Talia Novos (Presenter)
Prince of Wales Hospital, SEALS
Bio: Talia Novos currently works as a Hospital Scientist at Prince of Wales Hospital in the Mass spectrometry, Chemistry and Endocrinolgy department where she performs analysis on both HPLC and Liquid chromatography Mass Spectrometry (LC-MS) for a range of chemistry and endocrinology analytes. She has been involved in the areas of therapeutic drug monitoring, analysis of Clinical biomarkers and studies of endocrine tumors. Talia has a bachelors Degree majoring in Biochemistry from Sydney University and a Masters in forensic science from the University of Canberra. She is also member of the Australian association of clinical biochemists (AACB) committee, as well as the Biogenic Amines working party and Vitamins working party.
Authorship: Talia Novos (1), Michael Wright (1), Trisha Dwight (2), Edward Kim (2), Roderick Clifton-Bligh (2)
(1) Prince of Wales Hospital, SEALS, Sydney, Australia, (2) Kolling Institute of Medical Research, Sydney, Australia
| Short Abstract Paragangliomas and pheochromocytomas are endocrine tumours associated with mutations of the Succinate dehydrogenase (SDH) gene. SDH catalyses the conversion of succinate to fumarate in the Krebs cycle. Therefore a mutation in the SDHx gene will result in an accumulation of succinate and decreased production of fumarate. We have developed a novel method for the analysis of these metabolites by liquid chromatography mass spectrometry. Increased succinate:fumarate ratios correlate with patients that have been confirmed to have SDHx mutations with immunochemistry staining. This method will aid in the early detection of patients at risk of developing SDHx associated tumours. |
Long Abstract
Background
Succinate dehydrogenase (SDH) is a crucial enzyme, catalysing the oxidation of succinate to fumarate in the Krebs cycle. Mutations affecting any of the four genes (SDHA-D, collectively SDHx) that encode the SDH complex have been associated with the development of a number of tumours, including phaeochromocytomas, paragangliomas, gastrointestional stromal tumours and renal cell carcinomas. As a consequence of these SDHx mutations, a loss (or reduction) in SDH function occurs, which results in an accumulation of succinate and decreased production of fumarate.
Objectives
Develop a method for the rapid analysis of Krebs cycle metabolites, in particular succinate and fumarate, and assess the utility of this method to reliably predict SDHx mutations, as well as its applicability for assessment of archived tissue.
Methods
Fresh frozen (FF) tumour and formalin-fixed paraffin-embedded (FFPE) tissue was obtained from 48 patients with pheochromocytoma, paraganglioma, gastrointestional stromal tumour or renal cell carcinoma. Of these, 22 patients were SDHx-mutated, while the remaining 26 were SDHx-wildtype. Metabolites were extracted, and samples reconstituted in water prior to ultracentrifugation. Analytes were separated using an Ascentis Express RP-Amide column under isocratic conditions of 0.4% formic acid in water before detection on an SCIEX API 5500 using electrospray ionization in negative mode. Sample to sample run time was 3 minutes.
Results
Succinate:fumarate ratios observed in the SDHx-mutated tumors were higher when compared to SDHx-wildtype tumors (p<0.05). Succinate:Fumarate ratios were comparable between FF tumour and FFPE tissue samples. Additional analyses based on specific tumour types will be presented.
Discussion
We have developed a rapid method for the analysis of Krebs cycle metabolites (in both fresh frozen and formalin-fixed paraffin-embedded tissue), and have shown that elevated succinate:fumarate ratios can be used to effectively identify patients that are likely to harbor SDHx mutations. This method will aid in the early detection of patients at risk of developing SDHx-associated tumours and could be more broadly applied to assist in the identification of mutations occurring in other genes associated with the Krebs cycle. Further, this method could be applied to assist with the classification of gene variants of unknown clinical significance, helping identify those variants that are pathogenic.
References & Acknowledgements:
The authors would like to acknowledge the assistance of David Bell (Supelco, Bellefonte,PA, USA)and Chris Hodgkins (SCIEX, Sydney, Australia).
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