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


OBJECTIVE
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.


CONCLUSIONS
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.
Department of General Dermatology, Medical University Graz, Graz, Austria.
Richard
R
Ofner
Department of General Dermatology, Medical University Graz, Graz, Austria.|Translational Skin Cancer Research-TSCR, German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, German Cancer Research Center (DKFZ), University Hospital Essen, Essen, Germany.
Cathrin
C
Ritter
Department of Dermatology, University Hospital of Essen, Essen, Germany.
Selma
S
Ugurel
Department of General Dermatology, Medical University Graz, Graz, Austria.
Lorenzo
L
Cerroni
Translational Skin Cancer Research-TSCR, German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, German Cancer Research Center (DKFZ), University Hospital Essen, Essen, Germany.
Mathias
M
Stiller
Boehringer Ingelheim RCV, Vienna, Austria.
Thomas
T
Bogenrieder
Boehringer Ingelheim RCV, Vienna, Austria.
Flavio
F
Solca
Department of Dermatology, University Hospital of Würzburg, Würzburg, Germany.
David
D
Schrama
Department of General Dermatology, Medical University Graz, Graz, Austria. j.becker@dkfz.de.|Translational Skin Cancer Research-TSCR, German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, German Cancer Research Center (DKFZ), University Hospital Essen, Essen, Germany. j.becker@dkfz.de.|Department of Dermatology, University Hospital of Essen, Essen, Germany. j.becker@dkfz.de.
Jürgen C
http://orcid.org/0000-0001-9183-653X
JC
Becker
D016477:Artifacts; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde; D006801:Humans; D008545:Melanoma / Q000235:genetics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D066247:Receptor, ErbB-4 / Q000032:analysis / Q000235:genetics*; D016707:Tissue Fixation
D005557:Formaldehyde; D004247:DNA; C587689:ERBB4 protein, human; D066247:Receptor, ErbB-4
ERBB4; FFPE; Melanoma; Sanger sequencing; Sequencing artifacts