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Journal of Cancer Research and Clinical Oncology

, Volume 143, Issue 7, pp 1199–1207 | Cite as

Non-reproducible sequence artifacts in FFPE tissue: an experience report

  • Richard Ofner
  • Cathrin Ritter
  • Selma Ugurel
  • Lorenzo Cerroni
  • Mathias Stiller
  • Thomas Bogenrieder
  • Flavio Solca
  • David Schrama
  • Jürgen C. Becker
Original Article – Cancer Research
  • 399 Downloads

Abstract

Background

Recent advances in sequencing technologies supported the development of molecularly targeted therapy in cancer patients. Thus, genomic analyses are becoming a routine part in clinical practice and accurate detection of actionable mutations is essential to assist diagnosis and therapy choice. However, this is often challenging due to major problems associated with DNA from formalin-fixed paraffin-embedded tissue which is usually the primary source for genetic testing.

Objectives

Here we want to share our experience regarding major problems associated with FFPE DNA used for PCR-based sequencing as illustrated by the mutational analysis of ERBB4 in melanoma. We want to focus on two major problems including extensive DNA fragmentation and hydrolytic deamination as source of non-reproducible sequence artifacts. Further, we provide potential explanations and possible strategies to minimize these difficulties and improve the detection of targetable mutations.

Methods

Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis.

Results

Analysis of Sanger sequencing data revealed a total of 46 ERBB4 mutations in 27 of 96 samples including the identification of 11 mutations at three previously unknown mutational hotspots. Unfortunately, we were not able to confirm any assumed hotspot mutation within repeated sequencing of relevant amplicons suggesting the detection of sequence artifacts most likely caused by DNA lesions associated with FFPE tissues.

Conclusion

Since DNA from FFPE tissue is usually the primary source for mutational analyses, appropriate measures must be implemented in the workflow to assess DNA damage in formalin-fixed tissue to ensure accurate detection of actionable mutations and minimize the occurrence of sequence artifacts.

Keywords

Melanoma ERBB4 Sequencing artifacts Sanger sequencing FFPE 

Notes

Acknowledgements

We thank Isabella Fried for supporting this study, and Gerlinde Mayer and Ulrike Schmidbauer for excellent technical assistance (all Department of General Dermatology, Medical University Graz, Graz, Austria).

Compliance with ethical standards

Conflict of interest

The authors RO, CR, MS, LC, and DS state no conflict of interest. Author SU has received advisory board honorariums from Roche, BMS and Novartis. The authors TB and FS are employees of Boehringer Ingelheim. Author JCB has received speaker honorariums from Amgen, MerckSerono, and Pfizer, advisory board honorariums from Amgen, CureVac, Lytex, MerckSerono, Novartis, Rigontec, and Takeda as well as research funding from Boehringer Ingelheim, BMS and MerckSerono; none of these activities are related to the submitted report.

Ethics approval

The study was approved by the institutional review board of the Medical University of Graz (ethics votum 24-397 ex 11/12).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Funding

This study was funded in part by a Research Grant of Boehringer Ingelheim RCV, Vienna, Austria.

Supplementary material

432_2017_2399_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 20 KB)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of General DermatologyMedical University GrazGrazAustria
  2. 2.Translational Skin Cancer Research-TSCR, German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, German Cancer Research Center (DKFZ)University Hospital EssenEssenGermany
  3. 3.Department of DermatologyUniversity Hospital of EssenEssenGermany
  4. 4.Department of DermatologyUniversity Hospital of WürzburgWürzburgGermany
  5. 5.Boehringer Ingelheim RCVViennaAustria

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