title: Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions.
abstract: Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved.
  
  
  
  DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines.
  
  
  
  Libraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation -0.81; p < 1e-10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed.
  
  
  
  Reporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.
authors.affiliation: Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, 141700, Russia.|Atlas Biomed Group, Moscow, 121069, Russia.|Institute of Chemical Biology and Fundamental Medicine of SB RAS, Novosibirsk, 630090, Russia.
authors.forename: Maxim
authors.initials: M
authors.lastname: Ivanov
authors.affiliation: N.N. Blokhin Russian Cancer Research Center, Ministry of Health of the Russian Federation, Kashirskoe sh. 24, Moscow, 115478, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Laktionov
authors.affiliation: N.N. Blokhin Russian Cancer Research Center, Ministry of Health of the Russian Federation, Kashirskoe sh. 24, Moscow, 115478, Russia.
authors.forename: Valery
authors.initials: V
authors.lastname: Breder
authors.affiliation: N.N. Blokhin Russian Cancer Research Center, Ministry of Health of the Russian Federation, Kashirskoe sh. 24, Moscow, 115478, Russia.
authors.forename: Polina
authors.initials: P
authors.lastname: Chernenko
authors.affiliation: Federal State Budgetary Institution Russian Scientific Center of Roentgenoradiology (RSCRR) of the Ministry of Healthcare of the Russian Federation (Russian Scientific Center of Roentgenoradiology), Moscow, 117485, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Novikova
authors.affiliation: Federal State Budgetary Institution Russian Scientific Center of Roentgenoradiology (RSCRR) of the Ministry of Healthcare of the Russian Federation (Russian Scientific Center of Roentgenoradiology), Moscow, 117485, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Telysheva
authors.affiliation: Atlas Biomed Group, Moscow, 121069, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Musienko
authors.affiliation: Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, 141700, Russia. abaranov@gmu.edu.|Atlas Biomed Group, Moscow, 121069, Russia. abaranov@gmu.edu.|Research Centre for Medical Genetics, Moscow, 115478, Russia. abaranov@gmu.edu.|Center for the Study of Chronic Metabolic and Rare Diseases, School of System Biology, George Mason University, Fairfax, VA, USA. abaranov@gmu.edu.
authors.forename: Ancha
authors.initials: A
authors.lastname: Baranova
authors.affiliation: Atlas Biomed Group, Moscow, 121069, Russia.|Institute of Chemical Biology and Fundamental Medicine of SB RAS, Novosibirsk, 630090, Russia.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Mileyko
mesh_terms: D016477:Artifacts; D004247:DNA / Q000235:genetics; D056915:DNA Copy Number Variations / Q000235:genetics; D005091:Exons / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry*; D005787:Gene Frequency / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards*; D006801:Humans; D033162:Incidental Findings; D008495:Medical Oncology; D009154:Mutation / Q000235:genetics; D016612:Paraffin Embedding; D012015:Reference Standards; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde; D004247:DNA
keywords: Cancer; NGS; Non-small cell lung cancer; Oncology; Precision oncology; Targeted therapy