LETTERS TO THE EDITOR
Mitotane serum level analysis; good agreement between two different assays
Mitotane (o,p’DDD) has been one of the main treatments available for adrenocortical carcinoma (ACC) for over 40 years. This drug is used for the treatment of inoperable, recurrent and/or metastatic ACC and even seems to have a role in adjuvant treatment after complete resection.1
The effect of o,p’DDD on the human adrenal cortex was first described in 1959.2 Subsequently, Moolenaar et al.3 described an assay for the analysis of o,p’DDD, developed in the Leiden Univer- sity Hospital (LUMC) in 1977. The implications of this determina- tion were studied by van Slooten, categorizing serum samples as low (<14 mg/l) or high (>14 mg/l).4 . Two groups confirmed this categorization and also demonstrated tumour response and signifi- cant improved survival in patients with levels above 14 mg/l.5,6
In 2004, the FIRM-ACT study (First International Randomized trial in locally advanced and Metastatic Adrenocortical Carcinoma Treatment, ISRCTN94256573) was set up, comparing the efficacy of Etoposide, Doxorubicin and Cisplatin and Sz (Streptozotocin) in combination with o,p’DDD. With this study, a new assay for mitotane serum level analysis was developed by PAREXEL (Paris). PAREXEL monitoring is part of a LYSOSAFE Service in which free o,p’DDD plasma level testing is available for all patients with ACC treated with Lysodren®.
As the assays used for mitotane serum level analysis are different from an analytical point of view, PAREXEL uses the High Perfor- mance Liquid Chromatography (HPLC) method, LUMC the Gas Liquid Chromatography (GLC), we assessed the agreement between the two methods.
From all serum samples collected for o,p’DDD level determina- tion in LUMC, we randomly selected 20 samples of patients with ACC treated with mitotane.
This blood was collected in Vacutainer tubes without additives, at least 12 h after the last dose was taken, were frozen at a tempera- ture of -20 ℃. Subsequently, serum was analysed at LUMC and after shipment also at PAREXEL.
For better understanding of the assay’s used, we will describe them in more detail in the following paragraphs.
Description of the PAREXEL assay for o,p’DDD serum level determination by HPLC
Sample pretreatment starts with the addition of 4,4-DDE [1-chloro-4-(dichloro-1)-4-chlorophenyl)vinyl benzene], used as the internal standard, to the serum sample (100 ul). After this, eth- anol (200 ul) is added and the sample is vortexed for 30 s. Next, the sample is centrifuged at 3000 g for 5 min and the clear superna- tant is transferred to an autosampler vial. The samples are cooled to 5 ℃. Subsequently, 15 ul of the sample is injected onto the
2-00
+2SD =- 1.525
0
1.00
Difference in mg/l
Mean =- 0-1261
0:00
-1-00
-2SD = 1-272
-2-00
0-00
5.00
10-00
15-00
20 00
25.00
Average in mg/l
HPLC Column. The retention time of the internal standard is 10-8 min and of mitotane is approximately 6-5 min. After injection, o,p’-DDD and the internal standard are detected by a UV detector Agilent 1100 series, at 225 nm. This detector is interfaced with a computer running HPLC2D ChemStation version A.09.03 software (Agilent Technologies, Johannesburg, South Africa).
Description of the LUMC assay used for o,p’DDD serum level determination by GLC
4,4-DDD [1,1-Dichloro-2,2-bis (4-chlorophenyl)ethane] (1,0 mg/ l) mixed with methanol is used as the internal standard solution. This internal standard is added to the serum sample (50 ul). After this, ethanol (200 ul)is added and the sample is vortexed for 30 s. Next, 2-0-ml n-hepatane is added and the sample is again vortexed for 30 s. Now, 0-2-g sodium sulphate is added and vortexed again for 30 s. This total solution is then centrifuged at 4000 g for 3 min. Subsequently, 1 ul of the sample is injected onto the GLC Column (Trace-GC-Ultra with Triplus Autosampler). The retention time of the internal standard is 6.1 min and mitotane is approximately 5-2 min. After injection, o,p’-DDD and internal standard are detected by an Electron Capture Detector (Ni63).
The mitotane serum levels in the samples determined at PAREX- EL ranged from 1.84 mg/1 to 21.77 mg/1 [mean (SD) 9-035 mg/l], whereas those in the samples analysed at LUMC ranged from 2-10 to 23-70 mg/l [mean (SD) 9-160 mg/1]. The correlation between the results obtained with the two assays was 0-995 (P = 0-000). The results were plotted according to Bland-Altman (Fig. 1). The 95% limits of agreement on the Bland-Altman plot were -1.524 to 1.272 mg/l. The plot revealed a good agreement, with a tendency for higher serum level measurements (in the range >15 mg/l) with the LUMC method.
In clinical practice, this means that if a serum level of 20 mg/l is found with one method, the other method could deviate from this by 2 mg/l (18-22 mg/l). What is the clinical implication of this given difference? In general, the dosage of mitotane in patients with serum levels >14 mg/l depends on the occurrence of side-effects, and efficacy. If side-effects occur, dosage will be lowered otherwise the dosage will be continued provided that side-effects are mild and acceptable. Differences of 2-0 mg/l in the higher range (20 mg/ l) seem large in a scientific setting, but have little clinical implica- tions and are therefore acceptable in clinical practice and are also acceptable according to Therapeutic Drug Monitoring.
Given these results, it may be concluded that although the assays differ, they show good agreement and therefore historical data from the Leiden series can be compared with present trials such as the FIRM-ACT trial.
I. G. C. Hermsen*, J. den Hartight and H. R. Haak* *Department of internal medicine Maxima Medical Centre Eindhoven +Department of clinical pharmacy and toxicology University Medical Centre Leiden E-mail: i.hermsen@mmc.nl.
References
1 Terzolo, M., Angeli, A., Fassnacht, M. et al. (2007) Adjuvant mito- tane treatment for adrenocortical carcinoma. New England Journal of Medicine 356, 2372-2380.
2 Bergenstal, D.M., Hertz, R., Lipsett, M.B. et al. (1960) Chemother- apy of adrenocortical cancer with o,p’-DDD. Annals of Internal Med- icine, 53, 672.
3 Moolenaar, A.J., van, S.H., van Seters, A.P. et al. (1981) Blood levels of o,p’-DDD following administration in various vehicles after a sin- gle dose and during long-term treatment. Cancer Chemotherapy and Pharmacology 7, 51-54.
4 van Slooten, H., Moolenaar, A.J., van Seters, A.P. et al. (1984) The treatment of adrenocortical carcinoma with o,p’-DDD: prognostic implications of serum level monitoring. European Journal of Cancer and Clinical Oncology 20, 47-53.
5 Baudin, E., Pellegriti, G., Bonnay, M. et al. (2001) Impact of moni- toring plasma 1,1-dichlorodiphenildichloroethane (o,p’DDD) levels on the treatment of patients with adrenocortical carcinoma. Cancer 92, 1385-1392.
6 Haak, H.R., Hermans, J., van de Velde, C.J. et al. (1994) Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients. British Journal of Cancer 69, 947- 951.
Indirect markers for detecting growth hormone abuse by athletes
We read with interest the article by Erotokritou-Mulligan et al.1 in which they report on the use of indirect markers for detecting growth hormone (hGH) doping. We feel that some points warrant more detailed discussion.
The Authors used IGF-I data obtained from four different types of studies. Three different methods were used for measur- ing IGF-I concentrations in serum: Nichols (now discontinued), DSL and Mediagnost assays. To use all the data from different assays which give quantitatively different measurements, several equations for adjusting data were applied, taking the Nichols assay as reference. The equation for comparing and harmonizing data between the Nichols and the Mediagnost assays was Adjusted IGF-I (ng/ml) = - 6-07 + Mediagnost IGF-I value (ng/ ml) × 11-37.
This appears to be an equation for a linear regression analysis, but the slope of 11.37 is unacceptable. In a previous article, when the same samples analysed in two different laboratories were com- pared by using the same two methods, it was reported that Mediag- nost overestimated IGF-I.2 By applying the proposed adjustment, it seems that Mediagnost underestimates when compared with Nic- hols.
The use of adjustments among different assays is not the best way to harmonize values for the same parameters; rather, the use of International Standards is the correct approach. There is a standard for IGF-I, the World Health Organization 2nd IS 87/518,3 and a recent study on a new standard has recently been published.4 Admittedly, the availability of an international standard does not solve all the problems of interassay comparability, and kit produc- ers do not always completely adhere to standardization. For exam- ple, Mediagnost suggests applying a conversion factor of 1-66 to