RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples
Abstract
:1. Introduction
2. Results
2.1. Cohort Characteristics, Tumor Cell Content and Sample Dropouts
2.2. Turnaround Time and RNA Yields
2.3. Detection and Validation of Gene Fusions
2.4. Gene Fusions in Different Cancer Types
2.5. Clinical and Diagnostic Implications of Gene Fusions
2.6. Gene Fusion Isoforms
2.7. Identification of Previously Unknown Gene Fusions
2.8. Individual Cases
3. Discussion
4. Material and Methods
4.1. RNA Extraction, Library Preparation and Semiconductor Sequencing
4.2. Library Preparation for the Oncomine™ Comprehensive RNA Panel v3
4.3. Library Preparation for AMP Based NGS (Archer Dx) Translocation Detection
4.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger Sequencing Validation
4.5. Immunohistochemistry
4.6. FISH Analyses
4.7. Research Ethics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Characteristics and Features | All | AMP | OCA |
---|---|---|---|
n | 517 | 285 | 232 |
age [y] | 61.5 (4–84) | 62.1 (6–84) | 60.4 (4–82) |
% females | 45.7 | 38.2 | 54.7 |
n different entities | 21 | 18 | 14 |
tumor cell content [%] | 70 (10–95) | 80 (10–95) | 70 (10–95) |
Turnaround time [days] | 9 (3-35) | 6 (3-44) | |
RNA [ng/µL] | 35.5 (2.4–880) | 41.5 (2.4–880) | 29.3 (2.6–499) |
% fusion positive | 6.58 | 8.42 | 4.31 |
Unique fragments > 150,000 | 465,740 (21,498–3,974,814) | ||
Unique fragments > 10% | 8.6 (0.7–48.4) | ||
Average unique RNA start sites | 171.88 (18.6–405.3) | ||
per GSPS control > 50 | |||
On target deduplication ratio < 40 | 12.2 (2.0–144.4) | ||
Usable reads | 579,492 (37,591–13,897,936) |
Sample ID | Fusion | Entity | Reads | Panel | Validation Successful with Any Method * |
---|---|---|---|---|---|
AMP-1 | AXL::CAPN15 (A19C2) | Gastric adenocarcinoma | 6 | AMP | no |
AMP-2 | BRD3::NUTM1 (B11N2) | NUT-midline carcinoma of the lung | 2968 | AMP | |
AMP-3 | ETV6::NTRK3 (E5N15) | MASC | 15,730 | AMP | |
AMP-4 | FGFR2::INA (F17I2) | Pancreatic blastoma | >100 | AMP | |
AMP-5 | GATM::RAF1 (G2R8) | Neuroendocrine tumor of the pancreas | 4186 | AMP | |
AMP-6 | GPBP1L1::MAST2 (G6M4) | Adenocarcinoma, pancreatobiliary type | 6 | AMP | no |
AMP-7 | HNRNPA2B1::ETV1 (H9E6) | Acinar adenocarcinoma of the prostate | >11,194 | AMP | |
AMP-8 | MTMR::MAML2 (M2M1) | Cholangiocarcinoma | 5 | AMP | no |
AMP-9 | MYB::NFIB (M11N9) | Adenoid cystic carcinoma | >100 | AMP | |
AMP-10 | MYB::NFIB (M12N9) | Adenoid cystic carcinoma | >1800 | AMP | |
AMP-11 | MYB::NFIB (M12N9) | Adenoid cystic carcinoma | >580 | AMP | |
AMP-12 | MYB::NFIB (M13N9) | Adenoid cystic carcinoma | >6000 | AMP | |
AMP-13 | MYB::NFIB (M13N9) | Adenoid cystic carcinoma | >100 | AMP | |
AMP-14 | MYB::NFIB (M14N10) | Adenoid cystic carcinoma | >310 | AMP | |
AMP-15 | MYB::NFIB (M14N10) | Adenoid cystic carcinoma | >5873 | AMP | |
AMP-16 | PTPRK::RSPO3 (P1R2) | Adenocarcinoma of the hepatopancreatic ampulla, pancreato biliary type | >800 | AMP | |
AMP-17 | SND1::BRAF (S9B9) | Pancreatic ductal adenocarcinoma | >3800 | AMP | |
AMP-18 | TMPRSS2::ERG (T1E2) | Acinar adenocarcinoma of the prostate | >974 | AMP | |
AMP-19 | TMPRSS2::ERG (T1E2) | Acinar adenocarcinoma of the prostate | >15,022 | AMP | |
AMP-20 | TMPRSS2::ERG (T2E4) | Acinar adenocarcinoma of the prostate | >3432 | AMP | |
AMP-21 | TMPRSS2::ERG (T2E4) | Acinar adenocarcinoma of the prostate | >177 | AMP | |
AMP-22 | TMPRSS2::ERG (T1E5) | Acinar adenocarcinoma of the prostate | >11,745 | AMP | |
AMP-23 | TMPRSS2::ERG (T2E4) | Acinar adenocarcinoma of the prostate | >2666 | AMP | |
AMP-24 | TMPRSS2::ERG (T1E4) | Acinar adenocarcinoma of the prostate | >1207 | AMP | |
AMP-25 | TMPRSS2::ERG (T1E4) | Adenocarcinoma of the prostate | >2000 | AMP | |
AMP-26 | VTCN1::NRG1 (V2N2) | Cholangiocarcinoma | >12,000 | AMP | |
AMP-27 | WHSC1L1::FGFR1 (W1F2) | Adenocarcinoma, pancreatobiliary type | >100 | AMP | |
OCA-1 | BRD4::NUTM1 (B11N2) | CUP | >102,528 | OCA | |
OCA-2 | EML4::ALK (E20A20) | CUP | 893 | OCA | |
OCA-3 | EML4::ALK (E6A20) | NSCLC | >6200 | OCA | |
OCA-4 | ESR1::QK1 (E2Q5) | CUP | 2857 | OCA | |
OCA-5 | FNDC3B::PIK3CA (F3P2) | Gallbladder carcinoma | 73 | OCA | no |
OCA-6 | KIF5B::RET (K15R12) | CUP | 744 | OCA | no |
OCA-7 | RNF130::SEPT14 (R3S10) | CUP | >2153 | OCA | |
OCA-8 | SND1::BRAF (S9B9) | Pancreatic ductal adenocarcinoma | 85,887 | OCA | |
OCA-9 | TBL1XR1::PIK3CA (T1P2) | Chordoma | 531 | OCA | no |
OCA-10 | TBL1XR1::PIK3CA (T1P2) | Colloid carcinoma of the pancreas | 973 | OCA | |
OCA-11 | TMPRSS2::ERG (T1E4) | CUP | 35,966 | OCA | |
OCA-12 | TRIM24::BRAF (T9B11) | CUP | 341,016 | OCA | |
OCA-13 | WHSC1L1::FGFR1 (W1F2) | Anaplastic carcinoma of the thyroid | >345 | OCA |
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Kirchner, M.; Neumann, O.; Volckmar, A.-L.; Stögbauer, F.; Allgäuer, M.; Kazdal, D.; Budczies, J.; Rempel, E.; Brandt, R.; Talla, S.B.; et al. RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples. Cancers 2019, 11, 1309. https://doi.org/10.3390/cancers11091309
Kirchner M, Neumann O, Volckmar A-L, Stögbauer F, Allgäuer M, Kazdal D, Budczies J, Rempel E, Brandt R, Talla SB, et al. RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples. Cancers. 2019; 11(9):1309. https://doi.org/10.3390/cancers11091309
Chicago/Turabian StyleKirchner, Martina, Olaf Neumann, Anna-Lena Volckmar, Fabian Stögbauer, Michael Allgäuer, Daniel Kazdal, Jan Budczies, Eugen Rempel, Regine Brandt, Suranand Babu Talla, and et al. 2019. "RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples" Cancers 11, no. 9: 1309. https://doi.org/10.3390/cancers11091309