Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing
Abstract Background The rapid adoption of next-generation sequencing provides an efficient system for detecting somatic alterations in neoplasms. The detection of such alterations requires a matched non-neoplastic sample for adequate filtering of non-somatic events such as germline polymorphisms. No...
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2016-10-01
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Series: | BMC Medical Genomics |
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Online Access: | http://link.springer.com/article/10.1186/s12920-016-0226-1 |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lei Wei Antonios Papanicolau-Sengos Song Liu Jianmin Wang Jeffrey M. Conroy Sean T. Glenn Elizabeth Brese Qiang Hu Kiersten Marie Miles Blake Burgher Maochun Qin Karen Head Angela R. Omilian Wiam Bshara John Krolewski Donald L. Trump Candace S. Johnson Carl D. Morrison |
spellingShingle |
Lei Wei Antonios Papanicolau-Sengos Song Liu Jianmin Wang Jeffrey M. Conroy Sean T. Glenn Elizabeth Brese Qiang Hu Kiersten Marie Miles Blake Burgher Maochun Qin Karen Head Angela R. Omilian Wiam Bshara John Krolewski Donald L. Trump Candace S. Johnson Carl D. Morrison Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing BMC Medical Genomics Somatic mutations Tumor contamination Adjacent normal tissues |
author_facet |
Lei Wei Antonios Papanicolau-Sengos Song Liu Jianmin Wang Jeffrey M. Conroy Sean T. Glenn Elizabeth Brese Qiang Hu Kiersten Marie Miles Blake Burgher Maochun Qin Karen Head Angela R. Omilian Wiam Bshara John Krolewski Donald L. Trump Candace S. Johnson Carl D. Morrison |
author_sort |
Lei Wei |
title |
Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
title_short |
Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
title_full |
Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
title_fullStr |
Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
title_full_unstemmed |
Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
title_sort |
pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing |
publisher |
BMC |
series |
BMC Medical Genomics |
issn |
1755-8794 |
publishDate |
2016-10-01 |
description |
Abstract Background The rapid adoption of next-generation sequencing provides an efficient system for detecting somatic alterations in neoplasms. The detection of such alterations requires a matched non-neoplastic sample for adequate filtering of non-somatic events such as germline polymorphisms. Non-neoplastic tissue adjacent to the excised neoplasm is often used for this purpose as it is simultaneously collected and generally contains the same tissue type as the neoplasm. Following NGS analysis, we and others have frequently observed low-level somatic mutations in these non-neoplastic tissues, which may impose additional challenges to somatic mutation detection as it complicates germline variant filtering. Methods We hypothesized that the low-level somatic mutation observed in non-neoplastic tissues may be entirely or partially caused by inadvertent contamination by neoplastic cells during the surgical pathology gross assessment or tissue procurement process. To test this hypothesis, we applied a systematic protocol designed to collect multiple grossly non-neoplastic tissues using different methods surrounding each single neoplasm. The procedure was applied in two breast cancer lumpectomy specimens. In each case, all samples were first sequenced by whole-exome sequencing to identify somatic mutations in the neoplasm and determine their presence in the adjacent non-neoplastic tissues. We then generated ultra-deep coverage using targeted sequencing to assess the levels of contamination in non-neoplastic tissue samples collected under different conditions. Results Contamination levels in non-neoplastic tissues ranged up to 3.5 and 20.9 % respectively in the two cases tested, with consistent pattern correlated with the manner of grossing and procurement. By carefully controlling the conditions of various steps during this process, we were able to eliminate any detectable contamination in both patients. Conclusion The results demonstrated that the process of tissue procurement contributes to the level of contamination in non-neoplastic tissue, and contamination can be reduced to below detectable levels by using a carefully designed collection method. A standard protocol dedicated for acquiring adjacent non-neoplastic tissue that minimizes neoplasm contamination should be implemented for all somatic mutation detection studies. |
topic |
Somatic mutations Tumor contamination Adjacent normal tissues |
url |
http://link.springer.com/article/10.1186/s12920-016-0226-1 |
work_keys_str_mv |
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doaj-b68b8cd9e13241639297e3c8e50735322021-04-02T15:17:45ZengBMCBMC Medical Genomics1755-87942016-10-019111010.1186/s12920-016-0226-1Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencingLei Wei0Antonios Papanicolau-Sengos1Song Liu2Jianmin Wang3Jeffrey M. Conroy4Sean T. Glenn5Elizabeth Brese6Qiang Hu7Kiersten Marie Miles8Blake Burgher9Maochun Qin10Karen Head11Angela R. Omilian12Wiam Bshara13John Krolewski14Donald L. Trump15Candace S. Johnson16Carl D. Morrison17Department of Biostatistics and Bioinformatics, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteDepartment of Biostatistics and Bioinformatics, Roswell Park Cancer InstituteDepartment of Biostatistics and Bioinformatics, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteDepartment of Cancer Genetics, Roswell Park Cancer InstituteDepartment of Pathology, Roswell Park Cancer InstituteDepartment of Biostatistics and Bioinformatics, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteDepartment of Biostatistics and Bioinformatics, Roswell Park Cancer InstituteDepartment of Pathology, Roswell Park Cancer InstituteDepartment of Pathology, Roswell Park Cancer InstituteDepartment of Pathology, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteDepartment of Medicine, Roswell Park Cancer InstituteDepartment of Pharmacology and Therapeutics, Roswell Park Cancer InstituteCenter for Personalized Medicine, Roswell Park Cancer InstituteAbstract Background The rapid adoption of next-generation sequencing provides an efficient system for detecting somatic alterations in neoplasms. The detection of such alterations requires a matched non-neoplastic sample for adequate filtering of non-somatic events such as germline polymorphisms. Non-neoplastic tissue adjacent to the excised neoplasm is often used for this purpose as it is simultaneously collected and generally contains the same tissue type as the neoplasm. Following NGS analysis, we and others have frequently observed low-level somatic mutations in these non-neoplastic tissues, which may impose additional challenges to somatic mutation detection as it complicates germline variant filtering. Methods We hypothesized that the low-level somatic mutation observed in non-neoplastic tissues may be entirely or partially caused by inadvertent contamination by neoplastic cells during the surgical pathology gross assessment or tissue procurement process. To test this hypothesis, we applied a systematic protocol designed to collect multiple grossly non-neoplastic tissues using different methods surrounding each single neoplasm. The procedure was applied in two breast cancer lumpectomy specimens. In each case, all samples were first sequenced by whole-exome sequencing to identify somatic mutations in the neoplasm and determine their presence in the adjacent non-neoplastic tissues. We then generated ultra-deep coverage using targeted sequencing to assess the levels of contamination in non-neoplastic tissue samples collected under different conditions. Results Contamination levels in non-neoplastic tissues ranged up to 3.5 and 20.9 % respectively in the two cases tested, with consistent pattern correlated with the manner of grossing and procurement. By carefully controlling the conditions of various steps during this process, we were able to eliminate any detectable contamination in both patients. Conclusion The results demonstrated that the process of tissue procurement contributes to the level of contamination in non-neoplastic tissue, and contamination can be reduced to below detectable levels by using a carefully designed collection method. A standard protocol dedicated for acquiring adjacent non-neoplastic tissue that minimizes neoplasm contamination should be implemented for all somatic mutation detection studies.http://link.springer.com/article/10.1186/s12920-016-0226-1Somatic mutationsTumor contaminationAdjacent normal tissues |