Whilst adequate for most existing pathological tests, formalin is generally considered a poor DNA preservative and use of alternative fixatives may prove advantageous for molecular testing of tumour material; an increasingly common approach to identify targetable driver mutations in lung cancer patients. We collected paired PAXgene®tissue-fixed and formalin-fixed samples of block-sized tumour and lung parenchyma, Temno-needle core tumour biopsies and fine needle tumour aspirates (FNAs) from non-small cell lung cancer resection specimens. Traditionally processed formalin fixed paraffin wax embedded (FFPE) samples were compared to paired PAXgene® tissue fixed paraffin-embedded (PFPE) samples. We evaluated suitability for common laboratory tests (H&E staining and immunohistochemistry) and performance for downstream molecular investigations relevant to lung cancer ,including RT-PCR and next generation DNA sequencing (NGS). Adequate and comparable H&E staining was seen in all sample types and nuclear staining was preferable in PAXgene® fixed Temno tumour biopsies and tumour FNA samples. Immuno histochemical staining was broadly comparable. PFPE samples enabled greater yields of less-fragmented DNA than FFPE comparators. PFPE samples were also superior for PCR and NGS performance, both in terms of quality control metrics and for variant calling. Critically we identified a greater number of genetic variants in the epidermal growth factor receptor gene when using PFPE samples and the Ingenuity® Variant Analysis pipeline. In summary, PFPE samples are adequate for histopathological diagnosis and suitable for the majority of existing laboratory tests. PAXgene® fixation is superior for DNA and RNA integrity, particularly in low-yield samples and facilitates improved NGS performance, including the detection of actionable lung cancer mutations for precision medicine in lung cancer samples.
This study was performed as part of the STRATfix study; ‘Enabling stratified medicine with novel fixatives for improved pre‐analytical pathology workflows’ and was funded by Innovate UK (Grant Ref. 38618‐281179). We also acknowledge the staff of the Royal Papworth Hospital Research Tissuebank for assistance with sample collection and patients for providing consent.
© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.
- DNA sequencing
- Lung Cancer
- Pathology and Forensic Medicine