Validation of multiplex steroid hormone measurements in prostate cancer using plasma for multimodality biomarker studies

Background Steroid hormones are essential signalling molecules in prostate cancer (PC). However, many studies focusing on liquid biomarkers fail to take the hormonal status of these patients into account. Steroid measurements are sensitive to bias caused by matrix effects, thus assessing potential matrix effects is an important step in combining circulating tumour DNA analysis with hormone status. Methods We investigated the accuracy of multi-steroid hormone profiling in mechanically-separated plasma (MSP) samples and in plasma from CellSave Preservative (CS) tubes, that are typically used to obtain circulating tumour DNA (ctDNA), compared to measurements in serum. We performed multiplex steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples obtained from ten healthy controls and ten castration-resistant prostate cancer (CRPC) patients. Results Steroid measurements were comparable between MSP and serum. A small but consistent decrease of 8 – 21% compared to serum was observed when using CS plasma, which was considered to be within the acceptable margin. The minimal residual testosterone levels of CRPC patients could be sensitively quantified in both MSP and CS samples. Conclusions We validated the use of MSP and CS samples for multi-steroid profiling by LC-MS/MS. The optimised use of these samples in clinical trials will allow us to gain further insight into the steroid metabolism in PC patients. ### Competing Interest Statement M. P. L. is the recipient of grants of Sanofi, Johnson & Johnson and Astellas. Other authors did not declare a conflict of interests ### Funding Statement Funding for this project was provided by the Daniel den Hoed foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Medical ethical committee of the Erasmus MC Cancer Institute, Rotterdam, The Netherlands. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. * ANOVA : Analysis of variance CRPC : Castration-resistant prostate cancer CS : CellSave Preservative CTC : Circulating tumour cell ctDNA : circulating tumour DNA DHEA : Dehydroepiandrosterone DHT : 5a-dihydrotestosterone HC : Healthy Control LC-MS/MS : Liquid-chromatography tandem mass spectrometry LLOQ : Lower limits of quantification mCRPC : Metastatic castration-resistant prostate cancer MS : Mass spectrometry MSP : Mechanically-separated plasma MTBE : Methyl-tert butyl ether PC : Prostate cancer PBS-BSA : phosphate-buffered saline with bovine serum albumin