PROSPECTIVE EVALUATION OF MITOTANE TOXICITY IN

ADRENOCORTICAL CANCER PATIENTS TREATED

ADJUVANTLY.

Fulvia Daffara MD, Silvia De Francia PhD, Giuseppe Reimondo MD, Barbara Zaggia

MD, Emiliano Aroasio ScD PhD, Francesco Porpiglia MD, Marco Volante MD, Angela

Termine, Francesco Di Carlo MD, Luigi Dogliotti MD, Alberto Angeli MD, Alfredo

Berruti MD, Massimo Terzolo MD.

Medicina Interna I (F.D., G.R., B.Z., A.T., A.A., M.T.); Farmacologia (S.D.F., F.D.C.);

Oncologia Medica (L.D., A.B.); Urologia (F.P.); Anatomia Patologica (M.V.): Dipartimento di

Scienze Cliniche e Biologiche, Università di Torino and Laboratorio Analisi (E.A.), A.S.O.

San Luigi, Orbassano, Italy.

Running title: Toxicity of adjuvant mitotane

Key words: adjuvant treatment, adrenocortical cancer, hypoadrenalism, mitotane, toxicity.

Address correspondence to:M. Terzolo, MDMedicina Interna IA.S.O. San Luigi,Regione Gonzole, 10,10043 Orbassano ITALYTel: ++ 39 011 9026292Fax: ++ 39 011 9038655 e-mail: terzolo@usa.net

Page 1 of 34 Accepted Preprint first posted on 29 September 2008 as Manuscript ERC-08-0103

Copyright © 2008 by the Society for Endocrinology.

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SUMMARY

Toxicity of adjuvant mitotane treatment is poorly known; thus, our aim was to assess

prospectively the unwanted effects of adjuvant mitotane treatment and correlate the findings

with mitotane concentrations. Seventeen consecutive patients who were treated with

mitotane after radical resection of adrenocortical cancer (ACC) from 1999 to 2005 underwent

physical examination, routine laboratory evaluation, monitoring of mitotane concentrations

and a hormonal work-up at baseline and every 3 months till ACC relapse or study end

(December 2007). Mitotane toxicity was graded using NCI CTCAE criteria. All biochemical

measurements were performed at our center and plasma mitotane was measured by an in-

house HPLC assay. All patients reached mitotane concentrations >14 mg/l and none of them

discontinued definitively mitotane for toxicity; 14 patients maintained consistently elevated

mitotane concentrations despite tapering of the drug. Side effects occurred in all patients but

were manageable with palliative treatment and adjustment of hormone replacement therapy.

Mitotane affected adrenal steroidogenesis with a more remarkable inhibition of cortisol and

DHEAS than aldosterone. Mitotane induced either perturbation of thyroid function mimicking

central hypothyroidism or, in male patients, inhibition of testosterone secretion. The

discrepancy between salivary and serum cortisol, as well as between total and free

testosterone, is due to the mitotane-induced increase in hormone binding proteins that

complicates interpretation of hormone measurements. A low-dose monitored regimen of

mitotane is tolerable and able to mantain elevated drug concentrations in the long term.

Mitotane exerts a complex effect on the endocrine system that may require multiple hormone

replacement therapy.

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INTRODUCTION

Treatment of adrenocortical carcinoma (ACC) is still a challenge due to the rarity of this

tumor that hampered the development of effective therapeutic options (Pommier & Brennan

1992; Wajchenberg et al. 2000; Dackiw et al. 2001; Schteingart et al. 2005; Allolio &

Fassnacht, 2006; Libè et al. 2007). Mitotane (o,p’-DDD), an analogue of the insecticide DDT,

has been used for treating advanced ACC since the sixthies (Bergenstal et al. 1960; Luton et

al. 1990; Wooten & King 1993), whereas its use as an adjunctive post-operative measure

remained controversial (Allolio & Fassnacht, 2006;Schteingart 2007;Terzolo & Berruti; 2008).

The very recent demonstration that adjuvant treatment with mitotane was associated with

beneficial effects on outcome in a large series of patients with ACC should renew interest in

adjuvant mitotane therapy, notwithstanding bias inherent in a retrospective analysis (Terzolo

et al. 2007). However, there is still limited information on the consequences of adjuvant

mitotane treatment, since safety data of mitotane were mostly generated from series of

patients with advanced ACC. Moreover, these data were largely obtained without monitoring

mitotane concentrations, that is currently become a standard of care (Allolio & Fassnacht,

2006; Terzolo & Berruti, 2008). Thus, the relationship between mitotane concentrations and

the unwanted effects of the drug remains uncertain.

Therefore, we thought of interest to assess prospectively either the clinical or biochemical

effects of chronic adjuvant mitotane treatment in a consecutive series of patients with ACC

who have been rendered disease-free by surgery. We focused on the first year of treatment,

when mitotane concentrations are steeply increasing, and we assessed the unwanted effects

of the drug in relationship with its circulating concentrations. The aim of the present study

was not to evaluate the effects of mitotane on patient outcome but to address the safety and

feasibility of adjunctive mitotane treatment, that may be questionable given that mitotane is

toxic and of complex use because of its narrow therapeutic index, the need of drug

monitoring and steroid replacement (Lee, 2007).

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SUBJECTS and METHODS

Subjects

Cases were drawn from a series of patients with ACC referred to and followed at our

center. They were 17 consecutive patients (7 men and 10 women) aged 39.2 ± 11.7 years

who underwent radical resection of ACC from 1999 to 2005; follow-up for this report was

closed in December 2007. Patients had to meet the following inclusion criteria: age 18 years

or older, histologically confirmed diagnosis of ACC, complete tumor resection, availability of

preoperative and postoperative computed tomography or magnetic resonance imaging.

Exclusion criteria were macroscopically incomplete resection, incomplete staging, history of

previous or concomitant malignancy, renal or liver insufficiency or any other severe acute or

chronic medical or psychiatric condition, and previous or current treatment with

chemotherapy or radiotherapy. Complete resection was defined as no evidence of

macroscopic residual disease based on surgical and histopathology reports, as well as post-

operative imaging. The Institutional Ethics Committee of the “Azienda Sanitaria Ospedaliera

San Luigi” approved the study. All participants volunteered for the study and provided written

informed consent.

During the study period, adjuvant mitotane treatment aimed at reaching target serum

concentrations of the drug (Haak et al. 1994, Baudin et al. 2001) has been standard of care

at our institution following removal of ACC. In the absence of untolerability to mitotane,

treatment was scheduled for at least 3 years, or longer in patients at perceived high-risk of

recurrence. Of 25 patients who were treated adjuvantly with mitotane after radical resection

of ACC, 17 met all entry criteria and 8 patients were excluded: 5 had incomplete

postoperative staging, 2 were on interferent medications (antiepilepsy drugs) and 1 had liver

failure. All histological diagnoses were re-evaluated according to the Weiss criteria (nuclear

atypia, atypical mitoses, frequent mitoses, small percentage of clear cells, diffuse

architecture, necrosis, invasion of venous, sinusoidal, or capsular structures) (Weiss 1984;

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Weiss et al. 1989) by an expert pathologist (M.V.). Staging at diagnosis was based on

imaging studies and was corroborated by the findings at surgery. Staging was reported

according to the McFarlane-Sullivan criteria (stage I: tumor ≤ 5cm, stage II: tumor > 5cm,

stage III: tumor infiltration of neighbouring structures or positive lymphnodes, stage IV:

infiltration of neighbouring structures and positive lymphnodes or distant metastases)

(MacFarlane 1958; Sullivan et al. 1978). Disease recurrence was defined as radiologic

evidence of a new tumor lesion during follow-up.

Study protocol

Patient visits including imaging of both chest and abdomen were performed at our center

at baseline (before introducing mitotane) and thereafter every 3 months until disease

progression or end of the study period. At each visit, the patients underwent physical

examination, routine laboratory evaluation, monitoring of mitotane concentrations and a

hormonal work-up including determination of serum cortisol, aldosterone, 11-deoxycortisol,

DHEAS, TSH, FT4, FT3, PRL, FSH, LH, total and free testosterone, ACTH, PRA, cortisol

binding globulin (CBG), sex-hormone binding globulin (SHBG). Salivary cortisol was also

measured since 2006, when an in-house assay was established at our center. Moreover, a

careful interview of the patients was taken by the same physicians (F.D., B.Z.) to detect

subjective symptoms which may have occurred during treatment. Adverse effects were

retrieved by means of a questionnaire. Continous counseling was offered to the patients and

their primary care physicians by means of phone and e-mail contacts to cope with unwanted

effects; additional visits were also performed when necessary. We report herein the results

of the visits at baseline, +3, +6, +9, +12 months, and the last available follow-up for non-

recurring patients. For patients who experienced ACC recurrence, the follow-up immediately

preceding the detection of relapse was reported.

All patients received the same mitotane formulation (Lysodren®, 500-mg tablets) that was

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purchased by Bristol-Meyers, Squibb, New York, US till 2003 and thereafter by Laboratoire

HRA Pharma, Paris, France. Mitotane was administered orally at a starting dose of 1 g daily,

with progressive weekly increments up to 4-6 g daily, or the highest tolerated dose, aiming to

reach concentrations between 14–20 mg/L (Haak et al. 1994, Baudin et al. 2001). When

such or even higher concentrations were attained, doses were tapered with further individual

dose adjustments guided by the results of mitotane measurement and toxicity assessment.

In the event of unacceptable side effects, patients were allowed to return to a lower dose or

discontinue temporarily restarting with a lower dose. All patients received prophylactic

glucocorticoid replacement (Cortisone acetate, 25 mg daily) when mitotane was commenced.

Mitotane-related toxicity was graded using NCI CTCAE criteria (NCI 2003).

Assays

Blood samples were drawn between 0800 and 0900h from an antecubital vein that was

cannulated 30 minutes before after overnight fast and 24-hour discontinuation of any

hormone replacement. Saliva was collected in commercially available devices using a cotton

swab chewed for 2–3 minutes and inserted into a double-chamber plastic test tube. Salivary

cortisol was measured with the same assay used for serum (Radim, Italy) with the analyte

volume increased from 25–250 µl. The sensitivity of the method was 50 ng/dl (1.4 nmol/liter).

Routine clinical chemistry variables were determined using standard enzymatic methods.

Hormone variables were measured in-house using commercially available reagents. The

following hormones were measured with RIA: PRA and DHEAS (Sorin Biomedica, Italy),

aldosterone and 11-deoxycortisol (Adaltis, Italy), free testosterone (Radim, Rome) and CBG

(Biosource, Belgium). ACTH was measured with IRMA (Nichols Institute, US) and PRL, LH,

FSH, TSH, FT3, FT4, testosterone levels were determined using an automated

chemiluminescence system (Architect ci8200, Abbott Laboratories, USA); serum SHBG was

determined using an automated chemiluminescence system (Immulite, Siemens Healthcare

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Diagnostics, USA). All samples for an individual subject were determined in the same

laboratory and in duplicate. Intra- and interassay coefficients of variation for all hormone

variables were less than 8% and 12%, respectively. Plasma mitotane was measured in-

house by a HPLC assay, as described previously (De Francia et al. 2006).

Statistical analysis

Database management and all statistical analyses were performed by using the

“Statistica” for Windows software package (Statsoft Inc., Tulsa, OK, US). Rates and

proportions were calculated for categorical data, and median, ranges and percentiles for

continuous data. Normality of data was assessed by the Wilk-Shapiro’s test and since data

were not normally distributed two-tailed non-parametric tests were used. Differences for a

given variable at different follow-up times were analyzed by means of the. Correlation

analyses were determined by calculating the Spearman’s R coefficient. A matrix of simple

correlations was calculated between concentrations of mitotane and the other biochemical

variables measured at baseline and every 3 months for one year. Bonferroni adjustment for

multiple comparisons was performed. Missing data were dealt with by excluding patients

from particular analyses if their files did not contain data for the required variables. Levels of

statistical significance were set at p<0.05.

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RESULTS

The patient characteristics are provided in Table 1. The interval between tumor resection

and initiation of mitotane was 1-4 months (median 2). The first year follow-up was complete

for all the patients since none of them recurred in that period. During the follow-up (median

29 months, range 16-120), recurrence has been observed in 6 patients (37%) after 15-24

months. The characteristics of the patients with recurrent ACC are provided in Table 1.

Three patients died of ACC progression, whereas 14 patients are alive and 13 of them are

currently on mitotane. One patient with no evidence of disease discontinued treatment after 5

years for her willingness. Mitotane plasma concentrations increased progressively during the

first year of treatment (Figure 1) and levels greater than 14 mg/l were reached in 8 patients

(50%) after 3 months of treatment, in 4 patients (25%) after 6 months and in the remainders

after 9 months (25%), respectively. Three of the 6 patients with recurrent ACC reached target

mitotane concentrations after 6 months. The maximum mitotane dose ranged between 2 and

4 g daily (median 4). The maximum dose was reached after 2-6 months (median 4) of

treatment. There was no correlation between the maximum dose and the time elapsed to

reach the target mitotane concentrations. After reaching target mitotane levels, the dosage

was tapered over some months and then adjusted depending on mitotane monitoring. At the

last available follow-up, the patients were taking a mitotane dose ranging from 1 to 3 g/day.

Eleven patients (64%) maintained mitotane concentrations of 14 mg/l or greater, while 3 of

the remainders (18%) had concentrations between 10 and 14 mg/l and 3 (18%) less than 10

mg/l (2 of them had recurrent ACC).

The adverse events associated with mitotane therapy are listed in Table 2. All patients

experienced some toxicity and in 13 of them (76%) multiple side effects were recorded.

However, none of the patients discontinued mitotane definitively for toxicity but temporary

discontinuation was necessary in 2 patients for neurological toxicity and in one for

concomitant diagnosis of autoimmune hepatitis. The more common adverse event was a

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moderate increase in alkaline phospatase (aPh) and gamma glutamyl transpeptidase (γGT),

that was observed in all patients, but transaminases were elevated only in few patients

(Table 2). Constitutional and gastrointestinal symptoms were more frequently observed in the

first three to six months of therapy and in most cases resolved completely, or improved

consistently, after institution of palliative therapy and increase of glucocorticoid replacement.

No clear correlation between mitotane concentrations and patient complaints was evident.

Gastrointestinal symptoms relapsed at any increment in mitotane dose.

Serum cortisol levels decreased significantly on mitotane therapy (p=0.02) and a

remarkable reduction was already apparent after 3 months (Figure 2); a significant inverse

correlation was found between cortisol and mitotane concentrations (r= -0.36, p=0.003). At

the last available follow-up, serum cortisol concentrations were at their lowest value (1.8, 0.7-

9.4 µg/dl, p=0.02 vs. 12 month follow-up). Salivary cortisol was available for 9 patients at 12

months and the last available follow-up. At both time-points, salivary cortisol was in all

patients below the lower limit of normalcy (4.8 µg/l), that was established in 67 healthy

subjects. The daily dose of cortisone acetate was increased to 50-75 mg in 8 patients and

37.5 mg in 7 patients, while 2 patients were kept on 25 mg, the starting dose instituted

concomitantly with mitotane. Modification of glucocorticoid replacement was mostly based on

clinical assessment. DHEAS levels were greatly and progressively reduced on mitotane

therapy (p<0.0001) (Table 3). ACTH levels increased non significantly with a wide scattering

of data recorded at the different time-points (Table 3) being greater than 100 pg/ml in 15 of

17 patients (88%). ACTH levels were inversely correlated with cortisol (r= -0.41, p=0.005),

while there was no correlation with the dosage of cortisone acetate, even after adjustment for

body weight. In addition, ACTH concentrations did not change remarkably after any

increment in cortisone acetate in most patients and only one of the 2 patients on the lowest

cortisone acetate dosage had increased ACTH. CBG levels increased significantly during

treatment (p=0.04), peaking at 6 months to plateau thereatfer (Figure 2). Aldosterone and

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PRA levels did not change remarkably over the follow-up period (Table 3); however,

mineralocorticoid replacement was commenced in 11 patients (65%) after 6-9 months

because of orthostatic hypotension or dizziness. At the last available follow-up, only 7

patients were still on fludrocortisone replacement because of limited benefit. Levels of 11-

deoxycortisol increased non significantly on mitotane with a large interindividual variation at

any time-point (Table 3). In the light of the clear decrease in cortisol levels, the 11-

deoxycortisol to cortisol ratio was also calculated and showed a significant increase from

0.08 (0.03-0.18) at baseline to 0.46 (0.14-1.19) at 12-months (p=0.03).

FT4 levels decreased significantly on mitotane therapy (p=0.01) and a remarkable

reduction was already apparent after 3 months (Figure 3); however, TSH concentrations did

not change significantly on mitotane (Figure 3) and also FT3 was unmodified (Table 3). Four

patients were excluded from analysis because of current L-T4 treatment (n=3) or subclinical

hyperthyroidism (n=1). During the first year of treatment, FT4 levels dropped in the

hypothyroid range in 12 of 13 evaluable patients (92%) and in 4 of them substitutive therapy

was commenced after 9 months. A significant inverse correlation was found between FT4

and mitotane concentrations (r= -0.49, p=0.008). At the last available follow-up, FT4

concentrations were unchanged compared to the 12 month evaluation (0.77, 0.6-0.9 ng/dl)

but 3 additional patients were on thyroxine replacement .

LH, FSH and PRL levels did not change significantly in either sex (Table 3) even if slight

hyperprolactinemia was observed in 3 out of 10 women and 1 out of 7 men. In female

patients, levels of total and free testosterone did not change significantly on mitotane while in

male patients a biphasic behavior of total testosterone was observed, that was characterized

by a sharp increase at the 3 and 6 month time-points followed by a steep reduction; the

overall change was not statistically significant (Figure 4). Free testosterone levels decreased

significantly on mitotane therapy in male patients (p=0.009) (Figure 4) and a significant

inverse correlation was found between free testosterone and mitotane concentrations (r=

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-0.55, p=0.001). SHBG levels increased significantly in either sex (p=0.01) peaking at 3

months to plateau thereatfer (Table 3). Two male patients were put on testosterone

replacement after 12 months follow-up, and 2 additional patients were replaced in the

following months because they complained of sexual symptoms.

Either total or HDL cholesterol increased significantly during treatment with mitotane

(p=0.01 and p=0.03, respectively) while triglyceride levels did not change significantly (Table

3). A significant positive correlation was found between HDL cholesterol and mitotane

concentrations (r= 0.55, p=0.001). Four patients commenced statin treatment after 12

months, and 4 additional patients were treated during follow-up. At the last available follow-

up, total cholesterol decreased by an average 14% as a likely result of statin treatment, while

HDL and triglyceride were unmodified.

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DISCUSSION

Mitotane has been employed in the treatment of ACC over the last fifty years and it is

generally regarded as a toxic drug with a narrow therapeutic index (Pommier & Brennan

1992; Wajchenberg et al. 2000; Dackiw et al. 2001; Schteingart et al. 2005; Allolio &

Fassnacht, 2006;; Libè et al. 2007). Since ACC is a very aggressive tumor, the available

studies did not analyzed in detail the consequences of mitotane treatment and most of our

knowledge on mitotane toxicity comes from in vitro studies or small case series. Thus, there

is still limited knowledge on the unwanted effects associated with a chronic treatment, and

the dose-effect relationships remain unclear.

The present study demonstrates that adverse effects are to be expected in patients

treated adjuvantly with mitotane; however, toxicity is manageable and complaiance to

treatment can be attained proven that a close patient-physician relationship is established to

induce and mantain motivation to treatment and provide counseling to cope with unwanted

effects. The use of rather low doses of mitotane (up to 6 g daily) may explain why mitotane-

related toxicity was acceptable (Kasperlik-Zaluska et al. 1995; Dickstein et al. 1998), while

severe and disabling toxicity was reported in the studies where high, rapidly increasing, daily

doses of mitotane were employed (Vassilopoulou-Sellin et al. 1993; Schulick & Brennan

1999). In our series, all patients experienced elevation of aPH and γGT and most patients

reported also gastrointestinal symptoms, but gastrointestinal and hepatic toxicities were of

grade 1 in most cases. Adverse manifestations occurred early in the course of treatment but

usually subsided afterwards and did not require discontinuation of the drug in most patients.

Interestingly, gastrointestinal symptoms were particularly evident at the beginning of

treatment and relapsed at any increment in mitotane dose. It is possible to speculate that

patients may develop tolerance to the gradual effects of mitotane and indeed temporary

mitotane withdrawal was necessary in only 3 cases; however, such patients were then able

to resume treatment and to attain elevated drug levels without new episodes of toxicity. We

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disclose the limitation of a possible underreporting of mild adverse effects since the view that

mitotane is a toxic drug may have definitively influenced our approach of retrieving side

effects. In particular, neurological toxicity (Haak et al., 1994) should have been assessed

with specific neuropsychological examinations to detect subtle disturbances of memory,

language, and mental performance.

It is likely that the monitoring of plasma mitotane levels to guide dose adjustements has

been a key factor to predict and limit unwanted effects attaining better tolerance. Previously,

mitotane monitoring has been used in an adjunctive setting only by Haak et al. (1994) and

Baudin et al. (2001). We confirm our previous observation that a low-dose mitotane regimen

is able to consistently provide elevated mitotane levels (Terzolo et al. 2000), but the time lag

necessary for attaining mitotane levels greater than 14 mg/l was particularly long in some

patients, because we were very cautious in dose escalation, even if all patients eventually

reached such concentrations. The present findings may suggest the importance of attaining

elevated mitotane concentrations, since 3 out of 5 patients who reached the target levels

after 6 months suffered a relapse of ACC, while recurrent disease was observed in only 3 out

of 12 patients who reached those levels within 6 months. High dose regimens offer the

advantage of providing higher concentrations in less time but the trade-off may be less

tolerability and greater toxicity (Faggiano et al. 2006). During follow-up, most of the patients

maintained elevated mitotane concentrations despite tapering of the drug.

Hypoadrenalism was an expected consequence of mitotane treatment that occurred in

almost all patients in the first 12 months. Inhibition of cortisol secretion became more evident

while continuing the drug. Serum cortisol showed some variability but salivary cortisol, which

is an accurate index of free, biologically active cortisol, unaffected by CBG levels (Riad-

Fahmy et al., 1982), was more consistently reduced. Adrenal replacement therapy was

monitored best with careful clinical assessment and measurement of electrolytes, since

assessment of serum cortisol was confounded by current steroid supplementation and

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mitotane-induced increase in CBG. Monitoring of ACTH levels was also of little help because

of a great scattering of values among different subjects. Elevated ACTH concentrations may

imply insufficient glucocorticoid replacement even if we did not observe any clear correlation

between the dose of cortisone acetate and ACTH levels, that were little modified by any

further increment in the substitution dose.

We confirm that glucocorticoid replacement at higher doses than those currently used in

Addison’s disease is necessary (Dackiw et al. 2001; Schteingart et al. 2005; Allolio &

Fassnacht 2006; Libè et al. 2007) and doses as high as 50-75 mg cortisone acetate daily

had to be used and were of benefit in reducing gastrointestinal and constitutional symptoms.

The reportedly increased metabolic clearance rate of glucocorticoids (Hague et al. 1989;

Haak et al. 1994; Kasperlik-Zaluska et al. 1995; Allolio & Fassnacht 2006) and the

remarkable increment in CBG induced by mitotane (Van Seters & Moolenaar 1991), which

we have confirmed in the present series, may contribute to the increased demand of steroid

supplementation.

We recorded an increase in 11-deoxycortisol levels that became significant when

calculating the 11-deoxycortisol to cortisol ratio to take into consideration the remarkable

decrease in cortisol levels. This finding supports the view that mitotane is able to inhibit

CYP11B1 activity, as previously observed in vitro (Brown et al. 1973; Lindhe et al. 2002).

Interestingly, metabolic activation of o,p’-DDD is partially dependent on CYP11B1 and this

biotransformation may be critical to determine the activity of the drug (Hahner & Fassnacht

2005; Schteingart 2007). In our study, mitotane treatment decreased DHEAS levels in

parallel with cortisol while changes in aldosterone and PRA were less evident. These findings

are in line with the result of autoradiography studies showing that irreversible binding of o,p’-

[14C]DDD was confined to both zona fasciculata and zona reticularis in normal human cortex

(Lindhe et al. 2002), thus supporting the view that the zona glomerulosa is relatively spared

by the cytotoxic effect of mitotane (Hahner & Fassnacht 2005). As a matter of fact,

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mineralocorticoid supplementation was not necessary in all patients and was of limited

benefit in some patients who later discontinued it.

We have observed a marked reduction in FT4 levels that were inversely correlated with

mitotane concentrations and dropped in the hypothyroid range in most evaluable patients;

conversely, TSH did not change accordingly. Mitotane treatment was previously found to

increase thyroxine-binding globulin (Van Seters & Moolenaar 1991) and to compete with

thyroxine for thyroxine-binding globulin sites (Marshall & Tompkins 1968). However, these

mechanisms may hardly account for a pattern characterized by low FT4, with roughly normal

FT3 and TSH levels. These findings mimick central hypothyroidism and some patients with

reduced FT4 levels were put on thyroxine. An alternative explanation may imply that

mitotane affect deiodase activity, thus changing the FT4 to FT3 ratio. Scanty information on

free thyroid hormone concentrations during mitotane treatment is available; anyway, it was

already reported that in some patients thyroxine replacement may become necessary (Allolio

et al. 2004). However, the clinical benefit of thyroxine replacement in our patients was

uncertain.

We observed a clinical picture suggestive of hypogonadism in most of our male patients

and, at the last available follow-up, 4 out of 7 men were replaced with testosterone. Overall,

the replacement was followed by an improvement in strength, mood and sexual drive but

worsened gynecomastia in 2 patients. The endocrine pattern was puzzling being

characterized by a progressive decline in free testosterone concentrations, that were

inversely correlated with mitotane concentrations, while total testosterone increased in the

short time to decrease thereafter being gonadotropin mostly unchanged. These findings may

be explained by a complex effect of mitotane, including inhibition of testosterone secretion by

the testes and induction of SHBG synthesis, which was confirmed in the present study. The

increase in liver release of binding proteins is likely more rapid than inhibition of testicular

steroidogenesis thus explaining the particular time course of total testosterone

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concentrations. Because of the similarity betweeen adrenocortical and testicular tissue,

mitotane could be expected to cause testicular damage; however, there is a sparse support

for this in the literature (Sparagana 1987). In a recent study, Nader et al. (2006) reported that

after more than 6 months of treatment with mitotane total and free testosterone

concentrations were reduced while hepatic production of SHBG was stimulated by an

estrogen receptor-dependent mechanism. The lack of LH increase following decrease in free

testosterone suggest that mitotane may also have an effect at the pituitary level, possibly

through its estrogen-like activity. In our female patients, gonadal function was mostly

preserved and this may be explained by the fact that mitotane has only a weak estrogen-like

activity that may likely explain the slight hyperprolactinemia observed in some patients. As a

matter of fact, mitotane showed a 1000-fold lower binding affinity than 17β-estradiol for

recombinant human estrogen receptor α (Chen et al 1997). In the literature, there are scanty

data on the effects of mitotane on female sexual function suggesting that mitotane does not

interfere with gonadal steroidogenesis (Ojima et al. 1988).

It has been already reported that mitotane increases serum cholesterol mainly by

increasing the LDL component (Maher et al. 1992; Terzolo et al. 2000; Allolio et al. 2004). In

the present study, we confirmed the increase in total cholesterol but the new finding was the

observation of a marked increase in HDL cholesterol, that was also directly correlated with

serum mitotane concentrations and may be possibly related to the estrogen-like activity of

the drug.

To summarize, a low-dose monitored mitotane regimen is able to provide target serum

concentrations of the drug with an acceptable toxicity in an adjuvant setting. The variability in

mitotane levels between different patients seems to depend more on individual factors than

on the amount of drug administered. Strenghts of the present investigation include the

prospective nature, assessment of a rather homogeneous cohort of consecutive patients and

use of a monitored mitotane treatment according to a predefined protocol. In our view,

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patients who are rendered disease-free by surgery and are submitted to adjunctive mitotane

treatment are the best model to assess the clinical and biochemical alterations associated

with mitotane use without confounding due to the systemic and endocrine effects of the

tumour. Conversely, most of the previous studies were retrospective and included patients

with advanced ACC, who frequently had a poor performance status or suffered the

consequences of concomitant therapies (i.e. other steroid synthesis inhibitors or

antineoplastic treatments), thus explaining the poor safety profile of mitotane that was often

observed (Pommier & Brennan 1992; Wajchenberg et al. 2000; Dackiw et al. 2001;

Schteingart et al. 2005; Allolio & Fassnacht 2006, Libè et al. 2007; Terzolo & Berruti 2008).

We acknowledge the limitation of a rather small cohort of patients owing to the rarity of ACC,

thus the relationship between mitotane concentrations and patient outcome should be

investigated in larger studies.

Side effects occurred frequently but well-informed and motivated patients are able to cope

with them without discontinuing permanently mitotane, proven that a careful tailoring of the

mitotane schedule is done. Patient well-being is improved by an accurate monitoring and

adjustement of hormone replacement, that is difficult to do depending mostly on a clinician’s

judgement since hormone measurement is of little value apart from determination of salivary

cortisol and free testosterone, that may reflect more accurately the impact of mitotane on

adrenal and testicular steroidogenesis. These aspects add to the complexity of adjunctive

mitotane treatment. However, the complexity of treatment should not argue against its use

because we have demostrated that chronic mitotane administration is feasible and rather

well tolerated whenever ACC patients are managed by physicians with specific expertise.

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DECLARATION OF INTEREST

There is no conflict of interest that could be perceived as prejudicing the impartiality of the

research reported.

FUNDING

This work was partially supported by Ministero dell’Università e della Ricerca Scientifica e

Tecnologica (grant n° 2002068252) and Regione Piemonte, Ricerca Sanitaria Finalizzata

2007. The funding source had no role in the design of the study or in the analysis and

interpretation of results.

ACKNOWLEDGEMENTS

We thank Mrs L. Saba and Mrs C. Sciolla for their skillful technical assistance.

Page 18 of 34

19

REFERENCES

Allolio B, Fassnacht M. Adrenocortical carcinoma: clinical update. 2006 Journal of Clinical

Endocrinology & Metabolism; 60:273-287.

Allolio, B, Hahner, S, Weismann, D, Fassnacht, M. Management of adrenocortical

carcinoma. Clinical Endocrinology 2004; 60:273-87.

Baudin E, Pellegriti G, Bonnay M, et al. 2001 Impact of monitoring plasma 1,1-

dichlorodiphenildichloroethane (o,p'DDD) levels on the treatment of patients with

adrenocortical carcinoma. Cancer; 92:1385-1392.

Bergenstal DM, Hertz R, Lipsett MB, Moy RH 1960 Chemotherapy of adrenocortical cancer

with o,p’ DDD. Annals of Internal Medicine; 53:672–682.

Brown RD. Nicholson WE. Chick WT. Strott CA. 1973 Effect of o,p'DDD on human adrenal

steroid 11 beta-hydroxylation activity. Journal of Clinical Endocrinology & Metabolism;

36:730-3.

Chen CW, Hurd C, Vorojeikina DP, Arnold SF, Notides AC 1997 Transcriptional activation of

the human estrogen receptor by DDT isomers and metabolites in yeast and MCF-7 cells.

Biochemistry & Pharmacology; 53:1161–1172.

Common Terminology criteria for adverse event V3.0 (CTCAE). 2003 Bethesda, MD:

National Cancer Insitute.

Dackiw AP, Lee JE, Gagel RF, Evans DB. 2001 Adrenal cortical carcinoma. World Journal of

Surgery; 25:914-926.

De Francia S., Pirro E., Zappia F., et al. 2006 A new simple HPLC method for measuring

mitotane and its two principal metabolites. Tests in animals and mitotane-treated patients.

Journal of Chromatogry B 837: 69-75.

Dickstein G, Shechner C, Arad E, Best L, Nativ O. 1998 Is there a role for low doses of

mitotane (o,p’-DDD) as adjuvant therapy in adrenocortical carcinoma? Journal of Clinical

Endocrinology & Metabolism; 83: 3100-3103.

Page 19 of 34

20

Faggiano A., Leboulleux S., Young J., Schlumberger M., Baudin E. 2006 Rapidly progressing

high o,p'DDD doses shorten the time required to reach the therapeutic threshold with an

acceptable tolerance: Preliminary results. Clinical Endocrinology; 64:110-113.

Haak HR, Hermans J, van de Velde CJ, 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.

Hague RV, May W, Cullen DR. 1989 Hepatic microsomal enzyme induction and adrenal

crisis due to o,p’DDD therapy for metastatic adrenocortical carcinoma. Clinical

Endocrinology; 31:51–57.

Hahner S & Fassnacht M. 2005 Mitotane for adrenocortical carcinoma treatment. Current

Opinion on Investigational Drugs; 6: 386–394.

Kasperlik-Zaluska AA, Migdalska BM, Zgliczynski S, Makowska AM. 1995 Adrenocortical

carcinoma. A clinical study and treatment results of 52 patients. Cancer; 75: 2587-2591.

Lee JE. 2007 Adjuvant mitotane in adrenocortical carcinoma. Letter to the Editor. New

England Journal of Medicine; 357: 1258.

Libe´ R, Fratticci A, Bertherat J. 2007Adrenocortical cancer: pathophysiology and clinical

management. Endocrine-Related Cancer; 14:13–28.

Lindhe O, Skogseid B., Brandt I. 2002 Cytochrome P450-catalyzed binding of 3-

methylsulfonyl-DDE and o,p'-DDD in human adrenal Zona Fasciculata/Reticularis. Journal of

Clinical Endocrinology & Metabolism; 87: 1319-1326.

Luton JP, Cerdas S, Billaud L, et al. 1990 Clinical features of adrenocortical carcinoma,

prognostic factors, and the effect of mitotane therapy. New England Journal of Medicine;

322:1195-2001.

MacFarlane, DA. 1958 Cancer of the adrenal cortex: the natural history, prognosis and

treatment in the study of 50 cases. Annals of Royal College of Surgeons in England; 109:

613-618.

Page 20 of 34

21

Maher VM, Trainer PJ, Scoppola A, Anderson JV, Thompson GR, Besser GM. 1992

Possible mechanism and treatment of o,p'DDD-induced hypercholesterolaemia. Quaterly

Journal of Medicine; 84:671-679.

Marshall, JS & Tompkins, LS. 1968 Effect of o, p'-DDD and similar compounds on thyroxine

binding globulin. Journal of Clinical Endocrinology & Metabolism; 28: 386-392.

Nader N, Raverot G, Emptoz-Bonneton A, et al. 2006 Mitotane has an estrogenic effect on

sex hormone-binding globulin and corticosteroid-binding globulin in humans. Journal of

Clinical Endocrinology & Metabolism; 91:2165–2170.

Ojima M, Hashimoto S, Itoh N, Kusano Y, et al. 1988 Effects of o,p'-DDD on pituitary-

gonadal function in patients with Cushing's disease Nippon Naibunpi Gakkai Zasshi; 64:451-

62.

Pommier RF, Brennan MF. 1992 An eleven-year experience with adrenocortical carcinoma.

Surgery; 112:963-970.

Riad-Fahmy D, Read GF, Walker RF, Griffiths K 1982 Steroids in saliva for assessing

endocrine function. Endocrine Review 3:367–395

Schteingart DE. 2007Adjuvant mitotane therapy of adrenal cancer - use and controversy.

Editorial. New England Journal of Medicine; 356:2415-2418.

Schteingart DE, Doherty GM, Gauger PG, et al. 2005 Management of patients with adrenal

cancer: recommendations of an international consensus conference. Endocrine-Related

Cancer; 12:667–680.

Schulick, RD & Brennan, MF. 1999 Adrenocortical carcinoma. World Journal of Urology; 17:

26-34.

Sparagana M. 1987 Primary hypogonadism associated with o,p' DDD (mitotane) therapy.

Journal of Toxicology and Clinical Toxicology; 25:463-72.

Sullivan, M, Boileau, M & Hodges, CV. 1978 Adrenal cortical carcinoma. Journal of Urology;

120: 660-665.

Page 21 of 34

22

Terzolo M, Angeli A, Fassnacht M et al. 2007 Adjuvant mitotane treatment for adrenocortical

carcinoma. New England Journal of Medicine; 356:2372-80.

Terzolo M & Berruti A. 2008 Adjunctive treatment of adrenocortical carcinoma. Current

Opinion in Endocrinology, Diabetes and Obesity; 15:221–226.

Terzolo, M, Pia, A, Berruti, A, et al. 2000 Low-dose monitored mitotane treatment achieves

the therapeutic range with manageable side effects in patients with adrenocortical cancer.

Journal of Clinical Endocrinology & Metabolism; 85: 2234-2238.

Van Seters A.P., Moolenaar A.J. 1991 Mitotane increases the blood levels of hormone-

binding proteins. Acta Endocrinologica; 124: 526-533.

Vassilopoulou-Sellin R, Guinee VF, Klein MJ, et al. 1993 Impact of adjuvant mitotane on the

clinical course of patients with adrenocortical cancer. Cancer; 71:3119-3123.

Weiss LM. 1984 Comparative histologic study of 43 metastasizing and nonmetastasizing

adrenocortical tumors. American Journal of Surgical Pathology; 8:163-169.

Weiss LM, Medeiros LJ, Vickery AL, Jr. 1989 Pathologic features of prognostic significance

in adrenocortical carcinoma. American Journal of Surgical Pathology; 13:202-206.

Wajchenberg BL, Albergaria Pereira MA, Medonca BB, et al. 2000 Adrenocortical carcinoma:

clinical and laboratory observations. Cancer; 88:711-736.

Wooten MD, King DK. 1993 Adrenal cortical carcinoma. Epidemiology and treatment with

mitotane and a review of the literature. Cancer. 72:3145–55.

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23

LEGENDS

Figure 1. Plasma mitotane concentrations (mg/l) recorded during one year of treatment

with a mitotane low-dose regimen in 17 patients. Data are expressed as median and 25-

75th percentile (p=0.001 at Friedman’s ANOVA for repeated measurements).

Figure 2. Cortisol concentrations (µg/dl) [left panel] and Cortisol-Binding-Globulin

concentrations (mg/l) [right panel] recorded during one year of treatment with a low-dose

mitotane regimen in 17 patients. Data are expressed as median and 25-75th percentile

(cortisol, p=0.02 and CBG p=0.04 at Friedman’s ANOVA for repeated measurements).

Figure 3. FT4 concentrations (ng/dl) [left panel] and TSH concentrations (mU/l) [right

panel] recorded during one year of treatment with a low-dose mitotane regimen in 13

evaluable patients. Data are expressed as median and 25-75th percentile (FT4, p=0.01 at

Friedman’s ANOVA for repeated measurements).

Figure 4. Total testosterone concentrations (ng/ml) [left panel] and free testosterone

concentrations (pg/ml) [left panel] recorded during one year of treatment with a low-dose

mitotane regimen in 7 male patients. Data are expressed as median and 25-75th

percentile (free testosterone, p=0.009 at Friedman’s ANOVA for repeated measurements).

Page 23 of 34

Table 1 – Baseline characteristics of the patients.

Overall series

(n=17)

Patients with

recurrent

ACC

(n=6)

Characteristic

Age (yr)

Median 36 29.5

range 22-58 22-55

Sex (N., %)

Men 7 (41%) 3 (50%)

Women 10 (59%) 3 (50%)

Tumor Stage (N., %)

I 1 (6%) 1 (17%)

II 12 (70%) 3 (50%)

III 3 (18%) 1 (17%)

IV 1 (6%) 1 (17%)

Tumor size (cm)

Median 11 14

Range 5.5-24.0 5.5-24.0

Functional status (N., %)

Functional tumors 11 (65%) 4 (67%)

Glucocorticoids ±

androgens

7 (64%) 3 (75%)

Androgens 3 (27%) 1 (25%)

Aldosterone 1 (9%) -

Nonfunctional tumors 6 (35%) 2 (33%)

Weiss score (N.)

Median (range) 6 (3-9) 7.5 (4-9)

Page 24 of 34

Table 2 - Mitotane toxicity graded according to NCI criteria

Toxicity Grade

1 2 3 4

Blood

Leukopenia 3 - - -

Constitutional

symptoms

Asthenia/Fatigue 5 4 3 -

Gastrointestinal

Anorexia 3 3 - -

Diarrhea 3 - - -

Nausea/Vomiting 7 2 1 -

Hepatic

Elevated GGT/aPh 14 - 3 -

Elevated AST/ALT 3 - - -

Neurology

Confusion - 3 - -

Ataxia 2 -

Vertigo/Dizziness - 3 - -

Other

Gynecomastia 4 1 - -

Impotence 1 3 - -

Orthostatic

hypotension

6 1 - -

Data are expressed as number of patients experiencing toxicity.

Due to the adrenolytic action of mitotane all patients received prophylactic

glucocorticoid replacement therapy. Detailed monitoring of mitotane-

induced adrenal insufficiency was, therefore, not performed.

Page 25 of 34

Table 3 – Biochemical effects of adjuvant mitotane treatment.

Variable baseline +3 months +6 months +9 months +12 months

DHEAS

(µµµµg/dl)

74§

15-425

33§

5.0-133

15§

5.0-36

15§

5.0-147

15§

9.0-83

Aldo

(pg/ml)

51

42-410

64

50-400

62

37-306

50

25-490

77

37-270

PRA

(ng/ml/h)

1.1

0.3-2.1

0.8

0.4-16

1.6

0.3-8.0

0.8

0.5-6.0

1.1

0.2-5.0

ACTH

(ng/ml)

34

10-148

159

14-1230

133

32-1472

152

26-1155

81

19-1006

S

(ng/ml)

1.0

0.6-2.6

4.3

2.0-15.4

4

0.6-13.8

3.6

0.5-16.4

3.4

0.5-9.5

FT3

(pg/ml)

3.1

2.4-6.9

2.8

2.2-4.2

2.6

1.6-3.5

2.6

2.2-4

2.6

1.9-3.6

SHBG

(nmol/l)

39*

13-85

164*

33-180

180*

97-180

171*

47-180

102*

25-180

PRL ♀

(ng/ml)

16

1.9-46

14

7.7-59

10

7.0-25

14

7.5-88

11

6.7-19

PRL ♂

(ng/ml)

7.7

5.2-8.3

12

5.7-25

10

3.7-15

9.0

5.3-26.5

9.0

5.5-21.6

FSH ♀

(U/l)

5.0

1.9-100

8.2

3.7-60

5.6

4.0-76

9.6

5.0-63

5.8

1.9-81

FSH ♂

(U/l)

1.7

1.3-12

2.2

0.7-17

4.8

1.2-16

7.8

1.2-15

7.5

1.2-13

LH ♀

(U/l)

8.7

1.4-46

14.3

4.0-40

14

3.4-51

16

6.0-52

21

1.4-51

LH ♂

(U/l)

6.3

3.0-16

6.0

3.0-13

12

5.6-49

12

3.6-51

19

6.6-46

Testo ♀

(ng/ml)

0.2

0.1-0.6

0.3

0.1-0.8

0.3

0.1-1.0

0.4

0.1-0.8

0.3

0.1-1.0

F testo ♀

(pg/ml)

0.6

0.6-0.6

0.3

0.2-0.8

0.2

0.2-0.7

0.3

0.2-0.5

0.4

0.2-1.3

Tot chol

(mg/dl)

192*

108-303

270*

163-361

240*

196-414

246*

150-326

243*

143-378

LDL chol

(mg/dl)

122¶

68-216

177¶

86-245

150¶

106-247

129¶

70-161

127¶

75-263

HDL chol

(mg/dl)

57¶

30-97

66¶

40-118

80¶

44-128

94¶

41-127

81¶

33-155

TG

(mg/dl)

95

49-339

133

61-283

88

53-339

117

55-397

121

51-282

Data are expressed as median and range.

Page 26 of 34

2

Abbreviations are as follows: Aldo, aldosterone; S, 11-deoxycortisol; testo, testosterone; F testo,

free testosterone; tot chol, total cholesterol; HDL chol, HDL cholesterol; TG, tryglicerides

(§p<0.0001, *p=0.01, ¶p=0.03, at Friedman’s ANOVA for repeated measurements).

Page 27 of 34

Figure 1

Median 25%-75%

0 3 6 9 12Months

0

4

8

12

16

20

24

Page 28 of 34

Figure 2, left panel

Median 25%-75%

0 3 6 9 12Months

0

4

8

12

16

20

Page 29 of 34

Figure 2, right panel

Median 25%-75%

0 3 6 9 12Months

0

40

80

120

160

200

Page 30 of 34

Figure 3, left panel

Median Min-Max

0 3 6 9 12Months

0,0

0,3

0,6

0,9

1,2

25%- 75%

Page 31 of 34

Figure 3, right panel

Median 25%-75%

0 3 6 9 12Months

0

1

2

3

4

5

Page 32 of 34

Figure 4, left panel

Median 25%-75%

0 3 6 9 12Months

0

4

8

12

16

Page 33 of 34

Figure 4, right panel

Median 25%-75%

0 3 6 9 12Months

0

5

10

15

20

25

30

35

Page 34 of 34