Variability in Testosterone Measurement Between Radioimmunoassay (RIA), Chemiluminescence Assay (CLIA) and Liquid Chromatography-Tandem Mass Spectrometry (MS) Among Prostate Cancer Patients on Androgen Deprivation Therapy (ADT)

Historically, the definition of castrate testosterone levels for men on androgen deprivation therapy was set at 50ng/dL (1.7nM). This threshold corresponded to the lowest limit of quantification (LLOQ) using the radio-immunoassays (RIA) available at that time.¹ To date, this level remains widely accepted as a castrate threshold in most guidelines.² Improvements in immunoassay technology have seen LLOQ levels dropping and newer thresholds for castration definitions being studied. Klotz et al. showed that nadir testosterone ≤ 20ng/dL (0.7nM) would correspond to a longer time to castrate resistant prostate cancer (CRPC).³ This was followed up with a Canadian consensus statement in 2018 that called for testosterone suppression to ≤ 20ng/dL as a clinical goal, with a recommendation of alternate medical or surgical therapy if that threshold was not reached.⁴

Immunoassays like RIA and CLIA have traditionally been the work horses of testosterone measurement in most medical centers worldwide as they are automatable, fast, and cost-effective. Despite this, they have significant limitations showing a lack of specificity and reproducibility, especially at low levels of testosterone.⁵ This has been explained by interference of immunoassay measurements by various steroids that remain elevated in low testosterone states which have analogous chemical structures to testosterone and therefore cause falsely elevated readings. Some examples include androstenedione, dihydrotestosterone, and androstenediol.⁶ Liquid chromatography-tandem mass spectrometry (MS) in contrast is regarded as the gold standard for testosterone measurement with the Canadian consensus statement of 2018 recommending external validation of all immunoassay detected testosterone measurements ≤20 ng/dL, with recommendations for alternate medical or surgical therapy if that threshold was not reached.⁴ Despite this MS remains infrequently used due to high costs, the need for specialized equipment, and skilled technicians. The aim of our study was to determine the discordance rates of testosterone measurements amongst men with RIA, CLIA, and MS and to illustrate the magnitude of immunoassay overestimation in low testosterone states.

Our retrospective study analyzed 95 patients who had prostate cancer from 2011-2019 on ADT for at least 3 months. Patients on second line androgen deprivation agents and chemotherapy were excluded. Serum samples were split in triplicate with immunoassays being analyzed at Oregon Health Sciences University and MS being performed at University Laval. Observational data was reported and testosterone measurements were analyzed for variability looking for categorical concordance and over/under-estimation rates.

The results indicated that there was significant variability between the various measures of testosterone. 95% of patients measured ≤20ng/dL on MS and CLIA as compared to 80% by RIA. After subdividing patients into sub-categories (≤20, 20-50, ≥50 ng/dL) concordance analysis showed that 4.3% and 18.9% of testosterone measured by MS would have a different category result when remeasured by CLIA (Kappa 0.84) or RIA (Kappa 0.50) respectively. Most significantly we demonstrated CLIA and RIA overestimated testosterone > 20 ng/dL in 66.7% of patients who measured ≤ 20ng/dL with MS. The converse much smaller immunoassay underestimation rate of 4.4% was seen when compared with MS.

To our knowledge, this study is the first to study a triplicate analysis of 2 immunoassays (CLIA, RIA) and MS on the same batch of specimens. The significant variability amongst 3 measures, in particular at low testosterone levels ≤ 20 ng/dL is clear and clinical implications would include misdiagnosing truly castrated patients instead as being inadequately treated and then performing unnecessary treatment switches with their related costs and morbidity. This points to a need for repeat verification with MS in any patient on ADT who has immunoassay measurements above 20 ng/dL. We do acknowledge the practical challenges of making MS more widespread, foremost of which are cost related. Future studies on comparative cost-effectiveness between these methods would provide us with a better understanding of the implications of more wide-spread testing with MS.

Written by: Raj Tiwari, MD, FRCS, Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada

References:

The Leuprolide Study Group. Leuprolide versus diethylstilbestrol for metastatic prostate cancer. N Engl J Med 1984;311:1281-6
Mottet N, van der Bergh RCN, Briers E, et al. EAU-EANM-ESTRO-SIOG guidelines on prostate cancer-2020 update. Part 1: Screening, diagnosis, and local treatment with curative intent. Eur Urol 2021; 79(2): 243-62
Klotz L, O’Callaghan C, Ding K et al. Nadir testosterone within first year of androgen deprivation therapy (ADT) predicts for time to castration resistant progression: A secondary analysis of the PR-7 trial of intermittent vs continuous ADT. J Clin Oncol 2015;33:1151-6
Klotz L, Shayegan B, Guillemette C et al. Testosterone suppression in the treatment of recurrent or metastatic prostate cancer – a Canadian consensus statement. Canadian Urol Assoc J 2018;12:30-6
Wang C, Catlin DH, Demers LM et al. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2007;92:405-13
Krasowski MD, Drees D, Morris CS et al. Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction. BMC Clin Pathol 2014;14:14-33

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