | YES | 0.12% | |
EGFR | p.V769-D770insASV | 0.10% | NO | YES | 0.12% | |
KRAS | p.G12D | 0.13% | YES | 0.14% | YES | 0.16% |
NRAS | p.Q61K | 0.13% | YES | 0.26% | YES | 0.13% |
NRAS | p.A59T | 0.13% | YES | 0.19% | NO | |
PIK3CA | p.E545K | 0.13% | NO | YES | 0.13% | |
Sample with an Expected VAF of 0.5%, Diluted from 1% Multiplex I cfDNA Reference Standard (Part No.: HD778) | ||||||
Gene | Variant | Expected VAF (%) | AVENIO Results | |||
Detected | VAF | |||||
EGFR | p.L858R | 0.50% | YES | 0.49% | ||
EGFR | p.ΔE746-A750 | 0.50% | YES | 0.42% | ||
EGFR | p.T790M | 0.50% | YES | 0.30% | ||
EGFR | p.V769-D770insASV | 0.50% | YES | 0.29% | ||
KRAS | p.G12D | 0.65% | YES | 0.40% | ||
NRAS | p.Q61K | 0.65% | YES | 0.71% | ||
NRAS | p.A59T | 0.65% | YES | 0.72% | ||
PIK3CA | p.E545K | 0.65% | YES | 0.43% | ||
HORIZON Structural Multiplex cfDNA Reference Standard | ||||||
Gene | Variant | Expected VAF (%) | AVENIO Results | |||
Detected | VAF | |||||
GNA11 | p.Q209L | 5.6 | YES | 4.17% | ||
AKT1 | p.E17K | 5.0 | YES | 3.49% | ||
PIK3CA | p.E545K | 5.6 | YES | 4.63% | ||
EGFR | p.V769_D770insASV | 5.6 | YES | 4.50% | ||
EGFR | p.∆E746-A750 | 5.3 | YES | 4.70% | ||
ROS1 | SLC34A2/ROS1 fusion | 5.6 | YES | |||
RET | CCDC6/RET fusion | 5.0 | YES | |||
MET | amplification | 4.5 copies | YES | |||
KRAS | p.G13D | 5.6 | YES | 4.41% | ||
MET | p.V237fs | 2.5 | DEL NOT COVERED BY THE PANEL | |||
FLT3 | p.S985fs | 5.6 | DEL NOT COVERED BY THE PANEL | |||
BRCA2 | p.A1689fs | 5.6 | DEL NOT COVERED BY THE PANEL | |||
FBXW7 | p.G667fs | 5.6 | DEL NOT COVERED BY THE PANEL | |||
EGFR | p.G719S | 5.3 | YES | 5.07% | ||
BRAF | p.V600E | 18.2 | YES | 15.58% | ||
PIK3CA | p.H1047R | 16.7 | YES | 15.73% | ||
MYC-N | amplification | 9.5 copies | GENE NOT INCLUDED IN THE PANEL | |||
NOTCH1 | p.P668S | 5.0 | GENE NOT INCLUDED IN THE PANEL |
Sample | Ng Inpu | Gene | SNV | VAF (%) | Mean | St dev | ||
---|---|---|---|---|---|---|---|---|
RUN 1 | RUN 2 | RUN 1 | RUN 2 | |||||
1 | 50 | 50 | KRAS | p.Gly12Ala | 52.88 | 54.63 | 53.76 | 1.24 |
TP53 | p.Arg306 * | 41.22 | 41.18 | 41.20 | 0.03 | |||
FLT1 | p.Glu201Asp | 0.86 | 0.90 | 0.88 | 0.03 | |||
MSH2 | p.Arg621Leu | 16.17 | 16.95 | 16.56 | 0.55 | |||
MTOR | p.Ala1792Pro | 0.70 | 0.74 | 0.72 | 0.03 | |||
PTCH1 | p.Val1386Ile | 0.50 | 0.48 | 0.49 | 0.01 | |||
2 | 50 | 50 | KRAS | p.Gly12Ala | 8.91 | 8.81 | 8.86 | 0.07 |
TP53 | p.Arg306 * | 5.79 | 6.31 | 6.05 | 0.37 | |||
MSH2 | p.Arg621Leu | 2.89 | 3.11 | 3.00 | 0.16 | |||
3 | 50 | 27 | TP53 | p.Gln331 * | 1.92 | 1.72 | 1.82 | 0.14 |
TP53 | p.Arg248Gln | 0.95 | 0.95 | 0.95 | 0.00 | |||
APC | p.Gln1611Glu | 1.70 | 1.89 | 1.80 | 0.13 | |||
4 | 17,5 | 23 | BRAF | p.Gly596Arg | 5.53 | 5.54 | 5.54 | 0.01 |
TP53 | p.Gly245Val | 3.71 | 3.26 | 3.49 | 0.32 | |||
DDR2 | p.Glu583Lys | 2.26 | 2.34 | 2.30 | 0.06 | |||
KEAP1 | p.Arg536Pro | 4.71 | 4.43 | 4.57 | 0.20 | |||
TSC2 | p.Ser740Cys | 3.94 | 4.68 | 4.31 | 0.52 | |||
5 | 50 | 20 | RB1 | p.Arg262Gln | 15.61 | 16.80 | 16.21 | 0.84 |
TP53 | p.His179Arg | 33.71 | 36.57 | 35.14 | 2.02 | |||
KDR | p.Ser968Ile | 2.72 | 3.00 | 2.86 | 0.20 | |||
MSH6 | p.Leu1264Val | 15.64 | 16.15 | 15.90 | 0.36 | |||
TERT | c.-146C > T | 6.50 | 7.07 | 6.79 | 0.40 | |||
6 | 50 | 50 | TP53 | c.993 + 1G > A | 5.09 | 4.98 | 5.04 | 0.08 |
Sample | Gene | SNV | AVENIO cfDNA Expanded Assay | AVENIO Tumor Tissue Expanded Assay |
---|---|---|---|---|
1 | PTCH1 | p.Ala793Ser | detected | detected |
SMO | p.Asp25Gly | detected | detected | |
MLH1 | c.1558 + 3A> | detected | detected | |
2 | TP53 | p.Ile255Phe | detected | detected |
RB1 | p.Arg251 * | not detected | detected | |
3 | KRAS | p.Gly12Ala | detected | detected |
TP53 | p.Arg306 * | detected | detected | |
MSH2 | p.Arg621Leu | detected | detected | |
MTOR | p.Ala1792Pro | detected | not detected | |
MSH6 | p.Trp55Arg | not detected | detected |
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Zulato, E.; Tosello, V.; Nardo, G.; Bonanno, L.; Del Bianco, P.; Indraccolo, S. Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients. Diagnostics 2021, 11, 1468. https://doi.org/10.3390/diagnostics11081468
Zulato E, Tosello V, Nardo G, Bonanno L, Del Bianco P, Indraccolo S. Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients. Diagnostics. 2021; 11(8):1468. https://doi.org/10.3390/diagnostics11081468
Chicago/Turabian StyleZulato, Elisabetta, Valeria Tosello, Giorgia Nardo, Laura Bonanno, Paola Del Bianco, and Stefano Indraccolo. 2021. "Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients" Diagnostics 11, no. 8: 1468. https://doi.org/10.3390/diagnostics11081468