Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing

0 Department of Pathology, The University of Melbourne , Parkville, Victoria 3010 , Australia 1 Department of Pathology, Peter MacCallum Cancer Centre , East Melbourne, Victoria 3002 , Australia Background: Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood. Methods: The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days). Results: Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive. Conclusions: These findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes. - Background Advances in genomic technologies are improving the capability to arrive at a more informed decision on how to treat a patient [1,2]. In particular, a large number of genes can be screened for actionable changes using massively parallel sequencing (MPS) approaches [3,4]. Accurate and reliable interpretation of this new type of genomic data is therefore critical in deciding the appropriate course of management for patients. Most DNA for genetic testing from cancer patients is extracted from formalin-fixed paraffin-embedded tumour biopsies, where the primary intent is to preserve tumour cellular structure for histological examination and diagnosis [5]. However, the formalin fixation process is detrimental to downstream genomic applications, causing issues, such as DNA cross-linking to DNA and proteins, that can stall polymerases and DNA-DNA crosslinks that can inhibit denaturation [6]. A common form of DNA damage induced by formalin fixation is fragmentation, which can lead to low amounts of amplifiable template for PCR amplification. Fragmentation of DNA is caused by a number of factors during the fixation process, e.g. low pH formalin over time increases the rate of apurinic/apyrimidinic site formation and eventually decomposition and fragmentation [7]. Long-term storage of formalin-fixed blocks can also induce fragmentation due to exposure to environmental conditions [8-10]. Another prominent type of DNA damage that occurs commonly in formalin-fixed tissues is the hydrolytic deamination of cytosine to form uracil (or thymine if the cytosine is methylated). This results in non-reproducible C>T/G>A sequencing artefacts that are observed after PCR amplification when using formalin-fixed and paraffinembedded (FFPE) DNA [11-13]. Recently, we assessed an amplicon-based MPS technology and showed that C>T and G>A changes were the most prominent sequence errors in three formalinfixed lung squamous cell carcinoma samples [12]. While such artefacts occur in many formalin-fixed samples, we have found this to be more pronounced in highly fragmented samples. Due to stochastic effects, the low template numbers increase the probability of occurrence of template artefacts [14]. Many studies have reported the feasibility of using DNA from formalin-fixed material using both conventional PCR-based and MPS technologies [4,15,16]. Fragmentation can be a rate-limiting factor in ampliconbased approaches especially those that use longer amplicons. Shorter amplicons permit fragmented DNA from formalin-fixed material to be used more successfully. Some studies have reported elevated numbers of sequence artefacts [12,16-19] whereas others reported little evidence of artefacts appearing in FFPE samples [15,20]. The small number of samples assessed as well as the varying age of biopsies, degree of fixation and sequencing technologies used makes it difficult to know how important sequence artefacts are as a source of error in relatively fresh FFPE samples. This study assessed the prevalence of DNA fragmentation and sequencing artefacts from a large cohort of FFPE tumours using a uniform approach whereby all blocks were of similar age, were extracted in the same manner and were run through the same MPS platform. The originating anatomical pathology laboratory was also recorded to determine if variation in operating practices could affect sequencing artefacts and DNA fragmentation. Methods Patients and cell lines Cancer 2015 is a large-scale, prospective, longitudinal, multi-site cohort study of incident cancers in the Victorian population. The aim of the Cancer 2015 study is to classify cancers molecularly using MPS to promote more targeted treatment of cancer patients and improve patient survival and outcomes. An initial pilot phase was established to determine the feasibility of adopting MPS for the diagnostic mutational profiling of tumours. This study was approved by the Human Research Ethics Committees at the Peter MacCallum Cancer Centre, Royal Melbourne, Cabrini, Geelong and Warrnambool Hospitals, all located within the state of Victoria, Australia. All patients provided informed consent to participate in this study. Formalinfixed, paraffin-embedded (FFPE) tumour blocks or unstained sections from FFPE tumour blocks were acquired from anatomical pathology laboratories performing the diagnosis and sent to the Peter MacCallum Cancer Centre Pathology departm (...truncated)