An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Sequencing Results
2.2. Detection of SNVs and INDELs
2.3. Fusion Gene Detection
2.4. RNA Input Limit
2.5. Quality Criteria for Successful Mutation Detection
3. Discussion
4. Materials and Methods
4.1. Sample Information
4.2. RNA Isolation
4.3. Design of All-In-One Lung Cancer Assay
4.4. Library Preparation
4.5. NGS Data Analysis
4.6. Detection of Fusion Gene Transcripts by NanoString
4.7. Variant Detection by Droplet Digital (dd) PCR
4.8. Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample ID | Origin | DV200 | Known Variants Detected at DNA Level | Results of All-In-One Transcriptome-Based Assay a | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | Amino Acid Change | MD Test or Reference | Tool | Mutant Reads | Total Reads | VAF c | Status | |||
Variants Known at DNA Level | ||||||||||
P35 | PE | 81 | AKT1 | p.E17K | NGS | IGV | 81 | 292 | 28% | confirmed |
P13 | PE | 89 | ALK | p.G1269A | NGS | IGV | 44 | 240 | 18% | confirmed |
P13 | PE | 89 | ALK | p.I1171N | NGS | IGV and Pipeline | 331 | 336 | 99% | confirmed |
P07 | FFPE | 65 | ALK | p.L1196M | NGS | IGV | 1 | 3 | 33% | not confirmed |
P35 | PE | 81 | BRAF | p.V600E | NGS | IGV and Pipeline | 31 | 60 | 52% | confirmed |
P25 | FFPE | 71 | BRAF | p.V600E | NGS | IGV and Pipeline | 33 | 46 | 72% | confirmed |
H1650 | cell line | nd | EGFR | p.E746_A750del | NGS | IGV and Pipeline | 46 | 76 | 61% | confirmed |
H1975 | cell line | nd | EGFR | p.T790M | NGS | IGV and Pipeline | 347 | 425 | 82% | confirmed |
H1975 | cell line | nd | EGFR | p.L858R | NGS | IGV and Pipeline | 564 | 684 | 82% | confirmed |
H820 | cell line | 99 | EGFR | p.E746_A750del | NGS | IGV and Pipeline | 80 | 606 | 13% | confirmed |
H820 | cell line | 99 | EGFR | p.T790M | NGS | IGV and Pipeline | 127 | 660 | 19% | confirmed |
P04_S2 | PE | 88 | EGFR | p.L858R | NGS | IGV and Pipeline | 4661 | 4931 | 95% | confirmed |
P05 | PE | 17 | EGFR | p.E746_A750del | 22 | IGV | 0 | 0 | not confirmed | |
P04_S1 | FFPE | 26 | EGFR | p.L858R | 19,22 | IGV and Pipeline | 69 | 72 | 96% | confirmed |
P06 | FFPE | 37 | EGFR | p.L747_P753delinsS | 22 | IGV and Pipeline | 8 | 17 | 47% | confirmed |
P06 | FFPE | 37 | EGFR | p.T790M | 19,22 | IGV | 0 | 15 | 0% | not confirmed |
P15 | FFPE | 40 | EGFR | p.E746_A750del | NGS | IGV and Pipeline | 51 | 76 | 67% | confirmed |
P15 | FFPE | 40 | EGFR | p.T790M | NGS | IGV and Pipeline | 22 | 88 | 25% | confirmed |
P17 | FFPE | 57 | EGFR | p.E746_A750del | NGS | IGV and Pipeline | 128 | 182 | 70% | confirmed |
P17 | FFPE | 57 | EGFR | p.T790M | NGS | IGV and Pipeline | 62 | 127 | 49% | confirmed |
P22 | FFPE | 69 | EGFR | p.E746_A750del | 19,22 | IGV | 13 | 38 | 34% | confirmed |
P26 | FFPE | nd | EGFR | p.L858R | NGS | IGV | 0 | 6 | 0% | not confirmed |
A549 | cell line | nd | KRAS | p.G12S | 18 | IGV and Pipeline | 512 | 513 | 100% | confirmed |
HCT116 | cell line | nd | KRAS | p.G13D | 24 | IGV and Pipeline | 223 | 456 | 49% | confirmed |
KOPN-8 | cell line | 99 | KRAS | p.G12D | 29 | IGV and Pipeline | 99 | 177 | 56% | confirmed |
P01 | PE | 90 | KRAS | p.G12D | NGS | IGV and Pipeline | 14 | 111 | 13% | confirmed |
P03 | FFPE | nd | KRAS | p.G12A | NGS | IGV and Pipeline | 8 | 8 | 100% | confirmed |
P23 | FFPE | 44 | KRAS | p.G12C | NGS | IGV | 0 | 1 | not confirmed | |
P28 | FFPE | 38 | KRAS | p.G12A | NGS | IGV and Pipeline | 8 | 22 | 36% | confirmed |
P31 | FFPE | 66 | KRAS | p.Q61H | NGS | IGV and Pipeline | 60 | 123 | 49% | confirmed |
P39 | FFPE | 65 | KRAS | p.G12D | NGS | IGV and Pipeline | 8 | 12 | 67% | confirmed |
P40 | FFPE | 68 | KRAS | p.G12F | NGS | IGV | 0 | 1 | not confirmed | |
P32 | FFPE | 32 | KRAS | p.G12D | NGS | IGV | 0 | 2 | not confirmed | |
H596 | cell line | nd | MET | Exon skipping mut. | 27 | IGV | 1116 | 1196 b | 93% | confirmed |
Hs746T | cell line | 97 | MET | Exon skipping mut. | 30 | IGV | 8744 | 8774 b | 100% | confirmed |
P21 | FFPE | 34 | MET | Exon skipping mut. | NGS | IGV | 50 | 56 b | 89% | confirmed |
H1299 | cell line | nd | NRAS | p.Q61K | 20 | IGV and Pipeline | 1107 | 2549 | 43% | confirmed |
H596 | cell line | nd | PIK3CA | p.E545K | 28 | IGV and Pipeline | 156 | 330 | 47% | confirmed |
HCT116 | cell line | nd | PIK3CA | p.H1047R | 25 | IGV and Pipeline | 69 | 115 | 60% | confirmed |
P02 | FFPE | 51 | PIK3CA | p.H1047L | NGS | IGV and Pipeline | 12 | 31 | 39% | confirmed |
P26 | FFPE | nd | PIK3CA | p.E542K | NGS | IGV | 0 | 0 | not confirmed | |
P37 | PE | 21 | ROS1 | p.D2033N | NGS | IGV and Pipeline | 3 | 3 | 100% | confirmed |
Overview of Additional Variants that were Not Reported by MD | ||||||||||
P03 | FFPE | nd | EGFR | p.V834L | NGS; FISH | Pipeline | 9 | 64 | 14% | na |
P34_S1 | FFPE | 76 | KRAS | p.L56fs | NGS; FISH | Pipeline | 7 | 33 | 21% | na |
P39 | FFPE | 65 | NRAS | p.G15fs | NGS; FISH | Pipeline | 5 | 22 | 23% | na |
P40 | FFPE | 68 | BRAF | p.Y472fs | NGS; FISH | Pipeline | 6 | 13 | 46% | na |
Sample ID | Origin | DV200 | MD Variant | Results of All-In-One Transcriptome-Based Assay | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | IHC | FISH | Fusion Transcript | IGV (Splitting Reads in Gene 1, Splitting Reads in Gene 2) | Fusion Catcher (Spanning, Splitting Reads) | Strand NGS (Splitting Reads) | Status | |||
H2228 | cell line | nd | ALK | nd | nd | EML4_E6-ALK_E20 | 104, 59 | 41, 107 | 84 | confirmed |
P07 | FFPE | 65 | ALK | + | + | not confirmed | ||||
P08 | FFPE | 67 | ALK | + | + | KIF5B_E24-ALK_E20 | 5, 9 | confirmed | ||
P13 | PE | 89 | ALK | + | nd | EML4_E6-ALK_E20 | 83, 238 | 58, 185 | 170 | confirmed |
P14 | PE | 86 | ALK | + | nd | DCTN1_E26-ALK_E20 | 76, 21 | 20, 41 | 51 | confirmed |
P18 | Frozen | 86 | ALK | + | + | EML4_E6-ALK_E20 | 230, 143 | 74, 290 | 789 | confirmed |
P33 | FFPE | 58 | ALK | + | + | EML4_E6-ALK_E20 | 6, 4 | confirmed | ||
P34_S1 | Frozen | 82 | ALK | + | + | EML4_E6-ALK_E20 | 62, 41 | 44, 156 | 77 | confirmed |
P34_S1 | FFPE | 76 | ALK | + | + | EML4_E6-ALK_E20 | 7, 3 | 2, 3 | 2 | confirmed |
P34_S2 | FFPE | 70 | ALK | + | + | EML4_E6-ALK_E20 | 38, 17 | 10, 3 | 2 | confirmed |
P36 | PE | 93 | ALK | + | + | EML4_E6-ALK_E18 | 49, 105 | 35, 156 | 86 | confirmed |
P42 | PE | 53 | ALK | + | + | MPRIP_E21-ALK_E20 | 8, 20 | 5, 26 | 27 | confirmed |
KM12 | cell line | 94 | NTRK1 | nd | nd | TPM3_E7-NTRK1_E9 | 188, 87 | 41, 153 | 340 | confirmed |
P08 | FFPE | 67 | RET | nd | + | true negative | ||||
P11 | FFPE | 81 | RET | nd | + | KIF5B_E15-RET_E12 | 2, 3 | confirmed | ||
P37 | PE | 21 | ROS1 | nd | + | CD74_E6-ROS1_E34 | 0, 3 | 2, 3 | 1 | confirmed |
P38 | FFPE | 55 | ROS1 | nd | + | EZR_E10-ROS1_E34 | 11, 2 | 4, 3 | confirmed | |
P41 | FFPE | nd | ROS1 | nd | + | EZR_E10-ROS1_E34 | 19, 0 | 10, 9 | confirmed |
Variant Type | All Variants (Sensitivity) | Variants in Samples with DV200 >30 and Unique Read Count >50 K (Sensitivity) |
---|---|---|
SNVs/INDELs | 32/39 (82%) | 19/19 (100%) |
MET exon skipping | 3/3 (100%) | 3/3 (100%) |
Fusions | 17/18 (94%) | 13/13 (100%) |
Overall | 52/60 (87%) | 35/35 (100%) |
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Wei, J.; Rybczynska, A.A.; Meng, P.; Terpstra, M.; Saber, A.; Sietzema, J.; Timens, W.; Schuuring, E.; Hiltermann, T.J.N.; Groen, H.J.M.; et al. An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients. Cancers 2020, 12, 2843. https://doi.org/10.3390/cancers12102843
Wei J, Rybczynska AA, Meng P, Terpstra M, Saber A, Sietzema J, Timens W, Schuuring E, Hiltermann TJN, Groen HJM, et al. An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients. Cancers. 2020; 12(10):2843. https://doi.org/10.3390/cancers12102843
Chicago/Turabian StyleWei, Jiacong, Anna A. Rybczynska, Pei Meng, Martijn Terpstra, Ali Saber, Jantine Sietzema, Wim Timens, Ed Schuuring, T. Jeroen N. Hiltermann, Harry. J.M. Groen, and et al. 2020. "An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients" Cancers 12, no. 10: 2843. https://doi.org/10.3390/cancers12102843
APA StyleWei, J., Rybczynska, A. A., Meng, P., Terpstra, M., Saber, A., Sietzema, J., Timens, W., Schuuring, E., Hiltermann, T. J. N., Groen, H. J. M., van der Wekken, A. J., van den Berg, A., & Kok, K. (2020). An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients. Cancers, 12(10), 2843. https://doi.org/10.3390/cancers12102843