1844 jcem.endojournals.org J Clin Endocrinol Metab, June 2011, 96(6):

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1 ORIGINAL Endocrine ARTICLE Research Plasma Concentrations of o,p DDD, o,p DDA, and o,p DDE as Predictors of Tumor Response to Mitotane in Adrenocortical Carcinoma: Results of a Retrospective ENS@T Multicenter Study Ilse G. Hermsen, Martin Fassnacht, Massimo Terzolo, Saskia Houterman, Jan den Hartigh, Sophie Leboulleux, Fulvia Daffara, Alfredo Berruti, Rita Chadarevian, Martin Schlumberger, Bruno Allolio, Harm R. Haak, and Eric Baudin Department of Internal Medicine (I.G.H., H.R.H.) and MMC Academy (S.H.), Máxima Medical Centre, 5600 PD Eindhoven/Veldhoven, The Netherlands; Department of Internal Medicine (M.F., B.A.), University of Würzburg, Würzburg, Germany; Department of Clinical and Biological Sciences (M.T., F.D., A.B.), University of Turin, Turin, Italy; Department of Clinical Pharmacy and Toxicology (J.d.H.), University Medical Centre Leiden, 2300 RC Leiden, The Netherlands; Department of Nuclear Medicine and Endocrine Oncology (S.L., M.S., E.B.), Institute Gustave-Roussy, University Paris-Sud 11, Villejuif, France; and HRA-Pharma Paris (R.C.), Paris, France Context: In patients with adrenocortical carcinoma (ACC) mitotane activity has been suggested to depend on plasma levels 14mg/liter or greater and metabolite formation. Objective: The study was performed to confirm the correlation of the currently used mitotane (o,p DDD) threshold of 14 mg/liter with tumor response and to evaluate the additional value of 1,1-(o,p -dichlorodiphenyl) acetic acid (o,p DDA) and o,p DDE (1,1-(o,p -dichlorodiphenyl)-2,2 dichloroethene) levels for predicting tumor response. Subjects/Methods: Plasma samples collected within 3 months of best response from 91 patients on mitotane therapy for advanced ACC were analyzed retrospectively. O,p DDD and metabolites were assessed and related to tumor response and survival. Receiver operating characteristic curves were used. Sensitivity and specificity were calculated for different cutoff levels of o,p DDD and metabolites. Results: Objective tumor response was observed in 19% of patients. Median o,p DDD level was higher in responders (P 0.03). More responders were found among patients achieving o,p DDD levels 14 mg/liter or greater (P 0.02). Univariate and multivariate analysis showed significantly longer survival for patients with o,p DDD levels 14 mg/liter or greater (hazard ratio 0.52, P 0.04, hazard ratio 0.46, P 0.03). An o,p DDD cutoff value of 14 mg/liter was associated with a sensitivity of 65% and specificity 69%. An o,p DDD level 20 mg/liter or greater or 14 mg/liter or greater combined with o,p DDA level 92 mg/liter or greater was associated with a specificity of 90 and 92%, respectively. Conclusions: Our data confirm the value of o,p DDD plasma monitoring as well as targeting the 14 mg/liter cutoff level in the therapeutic management of ACC patients. Furthermore, our results suggest additional benefit of higher levels of o,p DDD and combined measurement of o,p DDD and o,p DDA. (J Clin Endocrinol Metab 96: , 2011) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2011 by The Endocrine Society doi: /jc Received November 12, Accepted March 18, First Published Online April 6, 2011 Abbreviations: ACC, Adrenocortical carcinoma; AUC, area under the curve; CR, complete response; HR, hazard ratio; o,p DDA, 1,1-(o,p -dichlorodiphenyl) acetic acid; o,p DDD, mitotane; o,p DDE, 1,1-(o,p -dichlorodiphenyl)-2,2 dichloroethane; PD, progressive disease; p,p DDA, structural isomer of o,p DDA; PR, partial response; ROC, receiver operating characteristic; SD, stable disease jcem.endojournals.org J Clin Endocrinol Metab, June 2011, 96(6):

2 J Clin Endocrinol Metab, June 2011, 96(6): jcem.endojournals.org 1845 Adrenocortical carcinoma (ACC) is a rare and aggressive tumor with limited therapeutic options. The only chance of cure is complete resection of the tumor (1 4). However, ACC is often diagnosed at a late stage, and approximately 30% of the patients already have metastasis at diagnosis (1, 5, 6). The prognosis for advanced disease is particularly poor with less than 15% of patients with metastatic ACC alive 5 yr after diagnosis (7, 8). Mitotane (o,p DDD), an isomer of the insecticide dichlorodiphenyltrichloroethane, remains one of the most active treatments for advanced ACC. Mitochondrial degeneration and destruction of the adrenal cortex after administration of analog of dichlorodiphenyltrichloroethane was described in dogs (9) and later also by Bergenstal et al. in 1960 (10). Eventually it was demonstrated that o,p DDD is the active component within the analog of dichlorodiphenyltrichloroethane solution (11) and that metabolic conversion as well as covalent binding to unknown mitochondrial protein targets is required for adrenolytic activity (12). In terms of an antitumor effect, the drug was first used for the treatment of inoperable, locoregional, and/or metastatic ACC; objective responses were demonstrated in 13 33% of patients (6, 13 15). More recently an impact of adjuvant mitotane on ACC survival was suggested (3, 16), and an international panel of experts recommend adjuvant mitotane in patients at high risk of recurrence (17). However, one has to acknowledge that not all centers agree on this concept (18, 19). The influence of o,p DDD plasma concentration on tumor response has been suggested in two studies: in case of plasma mitotane levels 14 mg/liter or greater, an objective response rate of 55 66% has been reported (13, 15), whereas only occasional responses have been reported when plasma mitotane levels remain below this threshold (20). However, due to the limited number of studies, the impact on survival of attaining this threshold is still questioned. In addition, a significant increase in neurological toxicity has been reported when plasma mitotane levels exceed 20 mg/liter (15, 21). Therefore, o,p DDD plasma monitoring is currently recommended for targeting and maintaining plasma levels between 14 and 20 mg/liter. Metabolic activation of o,p DDD within the liver results in the formation of two metabolites, 1,1-(o,p -dichlorodiphenyl) acetic acid (o,p DDA) and 1,1-(o,p -dichlorodiphenyl)-2,2 dichloroethene (o,p DDE), which derive from -or -hydroxylation of o,p DDD, respectively (21). Previous studies have suggested a role for o,p DDA as the active metabolite because -hydroxylation of o,p DDD results in an adrenolytic effect, whereas -hydroxylation of o,p DDD results in deactivation. Thus, it could be hypothesized that, for ACC patients, measurement of o,p DDA may help to refine the prediction of response to mitotane therapy. The objective of this retrospective multicenter study was (1) to confirm the correlation of the currently used o,p DDD threshold of 14 mg/l with tumor response and survival and (2) to evaluate the (additional) value of o,p DDA and o,p DDE levels for predicting tumor response. Patients and Methods Patients We retrospectively reviewed data on ACC patients treated with o,p DDD between 2001 and 2007 in two European centers (University of Turin, Turin, Italy; Institut Gustave Roussy, Villejuif, France,) and between 2005 and 2007 in three additional European centers (University of Würzburg, Würzburg, Germany; Máxima Medical Centre, Eindhoven, The Netherlands; and Leiden University Hospital, Leiden, The Netherlands). All patients met the following inclusion criteria: 1) histologically confirmed unresectable and/or metastatic adrenal carcinoma (ENS@T stage 3 and 4 disease) at o,p DDD initiation (22); 2) first-line therapy with o,p DDD and/or chemotherapy; 3) age older than 18 yr; 4) available o,p DDD plasma monitoring and banking within 3 months before the observation of best response or progression; and 5) tumor morphological evaluation every 2 3 months from diagnosis until progression. None of the patients enrolled in the two previous studies (13, 15) defining the 14 mg/liter plasma cutoff as a potential predictor of response to o,p DDD therapy were included in this study. One hundred fifty patients were considered for enrollment in the study representing 32 75% of the total number of ACC patients referred to each institution during the period under study. After careful review of all files, 40 patients were excluded because of lack of sample availability at the time of best response, previous adjuvant o,p DDD therapy (n 10), or incomplete clinical follow-up data (n 9). Finally, a total of 91 patients was included in the study. For each patient, informed consent and additional data were obtained by trained medical personnel including: demographic characteristics (age, sex), ACC characteristics (ENS@T tumor stage, tumor related hormone secretion status of the tumor), o,p DDD characteristics (maximum dose, plasma levels at time of best response or progression), morphological evaluation (best morphological response), associated chemotherapy, and last vital status. Morphological evaluation Evaluation of tumor response was reviewed in each center based on related to tumor response criteria (23): partial response (PR) requires at least a 30% decrease in the sum of the longest diameter of all target lesions; progressive disease (PD) is defined as at least a 20% increase in the sum of the longest diameter of all target lesions, progression of nontarget lesions, or the appearance of one or more new lesions; and stable disease (SD) is defined as neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease. Subsequently, patients were divided into three groups according to the observed response: group 1 responders (CR PR); group 2 SD, and group 3 nonresponders (PD).

3 1846 Hermsen et al. O,p DDD Metabolites in Adrenocortical Carcinoma J Clin Endocrinol Metab, June 2011, 96(6): Measurement of o,p DDD and its metabolites Plasma o,p DDD samples were prospectively stored and frozen at a temperature of 20 C at the time of plasma mitotane monitoring during follow-up. All available plasma samples from a given patient were collected. In case of availability of multiple samples, the sample closest to the date of best response or progression was selected (median 4 wk, range 0 12 wk). The selected frozen plasma samples were shipped to PAREXEL (bioanalytical services division of PAREXEL, Bloemfontein, South Africa) for measurements. o,p DDD, o,p DDA, and o,p DDE plasma levels were determined in the same run. The analytical method for assessment of o,p DDD and its metabolites in plasma depends on protein precipitation followed by HPLC with UV detection (24). Due to the unavailability of a reference standard for o, p DDA, structural isomer of o,p DDA (p,p DDA) was used as reference standard. The lower limit of quantification of this method is mg/ml for both o,p DDD and o,p DDE and 3.79 mg/ml for o,p DDA. Coefficient of variation is 20% at the lower limit of quantification and within 15% at higher concentrations ( 0.5 ng/ml). Statistical analysis Clinical characteristics of the patients are presented as mean SD or median with range as appropriate. Spearman rank correlation was used to assess correlations between o,p DDD and its metabolites o,p DDE and o,p DDA, respectively. The relationship between (mean, median) plasma level of o,p DDD, o,p DDE, and o,p DDA and tumor response was determined using ANOVA. Receiver operating characteristic (ROC) curves were used to define cutoff values as defined by the lowest number of both false-positive and false-negative results for each metabolite. Results of the ROC curves are expressed in terms of sensitivity and specificity. Sensitivity was defined as the proportion of responders above a given threshold divided by the total number of responders, and specificity was defined as the proportion nonresponders below a given threshold divided by the total number of nonresponders. After defining cutoff values for o,p DDD, o,p DDA, and o,p DDE plasma levels, 2 analysis was performed to assess the relationship with the best cutoff level of each compound, as defined by ROC analysis, and tumor response. The 2 analysis was also performed to assess tumor response in relation to the predefined o,p DDD plasma level less than 14 mg/liter vs. 14 mg/liter or greater. Kaplan-Meier curves were calculated to determine the effect of o,p DDD levels 14 mg/liter or greater on survival. Survival was calculated from time of first o,p DDD dose. Survival of patients who reached o,p DDD levels 14 mg/liter or greater at the time of best response was compared with patients who did not. Cox regression analysis was used to evaluate the effect of, sex, stage, tumor-related hormone secretion (functional vs. nonfunctional tumors), and concomitant chemotherapy on survival. A P 0.05 was considered significant. All analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL). Results Patient characteristics Table 1 summarizes the clinical characteristics of the 91 ACC patients included in this study. All patients received TABLE 1. Patient characteristics of the retrospective ENS@T multicenter study (n 91) Age (yr) Mean 51 Range Sex Male 35% (n 32) Female 65% (n 59) Stage Stage 3 7% (n 7) Stage 4 93% (n 84) Tumor function Cushing 47.3% (n 43) Virilization 12.1% (n 11) Feminization 2.2% (n 2) Conn 1% (n 1) Nonfunctioning 36.3% (n 33) Unknown 1% (n 1) Therapy Mitotane monotherapy 29.7% (n 27) Sz mitotane 23.1% (n 21) EDP mitotane 31.9% (n 29) Other 15.3% (n 14) o,p DDD serum level (mg/liter) Median 12.7 Range o,p DDA serum level (mg/liter) Median 58.7 Range o,p DDE serum level (mg/liter) Median 0.71 Range Response (RECIST) PD 53.8% (n 49) SD 27.5% (n 25) PR 17.6% (n 16) CR 1.1% (n 1) Last status ( ) Death 67.0% (n 61) LWD 29.7% (n 30) NED 3.3% (n 3) Sz, Streptozotocin; EDP, etoposid doxorubicin cisplatin; RECIST, response evaluation criteria in solid tumors; LWD, living with disease; NED, no evidence of disease. o,p DDD for treatment of nonoperable, localized or metastatic disease. o,p DDD was given as monotherapy (n 27) or in combination with chemotherapy (n 64). Nineteen percent of patients (n 17) were classified as responders (PR and CR), including one patient with complete response, 28% (n 25) as SD, and 54% (n 49) as nonresponders (PD). Of the 17 patients with tumor response, three patients received mitotane monotherapy, and 14 received mitotane in combination with chemotherapy. Median o,p DDD plasma level was 12.7 mg/liter (range ). Median plasma levels of o,p DDA and o, p DDE were 58.7 mg/liter (range ) and 0.71 mg/liter (range ), respectively. Correlation of o,p DDD with o,p DDD metabolite plasma levels Plasma levels of o,p DDD showed a significant correlation with o,p DDE (r 0.61, 0.001) as well as o, p DDA (r 0.67, P 0.001).

4 J Clin Endocrinol Metab, June 2011, 96(6): jcem.endojournals.org 1847 TABLE 2. Cutoff values of o,p DDD, o,p DDA and o,p DDE with corresponding sensitivity and specificity Cutoff Sensitivity (%) Specificity (%) ROC results o,p DDD (mg/liter) ROC results o,p DDA (mg/liter) ROC results o,p DDE (mg/liter) O,p DDD plasma level, correlation with tumor response, and survival Median o,p DDD levels were 16.3 mg/liter (range mg/liter) for responders, 11,6 mg/liter (range mg/liter) for patients with stable disease, and 11.0 mg/liter (range mg/liter) for nonresponders (P 0.03).The area under the curve (AUC) for o,p DDD was After ROC curve analysis, the cutoff levels for o,p DDD were determined (Table 2). The currently used cutoff level of 14 mg/liter resulted in a sensitivity of 65% (11 of 17 responders with o,p DDD above 14 mg/liter) and a specificity of 69% (34 of 49 nonresponders with o,p DDD below 14 mg/liter). Using the 14 mg/liter cutoff level, 15 of 49 nonresponders (33%) had o,p DDD plasma levels 14 mg/liter or greater vs. 11 of 17 responders (65%) ( 2, P 0.02). Positive and negative predictive values of the o,p DDD 14 mg/liter cutoff value were 42 and 85%, respectively. All responders with o,p DDD levels below 14 mg/liter (n 6) received concomitant chemotherapy. Cutoff values of 16, 18, and 20 mg/liter were associated with a lower sensitivity of 53, 32, and 26%, respectively, but a higher specificity of 76, 80, and 90%, respectively. Survival was significantly longer for patients achieving the 14 mg/liter threshold [hazard ratio (HR) 0.52, , P 0.04] (Fig. 1A). The observed survival benefit was even greater for the patients on o,p DDD monotherapy (n 27) in both univariate (HR 0.26, P 0.03) (Fig. 1B) and multivariate analysis (HR 0.26, P 0.02). No significant influence of age [HR 1.01 ( )], sex [HR 1.64 ( )], stage [HR 1.00 ( )], or FIG. 1. Survival from time of first o,p -DDD dose for 91 patients on o,p -DDD therapy and/or chemotherapy according to o,p DDD plasma level less than 14 mg/liter or 14 mg/liter or greater. B, Survival from time of first o,p -DDD dose for 27 patients on o,p -DDD monotherapy therapy according to o,p DDD plasma level less than 14 mg/liter or 14 mg/liter or greater. tumor-related hormone secretion [HR 1.29 ( )] on overall survival was observed. O,p DDA and o,p DDE plasma levels and tumor response Median o,p DDA levels were 70.2 mg/liter (range mg/liter) for responders, 44.8 mg/liter (range mg/liter) for nonresponders, and 58.0 mg/liter (range mg/liter) for patients with stable disease (P 0.2). The AUC for o,p DDA was After obtaining the ROC curves, cutoff levels were identified, demonstrating

5 1848 Hermsen et al. O,p DDD Metabolites in Adrenocortical Carcinoma J Clin Endocrinol Metab, June 2011, 96(6): an overall low sensitivity together with a high specificity (Table 2). For instance, an o,p DDA cutoff level of 92 mg/liter resulted in a sensitivity of 35% (six of 17 responders with o,p DDA 92 mg/liter) and a specificity of 84% (41 of 49 nonresponders with o,p DDA below 92 mg/ liter). Using the cutoff value of 92 mg/liter for o,p DDA, six of 16 patients (38%) were responders above this level vs. 15% (11 of 75) below that level (P 0.02). Positive and negative predictive values of o,p DDA with a cutoff value of 92 mg/liter were 43 and 79%, respectively. Note that, in the subgroup of ACC patients on o,p DDD monotherapy (n 17), a significantly higher median o,p DDA level was found for responding patients (n 3); median levels were mg/liter (range mg/liter) in responders, 78,0 mg/liter (range mg/liter) for patients with stable disease, and 62.5 mg/liter (range mg/ liter) for nonresponding patients (P 0.03). Median o,p DDE levels were 0.92 mg/liter (range mg/liter) for responders, 0.65 mg/liter (range mg/liter) for patients with SD, and 0.50 mg/liter (range mg/liter) for nonresponders (P 0.2). The AUC for o,p DDE was 0.61, indicating an overall low sensitivity together with a high specificity. For instance a cutoff value of 1.3 mg/liter resulted in a sensitivity of 35% (six of 17) and a specificity of 80% (39 of 49). When a cutoff level of 1.5 mg/liter was used, the sensitivity and specificity were 35 and 82%, respectively. No significant relationship was demonstrated with tumor response (P 0.16). Combined analysis of o,p DDD and o,p DDA Of 36 patients with o,p DDD levels 14 mg/liter or greater and 16 patients with o,p DDA levels 92 mg/liter or greater, the levels of both compounds were above these levels in 11 cases. Of these 11 patients, 45% (five of 11) had a response. The combination of o,p DDD levels 14 mg/liter or greater and o,p DDA levels 92 mg/liter or greater resulted in a sensitivity of 29%, specificity of 92%, a positive predictive value of 55%, and a negative predictive value of 68%. One of five patients with o,p DDA levels 92 mg/liter or greater but o,p DDD below 14 mg/liter experienced a response. This patient received o,p DDD in combination with chemotherapy. Five of 51 patients (10%) with both o,p DDD levels less than 14 mg/liter and o,p DDA levels less than 92 mg/liter had a response. Discussion The present study demonstrates the importance of o, p DDD plasma monitoring as well as the importance of reaching the therapeutic o,p DDD threshold of (at least) 14 mg/liter. An o,p DDD level above 14 mg/liter was associated significantly with tumor response and survival. In contrast, we observed a relationship only between o, p DDA and tumor response in a subgroup of patients on mitotane monotherapy. Fifty years after its first use, o,p DDD is still considered the most active drug for ACC. Nonetheless, a number of limitations exist including poor bioavailability, significant side effects, and a narrow range of optimal efficacy, making progress in the use of this drug highly important. In this setting, improved prediction of tumor response to o,p DDD constitutes an attractive field of research. Therefore, our study was aimed at progress in the field of o,p DDD pharmacokinetics and its attendant impact on patient outcome. Findings of previous studies on the relevance of o, p DDD plasma level 14 mg/liter or greater are confirmed in the present study because significantly higher median levels were found in responding patients compared with nonresponding patients and patients with stable disease (P 0.03). Importantly, it confirms the currently used 14 mg/liter threshold for o,p DDD as a good compromise for patients with unresectable ACC associated with a sensitivity of 65% and a specificity of 69%. Whether this threshold is also applicable for ACC patients in the adjuvant setting remains to be demonstrated; however, because the same objectives applied in both conditions, we suggest to use the same strategy (17). Furthermore, the observation of response among patients with plasma levels below 14 mg/liter was also confirmed. However, all responders with plasma levels of o,p DDD below 14 mg/liter received concomitant cytotoxic chemotherapy. Thus, the response found for these patients might be the result of chemotherapy or the combination of drugs rather than o,p DDD alone. Indeed, o,p DDD has been shown to antagonize multidrug resistance-1 in vitro (25, 26), which is involved in resistance to doxorubicin and etoposide agents. However, the clinical relevance of this observation for ACC patients remains to be confirmed. Regarding the specificity of o,p DDD associated with a 14 mg/liter cutoff plasma level, it was intermediate at 69% but increased up to 80% when the 18 mg/liter cutoff value was chosen, suggesting that for some patients reaching plasma levels of o,p DDD above 18 mg/liter may become a new therapeutic target, if tolerated without major toxicity. Although not considered as an anticipated objective in the population of patients under study, o,p DDD levels above 18 or 20 mg/liter were observed in 20 or 11% of cases, respectively. Although some authors have already investigated the efficacy of mitotane plasma levels (13, 27), the impact of

6 J Clin Endocrinol Metab, June 2011, 96(6): jcem.endojournals.org 1849 these levels on survival is still uncertain. Improved survival of patients with o,p DDD plasma levels within the therapeutic range has been suggested (13, 28, 29) but not always confirmed (25, 30 32). Furthermore, survival was not always determined from the time of first o,p DDD dose, which is the most appropriate for calculating o, p DDD-associated survival. From the time of first o, p DDD dose, we found a significantly improved survival for patients with mitotane plasma levels 14 mg/liter or greater compared with patients with plasma levels below 14 mg/liter. This finding emphasizes the importance of targeting plasma levels 14 mg/liter or greater, even for patients with progressive disease. In addition, a recent study suggested the importance of maintaining mitotane plasma levels 14 mg/liter or greater (29). We did not find a significant influence of age and tumor-related hormone secretion on overall survival, in contrast to one previous study (33). However, populations under study as well as the modality of o,p DDD analysis were different, making direct comparison impossible. Further progress in the scope of predictors of response to o,p DDD is still needed; therefore, the second focus of our study was the evaluation of the predictive roles of the o,p DDD metabolites, o,p DDA and o,p DDE. The present study for the first time evaluated the metabolites as potential predictors of antitumor activity compared with o,p DDD. Indeed, in preclinical studies, o,p DDD has been demonstrated to be activated by the liver via transformation to o,p DDA (21, 34). Tumor response was found to be correlated to o,p DDA but only in a subgroup of patients on mitotane monotherapy (n 27). Furthermore, an o,p DDA cutoff value of 92 mg/liter was found to be correlated with response to therapy and was associated with a 84% specificity rate in this subgroup of patients. However, only three responders were found in this subgroup of patients, which precludes definitive conclusions. Regarding the entire population under study, the AUC of the ROC curves of o,p DDA and o,p DDD were in the same range due to a lower sensitivity but higher specificity of o,p DDA compared with o,p DDD. Taken together, these results suggest that a combination of both metabolite measurements may be a logical next step to be investigated in future prospective studies. Indeed, the combination of o,p DDA 92 mg/liter or greater and o,p DDD 14 mg/liter or greater yields a 92% specificity and 55% positive predictive value. These data are consistent with the positive but not perfect correlation between plasma values of o,p DDA and o,p DDD, found in our study, suggesting that for some patients both compounds can provide complementary information to some extent. Note that a similar range of specificity and positive predictive results were obtained than by setting the cutoff value of o,p DDD at 18 mg/liter. Indeed combining o,p DDD and o,p DDA measurements may help to refine the predictions of o,p DDD antitumor activity and avoid the risk of an increased toxicity rate. Patients with o,p DDA 92 mg/liter or greater and o,p- DDD 14 mg/liter or greater may not require an increase in mitotane dose. In contrast to o,p DDA, our study does not confirm a correlation of o,p DDE with tumor response as previously hypothesized in one study (35). As stated earlier, reaching the therapeutic threshold of 14 mg/liter is very important. However, only 40% of the ACC patients in this study reached an o,p DDD level 14 mg/liter or greater. Low bioavailability of o,p DDD treatment is a well-known drawback of this drug. Both the low absorption rate of o,p DDD and its lipophilic nature partly explain this characteristic (36). We acknowledge that our article has important limitations. Its retrospective nature, the still relatively low number of patients, and the use of o,p DDD in combination with other chemotherapy regimens in most cases constitute major limitations. In addition, we analyzed selected unresectable ACC patients, mainly based on the availability of stored plasma o,p DDD samples, in which an unrecognized bias cannot be excluded. However, to date these limitations cannot be overcome due to the scarcity of the disease and the delayed action of o,p DDD. In addition, p,p DDA but not o,p DDA was measured in the plasma. Whether the o,p DDA peak at HPLC is similar to the p,p DDA peak remains to be proven. In conclusion, our data confirm the value of o,p DDD plasma monitoring as well as targeting the 14 mg/liter cutoff level of o,p DDD in the therapeutic management of ACC patients. Furthermore, our results suggest some additional benefit of a targeting of higher plasma level of o,p DDD and combined measurement of o,p DDD and o,p DDA, which has to be confirmed in a prospective study that is currently running as a substudy of the First International Randomized Trial in Locally Advanced and Metastatic Adrenocortical Carcinoma Treatment trial. Acknowledgments Address all correspondence and requests for reprints to: Ilse Hermsen, Department of Internal Medicine, Máxima Medical Centre, P.O. Box 90052, 5600 PD Eindhoven, The Netherlands. i.hermsen@mmc.nl. Disclosure Summary: I.G.H., S.H., J.d.H., S.L., F.D., A.B., M.S., B.A., and H.R.H. have nothing to declare. M.F. received

7 1850 Hermsen et al. O,p DDD Metabolites in Adrenocortical Carcinoma J Clin Endocrinol Metab, June 2011, 96(6): a grant for a pharmakinetic trial of mitotane sponsored by HRA-Pharma. M.T. received fees for participating on the advisory board for HRA-Pharma. R.C. is employed by HRA- Pharma. E.B. received lecture fees from HRA-Pharma. Measurements of o,p DDD and metabolites were sponsored by HRA-Pharma. References 1. Crucitti F, Bellantone R, Ferrante A, Boscherini M, Crucitti P 1996 The Italian Registry for Adrenal Cortical Carcinoma: analysis of a multiinstitutional series of 129 patients. The ACC Italian Registry Study Group. Surgery 119: Schulick RD, Brennan MF 1999 Long-term survival after complete resection and repeat resection in patients with adrenocortical carcinoma. Ann Surg Oncol 6: Grubbs EG, Callender GG, Xing Y, Perrier ND, Evans DB, Phan AT, Lee JE 2010 Recurrence of adrenal cortical carcinoma following resection: surgery alone can achieve results equal to surgery plus mitotane. Ann Surg Oncol 17: Icard P, Chapuis Y, Andreassian B, Bernard A, Proye C 1992 Adrenocortical carcinoma in surgically treated patients: a retrospective study on 156 cases by the French Association of Endocrine Surgery. Surgery 112: ; discussion Wooten MD, King DK 1993 Adrenal cortical carcinoma. Epidemiology and treatment with mitotane and a review of the literature. Cancer 72: Icard P, Goudet P, Charpenay C, Andreassian B, Carnaille B, Chapuis Y, Cougard P, Henry JF, Proye C 2001 Adrenocortical carcinomas: surgical trends and results of a 253-patient series from the French Association of Endocrine Surgeons Study Group. World J Surg 25: Assié G, Antoni G, Tissier F, Caillou B, Abiven G, Gicquel C, Leboulleux S, Travagli JP, Dromain C, Bertagna X, Bertherat J, Schlumberger M, Baudin E 2007 Prognostic parameters of metastatic adrenocortical carcinoma. J Clin Endocrinol Metab 92: Allolio B, Hahner S, Weismann D, Fassnacht M 2004 Management of adrenocortical carcinoma. Clin Endocrinol (Oxf) 60: Nelson AA, Woodard G 1948 Adrenal cortical atrophy and liver damage produced in dogs by feeding 2,2-bis-(parachloro-phenyl)- 1,1-dichloroethane. Fed Proc 7: Bergenstal DM, Hertz R, Lipsett MB, Moy RH 1960 Chemotherapy of adrenocortical cancer with o,p -DDD. Ann Intern Med 53: Cueto C, Brown JH, Richardson Jr AP 1958 Biological studies on an adrenocorticolytic agent and the isolation of the active components. Endocrinology 62: Martz F, Straw JA 1980 Metabolism and covalent binding of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (o, p, -DDD). Correlation between adrenocorticolytic activity and metabolic activation by adrenocortical mitochondria. Drug Metab Dispos 8: Haak HR, Hermans J, van de Velde CJ, Lentjes EG, Goslings BM, Fleuren GJ, Krans HM 1994 Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients. Br J Cancer 69: Williamson SK, Lew D, Miller GJ, Balcerzak SP, Baker LH, Crawford ED 2000 Phase II evaluation of cisplatin and etoposide followed by mitotane at disease progression in patients with locally advanced or metastatic adrenocortical carcinoma: a Southwest Oncology Group Study. Cancer 88: Baudin E, Pellegriti G, Bonnay M, Penfornis A, Laplanche A, Vassal G, Schlumberger M 2001 Impact of monitoring plasma 1,1-dichlorodiphenildichloroethane (o,p DDD) levels on the treatment of patients with adrenocortical carcinoma. Cancer 92: Terzolo M, Angeli A, Fassnacht M, Daffara F, Tauchmanova L, Conton PA, Rossetto R, Buci L, Sperone P, Grossrubatscher E, Reimondo G, Bollito E, Papotti M, Saeger W, Hahner S, Koschker AC, Arvat E, Ambrosi B, Loli P, Lombardi G, Mannelli M, Bruzzi P, Mantero F, Allolio B, Dogliotti L, Berruti A 2007 Adjuvant mitotane treatment for adrenocortical carcinoma. N Engl J Med 356: Berruti A, Fassnacht M, Baudin E, Hammer G, Haak H, Leboulleux S, Skogseid B, Allolio B, Terzolo M 2010 Adjuvant therapy in patients with adrenocortical carcinoma: a position of an international panel. J Clin Oncol 28:e401 e402; author reply e Huang H, Fojo T 2008 Adjuvant mitotane for adrenocortical cancer a recurring controversy. J Clin Endocrinol Metab 93: Bodie B, Novick AC, Pontes JE, Straffon RA, Montie JE, Babiak T, Sheeler L, Schumacher P 1989 The Cleveland Clinic experience with adrenal cortical carcinoma. J Urol 141: Seki M, Nomura K, Hirohara D, Kanazawa M, Sawada T, Takasaki K, Demura H 1999 Changes in neoplastic cell features and sensitivity to mitotane during mitotane-induced remission in a patient with recurrent, metastatic adrenocortical carcinoma. Endocr Relat Cancer 6: van Slooten H, Moolenaar AJ, van Seters AP, Smeenk D 1984 The treatment of adrenocortical carcinoma with o,p -DDD: prognostic implications of serum level monitoring. Eur J Cancer Clin Oncol 20: Cai W, Counsell RE, Djanegara T, Schteingart DE, Sinsheimer JE, Wotring LL 1995 Metabolic activation and binding of mitotane in adrenal cortex homogenates. J Pharm Sci 84: Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG 2000 New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: Hermsen IG, den Hartigh HJ, Haak HR 2010 Mitotane serum level analysis; good agreement between two different assays. Clin Endocrinol (Oxf) 73: Abraham J, Bakke S, Rutt A, Meadows B, Merino M, Alexander R, Schrump D, Bartlett D, Choyke P, Robey R, Hung E, Steinberg SM, Bates S, Fojo T 2002 A phase II trial of combination chemotherapy and surgical resection for the treatment of metastatic adrenocortical carcinoma: continuous infusion doxorubicin, vincristine, and etoposide with daily mitotane as a P-glycoprotein antagonist. Cancer 94: Bates SE, Shieh CY, Mickley LA, Dichek HL, Gazdar A, Loriaux DL, Fojo AT 1991 Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/p-glycoprotein) which is also expressed by adrenocortical carcinomas. J Clin Endocrinol Metab 73: Baudin E, Docao C, Gicquel C, Vassal G, Bachelot A, Penfornis A, Schlumberger M 2002 Use of a topoisomerase I inhibitor (irinotecan, CPT-11) in metastatic adrenocortical carcinoma. Ann Oncol 13: Wängberg B, Khorram-Manesh A, Jansson S, Nilsson B, Nilsson O, Jakobsson CE, Lindstedt S, Odén,A, Ahlman H 2010 The long-term survival in adrenocortical carcinoma with active surgical management and use of monitored mitotane. Endocr Relat Cancer 17: Malandrino P, Al Ghuzlan A, Castaing M, Young J, Caillou B, Travagli JP, Elias D, de Baere T, Dromain C, Paci A, Chanson P, Schlumberger M, Leboulleux S, Baudin E 2010 Prognostic markers of survival after combined mitotane- and platinum-based chemotherapy in metastatic adrenocortical carcinoma. Endocr Relat Cancer 17:

8 J Clin Endocrinol Metab, June 2011, 96(6): jcem.endojournals.org Venkatesh S, Hickey RC, Sellin RV, Fernandez JF, Samaan NA 1989 Adrenal cortical carcinoma. Cancer 64: Hutter Jr AM, Kayhoe DE 1966 Adrenal cortical carcinoma. Results of treatment with o,p DDD in 138 patients. Am J Med 41: Lubitz JA, Freeman L, Okun R 1973 Mitotane use in inoperable adrenal cortical carcinoma. JAMA 223: Abiven G, Coste J, Groussin L, Anract P, Tissier F, Legmann P, Dousset B, Bertagna X, Bertherat J 2006 Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J Clin Endocrinol Metab 91: Cai W, Benitez R, Counsell RE, Djanegara T, Schteingart DE, Sinsheimer JE, Wotring LL 1995 Bovine adrenal cortex transformations of mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane; o,p - DDD] and its p,p - and m,p -isomers. Biochem Pharmacol 49: Kasperlik-Zaluska AA, Cichocki A 2005 Clinical role of determination of plasma mitotane and its metabolites levels in patients with adrenal cancer: results of a long-term follow-up. J Exp Ther Oncol 5: Watson AD, Rijnberk A, Moolenaar AJ 1987 Systemic availability of o,p -DDD in normal dogs, fasted and fed, and in dogs with hyperadrenocorticism. Res Vet Sci 43: Earn more than 100 hours of CME credit through ENDO 2010 Session Library.