BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

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Garvan Institute of Medical Research Sydney NSW Australia

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Institute of Pathology Fudan University Shanghai 200032 China

ITHACA European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability European Commission Lille France

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Kinghorn Centre for Clinical Genomics Garvan Institute of Medical Research Sydney NSW Australia

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Market and Application Development Bioinformatics Roche Sequencing Solutions Inc 4300 Hacienda Dr Pleasanton CA 94588 USA

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See more in PubMed

Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol. 2013;8(7):823–59. 10.1097/JTO.0b013e318290868f. PubMed

Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, McArthur GA, Hutson TE, Moschos SJ, Flaherty KT, Hersey P, Kefford R, Lawrence D, Puzanov I, Lewis KD, Amaravadi RK, Chmielowski B, Lawrence HJ, Shyr Y, Ye F, Li J, Nolop KB, Lee RJ, Joe AK, Ribas A. Survival in BRAF V600–mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012;366(8):707–714. doi: 10.1056/NEJMoa1112302. PubMed DOI PMC

Berger MF, Mardis ER. The emerging clinical relevance of genomics in cancer medicine. Nat Rev Clin Oncol. 2018;15(6):353–365. doi: 10.1038/s41571-018-0002-6. PubMed DOI PMC

Cordova-Delgado M, Pinto MP, Retamal IN, Muñoz-Medel M, Bravo ML, Fernández MF, Cisternas B, Mondaca S, Sanchez C, Galindo H, Nervi B, Ibáñez C, Acevedo F, Madrid J, Peña J, Koch E, Maturana MJ, Romero D, de la Jara N, Torres J, Espinoza M, Balmaceda C, Liao Y, Li Z, Freire M, Gárate-Calderón V, Cáceres J, Sepúlveda-Hermosilla G, Lizana R, Ramos L, Artigas R, Norero E, Crovari F, Armisén R, Corvalán AH, Owen GI, Garrido M. High proportion of potential candidates for immunotherapy in a Chilean cohort of gastric cancer patients: results of the FORCE1 study. Cancers. 2019;11(9):1275. doi: 10.3390/cancers11091275. PubMed DOI PMC

Heeke AL, Pishvaian MJ, Lynce F, Xiu J, Brody JR, Chen W-J, et al. Prevalence of homologous recombination–related gene mutations across multiple cancer types. JCO Precis Oncol. 2018;2:PO.17.00286. 10.1200/PO.17.00286. PubMed PMC

Kacew AJ, Harris EJ, Lorch JH, Haddad RI, Chau NG, Rabinowits G, LeBoeuf NR, Schmults CD, Thakuria M, MacConaill LE, Hanna GJ. Chromosome 3q arm gain linked to immunotherapy response in advanced cutaneous squamous cell carcinoma. Eur J Cancer. 2019;113:1–9. doi: 10.1016/j.ejca.2019.03.004. PubMed DOI

Hiley CT, Le Quesne J, Santis G, Sharpe R, de Castro DG, Middleton G, et al. Challenges in molecular testing in non-small-cell lung cancer patients with advanced disease. Lancet. 2016;388(10048):1002–1011. doi: 10.1016/S0140-6736(16)31340-X. PubMed DOI

Martin P, Shiau CJ, Pasic M, Tsao M, Kamel-Reid S, Lin S, Tudor R, Cheng S, Higgins B, Burkes R, Ng M, Arif S, Ellis PM, Hubay S, Kuruvilla S, Laurie SA, Li J, Hwang D, Lau A, Shepherd FA, le LW, Leighl NB. Clinical impact of mutation fraction in epidermal growth factor receptor mutation positive NSCLC patients. Br J Cancer. 2016;114(6):616–622. doi: 10.1038/bjc.2016.22. PubMed DOI PMC

Groopman EE, Rasouly HM, Gharavi AG. Genomic medicine for kidney disease. Nat Rev Nephrol. 2018;14(2):83–104. doi: 10.1038/nrneph.2017.167. PubMed DOI PMC

Cheng DT, Mitchell TN, Zehir A, Shah RH, Benayed R, Syed A, Chandramohan R, Liu ZY, Won HH, Scott SN, Brannon AR, O'Reilly C, Sadowska J, Casanova J, Yannes A, Hechtman JF, Yao J, Song W, Ross DS, Oultache A, Dogan S, Borsu L, Hameed M, Nafa K, Arcila ME, Ladanyi M, Berger MF. Memorial Sloan Kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn. 2015;17(3):251–264. doi: 10.1016/j.jmoldx.2014.12.006. PubMed DOI PMC

Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome Med. 2015;7(1):80. doi: 10.1186/s13073-015-0203-x. PubMed DOI PMC

Samstein RM, Lee C-H, Shoushtari AN, Hellmann MD, Shen R, Janjigian YY, Barron DA, Zehir A, Jordan EJ, Omuro A, Kaley TJ, Kendall SM, Motzer RJ, Hakimi AA, Voss MH, Russo P, Rosenberg J, Iyer G, Bochner BH, Bajorin DF, al-Ahmadie HA, Chaft JE, Rudin CM, Riely GJ, Baxi S, Ho AL, Wong RJ, Pfister DG, Wolchok JD, Barker CA, Gutin PH, Brennan CW, Tabar V, Mellinghoff IK, DeAngelis LM, Ariyan CE, Lee N, Tap WD, Gounder MM, D’Angelo SP, Saltz L, Stadler ZK, Scher HI, Baselga J, Razavi P, Klebanoff CA, Yaeger R, Segal NH, Ku GY, DeMatteo RP, Ladanyi M, Rizvi NA, Berger MF, Riaz N, Solit DB, Chan TA, Morris LGT. Tumor mutational load predicts survival after immunotherapy across multiple cancer types. Nat Genet. 2019;51(2):202–206. doi: 10.1038/s41588-018-0312-8. PubMed DOI PMC

Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med. 2017;9:34. 10.1186/s13073-017-0424-2. PubMed PMC

FoundationOne. FoundationOne CDx Technical Information. https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019C.pdf. Accessed 24 Feb. 2020.

Omics Core by NantHealth, Inc. 510(k) Premarket Notification. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm? ID=K190661. Accessed 24 Mar 2020.

PGDx elio tissue complete by Personal Genome Diagnostic, Inc. 510(k) Premarket Notification. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm? ID=K192063. Accessed 13 Dec 2020.

Lemery S, Keegan P, Pazdur R. First FDA approval agnostic of Cancer site — when a biomarker defines the indication. N Engl J Med. 2017;377(15):1409–1412. doi: 10.1056/NEJMp1709968. PubMed DOI

FDA approves third oncology drug that targets a key genetic driver of cancer, rather than a specific type of tumor. https://www.fda.gov/news-events/press-announcements/fda-approves-third-oncology-drug-targets-key-genetic-driver-cancer-rather-specific-type-tumor. Accessed 13 Mar 2020.

Meric-Bernstam F, Brusco L, Shaw K, Horombe C, Kopetz S, Davies MA, Routbort M, Piha-Paul SA, Janku F, Ueno N, Hong D, de Groot J, Ravi V, Li Y, Luthra R, Patel K, Broaddus R, Mendelsohn J, Mills GB. Feasibility of large-scale genomic testing to facilitate enrollment onto Genomically matched clinical trials. J Clin Oncol. 2015;33(25):2753–2762. doi: 10.1200/JCO.2014.60.4165. PubMed DOI PMC

Gray SW, Hicks-Courant K, Cronin A, Rollins BJ, Weeks JC. Physicians’ attitudes about multiplex tumor genomic testing. J Clin Oncol. 2014;32(13):1317–1323. doi: 10.1200/JCO.2013.52.4298. PubMed DOI PMC

Jameson GS, Petricoin EF, Sachdev J, Liotta LA, Loesch DM, Anthony SP, Chadha MK, Wulfkuhle JD, Gallagher RI, Reeder KA, Pierobon M, Fulk MR, Cantafio NA, Dunetz B, Mikrut WD, von Hoff DD, Robert NJ. A pilot study utilizing multi-omic molecular profiling to find potential targets and select individualized treatments for patients with previously treated metastatic breast cancer. Breast Cancer Res Treat. 2014;147(3):579–588. doi: 10.1007/s10549-014-3117-1. PubMed DOI

Tsongalis GJ, Peterson JD, de AFB, Tunkey CD, Gallagher TL, Strausbaugh LD, et al. Routine use of the ion torrent AmpliSeq™ Cancer hotspot panel for identification of clinically actionable somatic mutations. Clin Chem Lab Med. 2013;52(5):707–14. 10.1515/cclm-2013-0883. PubMed

Garofalo A, Sholl L, Reardon B, Taylor-Weiner A, Amin-Mansour A, Miao D, Liu D, Oliver N, MacConaill L, Ducar M, Rojas-Rudilla V, Giannakis M, Ghazani A, Gray S, Janne P, Garber J, Joffe S, Lindeman N, Wagle N, Garraway LA, van Allen EM. The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine. Genome Med. 2016;8(1):79. doi: 10.1186/s13073-016-0333-9. PubMed DOI PMC

Kuderer NM, Burton KA, Blau S, Rose AL, Parker S, Lyman GH, Blau CA. Comparison of 2 commercially available next-generation sequencing platforms in oncology. JAMA Oncol. 2017;3(7):996–998. doi: 10.1001/jamaoncol.2016.4983. PubMed DOI PMC

Garcia EP, Minkovsky A, Jia Y, Ducar MD, Shivdasani P, Gong X, Ligon AH, Sholl LM, Kuo FC, MacConaill LE, Lindeman NI, Dong F. Validation of OncoPanel: a targeted next-generation sequencing assay for the detection of somatic variants in cancer. Arch Pathol Lab Med. 2017;141(6):751–758. doi: 10.5858/arpa.2016-0527-OA. PubMed DOI

Jones WD. SEQC2 Oncopanel sequencing working group. A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency. Genome Biol. 10.1186/s13059-021-02316-z. PubMed PMC

MAQC Consortium The MicroArray quality control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol. 2006;24(9):1151–1161. doi: 10.1038/nbt1239. PubMed DOI PMC

SEQC/MAQC-III Consortium A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the sequencing quality control consortium. Nat Biotechnol. 2014;32(9):903–914. doi: 10.1038/nbt.2957. PubMed DOI PMC

MAQC Consortium. Shi L, Campbell G, Jones WD, Campagne F, Wen Z, et al. The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models. Nat Biotechnol. 2010;28(8):827–838. doi: 10.1038/nbt.1665. PubMed DOI PMC

Shi L, Perkins RG, Fang H, Tong W. Reproducible and reliable microarray results through quality control: good laboratory proficiency and appropriate data analysis practices are essential. Curr Opin Biotechnol. 2008;19(1):10–18. doi: 10.1016/j.copbio.2007.11.003. PubMed DOI

Xiao W, SEQC2 Somatic Mutation Working Group. Achieving reproducibility and accuracy in cancer mutation detection with whole-genome and whole-exome sequencing. bioRxiv. 2019. 10.1101/626440. Accessed 24 Feb 2020.

Fang LT, SEQC2 Somatic Mutation Working Group. Establishing reference samples for detection of somatic mutations and germline variants with NGS technologies. bioRxiv. 2019. 10.1101/625624. Accessed 24 Feb 2020.

Sha D, Jin Z, Budczies J, Kluck K, Stenzinger A, Sinicrope FA. Tumor mutational burden as a predictive biomarker in solid tumors. Cancer Discov. 2020;10(12):1808–1825. doi: 10.1158/2159-8290.CD-20-0522. PubMed DOI PMC

Novoradovskaya N, Whitfield ML, Basehore LS, Novoradovsky A, Pesich R, Usary J, Karaca M, Wong WK, Aprelikova O, Fero M, Perou CM, Botstein D, Braman J. Universal reference RNA as a standard for microarray experiments. BMC Genomics. 2004;5(1):20. doi: 10.1186/1471-2164-5-20. PubMed DOI PMC

Thermo Scientific. AcroMetrix Oncology Hotspot Control Package Insert. https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FCDD%2Fmanuals%2FMAN0010820-AMX-Oncology-Hotspot-Ctrl-EN.pdf&title=QWNyb01ldHJpeCBPbmNvbG9neSBIb3RzcG90IENvbnRyb2wgUGFja2FnZSBJbnNlcnQgW0VOXQ==. Accessed 24 Apr 2019.

Ellrott K, Bailey MH, Saksena G, Covington KR, Kandoth C, Stewart C, et al. Scalable open science approach for mutation calling of tumor exomes using multiple genomic pipelines. Cell Syst. 2018;6(3):271–81.e7. 10.1016/j.cels.2018.03.002. PubMed PMC

Campbell PJ, Getz G, Korbel JO, Stuart JM, Jennings JL, Stein LD, et al. Pan-cancer analysis of whole genomes. Nature. 2020;578(7793):82–93. 10.1038/s41586-020-1969-6. PubMed PMC

Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP. Sequencing depth and coverage: key considerations in genomic analyses. Nat Rev Genet. 2014;15(2):121–132. doi: 10.1038/nrg3642. PubMed DOI

Zhang Y, SEQC2 Oncopanel Sequencing Working Group. Deep oncopanel sequencing reveals fixation time- and within block position-dependent quality degradation in FFPE processed samples. bioRxiv. 2021. 10.1101/2021.04.06.438687. Accessed 8 Apr 2021.

Willey JC, Morrison T, Austermiller B, Crawford EL, Craig DJ, Blomquist TM, et al. Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA. bioRxiv. 2021. 10.1101/2021.04.06.438497. Accessed 8 Apr 2021. PubMed PMC

Allgäuer M, Budczies J, Christopoulos P, Endris V, Lier A, Rempel E, Volckmar AL, Kirchner M, von Winterfeld M, Leichsenring J, Neumann O, Fröhling S, Penzel R, Thomas M, Schirmacher P, Stenzinger A. Implementing tumor mutational burden (TMB) analysis in routine diagnostics—a primer for molecular pathologists and clinicians. Transl Lung Cancer Res. 2018;7(6):703–715. doi: 10.21037/tlcr.2018.08.14. PubMed DOI PMC

Buchhalter I, Rempel E, Endris V, Allgäuer M, Neumann O, Volckmar A-L, Kirchner M, Leichsenring J, Lier A, von Winterfeld M, Penzel R, Christopoulos P, Thomas M, Fröhling S, Schirmacher P, Budczies J, Stenzinger A. Size matters: dissecting key parameters for panel-based tumor mutational burden analysis. Int J Cancer. 2019;144(4):848–858. doi: 10.1002/ijc.31878. PubMed DOI

Budczies J, Allgäuer M, Litchfield K, Rempel E, Christopoulos P, Kazdal D, Endris V, Thomas M, Fröhling S, Peters S, Swanton C, Schirmacher P, Stenzinger A. Optimizing panel-based tumor mutational burden (TMB) measurement. Ann Oncol. 2019;30(9):1496–1506. doi: 10.1093/annonc/mdz205. PubMed DOI

Gong B, Xu J. SEQC2 Onco-panel Sequencing Working Group - PanCancer panel Study. BioProject PRJNA677997. NCBI. 2021. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA677997. Accessed 26 Feb 2021.

Gong B, Xu J. SEQC2 Onco-panel Sequencing Working Group - PanCancer panel Study. figshare. 2021. https://figshare.com/projects/SEQC2_Onco-panel_Sequencing_Working_Group_-_PanCancer_panel_Study/94520. Accessed 25 Feb 2021.

Deep oncopanel sequencing reveals within block position-dependent quality degradation in FFPE processed samples

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency