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Poster Display session

455 - Reliability of the detection of the mutation burden status by targeted next generation sequencing applying a large gene panel


14 Dec 2018


Poster Display session


Nicole Pfarr


Annals of Oncology (2018) 29 (suppl_10): x1-x10. 10.1093/annonc/mdy493


N. Pfarr1, M. Boxberg1, K. Riedmann2, M. Jesinghaus1, B. Konukiewitz1, H. Glimm3, P.J. Jost4, S. Fröhling5, W. Weichert6

Author affiliations

  • 1 Institute Of Pathology, Technische Universität München, 81675 - Munich/DE
  • 2 Medical Department Iii, Klinikum rechts der Isar, 81675 - Munich/DE
  • 3 Nct, National Center for Tumor Diseases, Dresden/DE
  • 4 Medical Department Iii, Klinikum rechts der Isar, Munich/DE
  • 5 Dkfz, HI-STEM gGmbH im Deutschen Krebsforschungszentrum, 69120 - Heidelberg/DE
  • 6 Institute Of Pathology, Technische Universität München, Munich/DE


Abstract 455


In the emerging field biomarker analysis the need for testing of tumor mutation burden in routine diagnostic becomes more and more important. Currently most analysis is done by exome sequencing but regarding time for diagnosis and limitation of tissue material for testing the applicability of large gene panels becomes of great value. To test the feasibility of targeted next generation sequencing using formalin-fixed paraffin-embedded tissue for assessing tumor mutation burden statuswe applied the 1.95 Mb Illumina TSO500 panel covering an exonic region of about 1.24 Mb.


A three-phase approach was applied: 1. Validation of the panel using DNA derived from 8 different cell lines (mutation high vs. mutation low). 2. Comparison of data from whole exome sequencing (mutation high vs. mutation low) vs.the TSO500 panel from 36 FFPE tumor samples. 3. Testing of ten samples with a) high PD-L1, or b) high MSI status or c) POLE mutation. Sequencing was performed on a NextSeq® system with the NextSeq® 500 hi-Output Kit v2 (300 cycles) and 40ng DNA as input. Data analysis was performed using a bioinformatics pipeline (“TSO500“) provided by Illumina with a limit of detection of 5% allele frequency.


The mutation burden status of all validation samples (phase 1) were confirmed applying our NGS panel approach achieving a concordance of 100%. In phase 2 we were able to confirm the mutation burden status high or low derived from exome sequencing for all except for two cases achieving a concordance of 94% between the different approaches. In further testing a UDG pre-treatment of DNA samples will be performed to decrease the amount of fixation artefacts and therefore improve the quality of the tested samples. Analysis of the samples from phase 3 confirmed a high mutation status for the MSI high and the POLE mutated cases all revealing a mutation burden values whereas the PD-L1 cases showed only borderline values.


We here show that data generated by targeted NGS data approaches can be used to determine the mutation burden status in tumor samples of different entities in high concordance with exome sequencing. Furthermore, additional genetic events (e.g. MSI) that may be clinically exploitable can be identified using a single assay.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

Nicole Pfarr.




N. Pfarr: Member of the immune oncology Consortium (Thermo Fisher Scientific). H. Glimm: Clinical studies: Pfizer, AstraZeneca; Honoraria and travel: Roche, Lilly, Amgen. S. Fröhling: Clinical studies: Pfizer, PharmaMar, AstraZeneca; Honoraria and travel: PharmaMar, Roche, Lilly, Amgen.  W. Weichert: Advisory board and/or speakers bureau: Lilly, BMS, Merck/Sharpe, Dome, Novartis, Pfizer, Roche, AstraZeneca, Boehringer, Merck; Research funding: BMS, Roche, MSD. All other authors have declared no conflicts of interest.

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