Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples

Piskorz, A.M. et al. (2016) Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples. Annals of Oncology, 27(3), pp. 532-539. (doi: 10.1093/annonc/mdv613) (PMID:26681675) (PMCID:PMC4769995)

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Background Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but requires high quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. Patients and Methods We have performed detailed comparison of DNA quality from matched samples isolated from high grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments as well as mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Results Using matched snap frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy number calling using shallow whole genome sequencing. These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and whole genome sequencing data. Conclusion We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Orange, Miss Clare and Ennis, Dr Darren and Mcneish, Professor Iain
Authors: Piskorz, A.M., Ennis, D., Macintyre, G., Goranova, T.E., Eldridge, M., Segui-Gracia, N., Valganon, M., Hoyle, A., Orange, C., Moore, L., Jimenez-Linan, M., Millan, D., Mcneish, I.A., and Brenton, J.D.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Journal Name:Annals of Oncology
Publisher:Oxford University Press
ISSN (Online):1569-8041
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Annals of Oncology 27(3):532-539
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
643981Personalised biomarkers of response in high-grade serious ovarian cancerIain McNeishCancer Research UK (CAN-RES-UK)C608/A15973RI CANCER SCIENCES