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Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions

BACKGROUND: 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 pro...

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Autores principales: Ivanov, Maxim, Laktionov, Konstantin, Breder, Valery, Chernenko, Polina, Novikova, Ekaterina, Telysheva, Ekaterina, Musienko, Sergey, Baranova, Ancha, Mileyko, Vladislav
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282851/
https://www.ncbi.nlm.nih.gov/pubmed/28137276
http://dx.doi.org/10.1186/s12967-017-1125-8
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author Ivanov, Maxim
Laktionov, Konstantin
Breder, Valery
Chernenko, Polina
Novikova, Ekaterina
Telysheva, Ekaterina
Musienko, Sergey
Baranova, Ancha
Mileyko, Vladislav
author_facet Ivanov, Maxim
Laktionov, Konstantin
Breder, Valery
Chernenko, Polina
Novikova, Ekaterina
Telysheva, Ekaterina
Musienko, Sergey
Baranova, Ancha
Mileyko, Vladislav
author_sort Ivanov, Maxim
collection PubMed
description BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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spelling pubmed-52828512017-02-03 Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions Ivanov, Maxim Laktionov, Konstantin Breder, Valery Chernenko, Polina Novikova, Ekaterina Telysheva, Ekaterina Musienko, Sergey Baranova, Ancha Mileyko, Vladislav J Transl Med Research BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. BioMed Central 2017-01-31 /pmc/articles/PMC5282851/ /pubmed/28137276 http://dx.doi.org/10.1186/s12967-017-1125-8 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Ivanov, Maxim
Laktionov, Konstantin
Breder, Valery
Chernenko, Polina
Novikova, Ekaterina
Telysheva, Ekaterina
Musienko, Sergey
Baranova, Ancha
Mileyko, Vladislav
Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title_full Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title_fullStr Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title_full_unstemmed Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title_short Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions
title_sort towards standardization of next-generation sequencing of ffpe samples for clinical oncology: intrinsic obstacles and possible solutions
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282851/
https://www.ncbi.nlm.nih.gov/pubmed/28137276
http://dx.doi.org/10.1186/s12967-017-1125-8
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