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. 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.

Distribution of TERT promoter mutations in primary and metastatic melanomas in Austrian patients.

BACKGROUND: TERT promoter mutations were detected at high frequencies in several cancer types including melanoma. Previous reports showed that these recurrent mutations increase TERT gene expression and the use of TERT mutation status as prognostic factor was suggested. OBJECTIVES: Here we screen a panel of 115 melanoma tumor samples from Austrian patients to evaluate the prevalence and distribution of TERT promoter mutations. The association with clinical and tumor characteristics and the effect on overall survival was analyzed. METHODS: Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis. RESULTS: We identified TERT promoter mutations in 63 of 115 (54.8%) tumor samples. No statistical significant difference in mutation frequency between primary (22/40 [55%]) and metastatic lesions (41/75 [54.7%]) was detected. BRAF-/NRAS-mutated tumors showed a higher frequency of TERT mutations (pT OR 2.24, 95% CI 0.56-9.02, p = 0.3) (met OR 2.74, 95% CI 0.98-7.66, p = 0.05). In primary melanoma, the presence of alterations in TERT was associated with the carrier status of a common single-nucleotide polymorphism rs2853669 (OR 4.55, CI 1.18-17.52, p = 0.03). In this patient cohort, TERT promoter mutations were not associated with clinical characteristics such as the presence of ulceration or Breslow thickness or showed an effect on overall survival. CONCLUSION: Alterations in the TERT promoter region are one of the most frequent mutations in melanoma. Based on this analysis and preliminary evidence, prospective studies will be needed to evaluate the reliability of TERT promoter mutations as prognostic factors in melanoma.