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.