BACKGROUND: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. METHODS: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. RESULTS: We describe a strong correlation between WES-based and panel-based TMBs; however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients; however, this significance decreases with the increasing cut-off values. CONCLUSIONS: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines.

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Department of Hematology University Hospital Schleswig Holstein 24105 Kiel Germany

Department of Pathology University Hospital Brno 62500 Brno Czech Republic

Department of Pediatric Oncology University Hospital Brno 613 00 Brno Czech Republic

Faculty of Medicine Masaryk University 62500 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital 65691 Brno Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 60200 Brno Czech Republic

Zobrazit více v PubMed

Stratton M.R., Campbell P.J., Futreal P.A. The cancer genome. Nature. 2009;458:719–724. doi: 10.1038/nature07943. PubMed DOI PMC

Chalmers Z.R., Connelly C.F., Fabrizio D., Gay L., Ali S.M., Ennis R., Schrock A., Campbell B., Shlien A., Chmielecki J., et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med. 2017;9:34. doi: 10.1186/s13073-017-0424-2. PubMed DOI PMC

Chang H., Sasson A., Srinivasan S., Golhar R., Greenawalt D.M., Geese W.J., Green G., Zerba K., Kirov S., Szustakowski J. Bioinformatic Methods and Bridging of Assay Results for Reliable Tumor Mutational Burden Assessment in Non-Small-Cell Lung Cancer. Mol. Diagn. Ther. 2019;23:507–520. doi: 10.1007/s40291-019-00408-y. PubMed DOI PMC

Lawrence M.S., Stojanov P., Polak P., Kryukov G.V., Cibulskis K., Sivachenko A., Carter S.L., Stewart C., Mermel C.H., Roberts S.A., et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature. 2013;499:214–218. doi: 10.1038/nature12213. PubMed DOI PMC

Hodi F.S., O’Day S.J., McDermott D.F., Weber R.W., Sosman J.A., Haanen J.B., Gonzalez R., Robert C., Schadendorf D., Hassel J.C., et al. Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 2010;363:711–723. doi: 10.1056/NEJMoa1003466. PubMed DOI PMC

Borghaei H., Paz-Ares L., Horn L., Spigel D.R., Steins M., Ready N.E., Chow L.Q., Vokes E.E., Felip E., Holgado E., et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2015;373:1627–1639. doi: 10.1056/NEJMoa1507643. PubMed DOI PMC

Motzer R.J., Escudier B., McDermott D.F., George S., Hammers H.J., Srinivas S., Tykodi S.S., Sosman J.A., Procopio G., Plimack E.R., et al. Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma. N. Engl. J. Med. 2015;373:1803–1813. doi: 10.1056/NEJMoa1510665. PubMed DOI PMC

Powles T., Eder J.P., Fine G.D., Braiteh F.S., Loriot Y., Cruz C., Bellmunt J., Burris H.A., Petrylak D.P., Teng S.L., et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 2014;515:558–562. doi: 10.1038/nature13904. PubMed DOI

Buchhalter I., Rempel E., Endris V., Allgauer M., Neumann O., Volckmar A.L., Kirchner M., Leichsenring J., Lier A., von Winterfeld M., et al. Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis. Int. J. Cancer. 2019;144:848–858. doi: 10.1002/ijc.31878. PubMed DOI

Rizvi N.A., Hellmann M.D., Snyder A., Kvistborg P., Makarov V., Havel J.J., Lee W., Yuan J., Wong P., Ho T.S., et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348:124–128. doi: 10.1126/science.aaa1348. PubMed DOI PMC

Snyder A., Makarov V., Merghoub T., Yuan J., Zaretsky J.M., Desrichard A., Walsh L.A., Postow M.A., Wong P., Ho T.S., et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 2014;371:2189–2199. doi: 10.1056/NEJMoa1406498. PubMed DOI PMC

Warr A., Robert C., Hume D., Archibald A., Deeb N., Watson M. Exome Sequencing: Current and Future Perspectives. G3 Genes Genomes Genet. 2015;5:1543–1550. doi: 10.1534/g3.115.018564. PubMed DOI PMC

Wu H.X., Wang Z.X., Zhao Q., Wang F., Xu R.H. Designing gene panels for tumor mutational burden estimation: The need to shift from ‘correlation’ to ‘accuracy’. J. Immunother. Cancer. 2019;7:206. doi: 10.1186/s40425-019-0681-2. PubMed DOI PMC

Gong J., Pan K., Fakih M., Pal S., Salgia R. Value-based genomics. Oncotarget. 2018;9:15792–15815. doi: 10.18632/oncotarget.24353. PubMed DOI PMC

Schumacher T.N., Schreiber R.D. Neoantigens in cancer immunotherapy. Science. 2015;348:69–74. doi: 10.1126/science.aaa4971. PubMed DOI

Zehir A., Benayed R., Shah R.H., Syed A., Middha S., Kim H.R., Srinivasan P., Gao J., Chakravarty D., Devlin S.M., et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat. Med. 2017;23:703–713. doi: 10.1038/nm.4333. PubMed DOI PMC

Hellmann M.D., Ciuleanu T.E., Pluzanski A., Lee J.S., Otterson G.A., Audigier Valette C., Minenza E., Linardou H., Burgers S., Salman P., et al. Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden. N. Engl. J. Med. 2018;378:2093–2104. doi: 10.1056/NEJMoa1801946. PubMed DOI PMC

Yarchoan M., Hopkins A., Jaffee E.M. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N. Engl. J. Med. 2017;377:2500–2501. doi: 10.1056/NEJMc1713444. PubMed DOI PMC

Endris V., Buchhalter I., Allgauer M., Rempel E., Lier A., Volckmar A.L., Kirchner M., von Winterfeld M., Leichsenring J., Neumann O., et al. Measurement of tumor mutational. burden (TMB) in routine molecular diagnostics: In silico and real-life analysis of three larger gene panels. Int. J. Cancer. 2019;144:2303–2312. doi: 10.1002/ijc.32002. PubMed DOI

Hellmann M.D., Nathanson T., Rizvi H., Creelan B.C., Sanchez-Vega F., Ahuja A., Ni A., Novik J.B., Mangarin L.M.B., Abu-Akeel M., et al. Genomic Features of Response to Combination Immunotherapy in Patients with Advanced Non-Small-Cell Lung Cancer. Cancer Cell. 2018;33:843–852. doi: 10.1016/j.ccell.2018.03.018. PubMed DOI PMC

Carbone D.P., Reck M., Paz-Ares L., Creelan B., Horn L., Steins M., Felip E., van den Heuvel M.M., Ciuleanu T.E., Badin F., et al. First-Line Nivolumab in Stage IV or Recurrent Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2017;376:2415–2426. doi: 10.1056/NEJMoa1613493. PubMed DOI PMC

Budczies J., Allgauer M., Litchfield K., Rempel E., Christopoulos P., Kazdal D., Endris V., Thomas M., Frohling S., Peters S., et al. Optimizing panel-based tumor mutational burden (TMB) measurement. Ann. Oncol. 2019;30:1496–1506. doi: 10.1093/annonc/mdz205. PubMed DOI

Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. 20131303.3997

Faust G.G., Hall I.M. SAMBLASTER: Fast duplicate marking and structural variant read extraction. Bioinformatics. 2014;30:2503–2505. doi: 10.1093/bioinformatics/btu314. PubMed DOI PMC

Poplin R., Ruano-Rubio V., DePristo M.A., Fennell T.J., Carneiro M.O., Van der Auwera G.A., Kling D.E., Gauthier L.D., Levy-Moonshine A., Roazen D., et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2017:201178.

Lai Z., Markovets A., Ahdesmaki M., Chapman B., Hofmann O., McEwen R., Johnson J., Dougherty B., Barrett J.C., Dry J.R. VarDict: A novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res. 2016;44:e108. doi: 10.1093/nar/gkw227. PubMed DOI PMC

Wang K., Li M., Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164. doi: 10.1093/nar/gkq603. PubMed DOI PMC

Ramos A.H., Lichtenstein L., Gupta M., Lawrence M.S., Pugh T.J., Saksena G., Meyerson M., Getz G. Oncotator: Cancer variant annotation tool. Hum. Mutat. 2015;36:E2423–E2429. doi: 10.1002/humu.22771. PubMed DOI PMC

Fang H., Bergmann E.A., Arora K., Vacic V., Zody M.C., Iossifov I., O’Rawe J.A., Wu Y., Jimenez Barron L.T., Rosenbaum J., et al. Indel variant analysis of short-read sequencing data with Scalpel. Nat. Protoc. 2016;11:2529–2548. doi: 10.1038/nprot.2016.150. PubMed DOI PMC

Tate J.G., Bamford S., Jubb H.C., Sondka Z., Beare D.M., Bindal N., Boutselakis H., Cole C.G., Creatore C., Dawson E., et al. COSMIC: The Catalogue of Somatic Mutations In Cancer. Nucleic Acids Res. 2019;47:D941–D947. doi: 10.1093/nar/gky1015. PubMed DOI PMC

Buttner R., Longshore J.W., Lopez-Rios F., Merkelbach-Bruse S., Normanno N., Rouleau E., Penault-Llorca F. Implementing TMB measurement in clinical practice: Considerations on assay requirements. ESMO Open. 2019;4:e000442. doi: 10.1136/esmoopen-2018-000442. PubMed DOI PMC

Deans Z.C., Costa J.L., Cree I., Dequeker E., Edsjo A., Henderson S., Hummel M., Ligtenberg M.J., Loddo M., Machado J.C., et al. Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL. Virchows Arch. 2017;470:5–20. doi: 10.1007/s00428-016-2025-7. PubMed DOI PMC

Van Krieken H., Deans S., Hall J.A., Normanno N., Ciardiello F., Douillard J.Y. Quality to rely on: Meeting report of the 5th Meeting of External Quality Assessment,c Naples 2016. ESMO Open. 2016;1:e000114. doi: 10.1136/esmoopen-2016-000114. PubMed DOI PMC