Received: 23 November 2019
Revised: 29 March 2020
AOGS Acta Obstetricia et Gynecologica Scandinavica
Ovarian cyst formation in women of reproductive age receiving mitotane as part of the treatment of adrenocortical carcinoma: Clinical and experimental observations
Gun Abrahamsson1 1 Erling Ekerhovd1 (D Per Olof Janson1 1 Svante Jansson 2
Håkan Ahlman2 | Bo Wängberg2 2 Anders Norström1
1Institution of Clinical Sciences, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
2Department of Surgery, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Correspondence
Erling Ekerhovd, Department of Fertility, Telemark Hospital, P.O. Box 2900 Kjørbekk, 3710 Skien, Norway. Email: erleke@sthf.no
Funding information
This work was supported by grants from The University of Göteborg, Stiftelsen Handlanden Hjalmars Svenssons Forskningsfond, and the Swedish Medical Research Council (MCR, grant no. 5220).
Abstract
Introduction: Mitotane is an adrenolytic drug that is used as an adjuvant to treat adren- ocortical carcinoma. This study aimed to evaluate the clinical course and pathogenetic mechanisms underlying ovarian cyst formation in women of reproductive age diagnosed with adrenocortical carcinoma and being treated with mitotane as an adjuvant to surgery.
Material and methods: Five women presented with stage III-IV adrenocortical car- cinoma and ovarian cyst formation during mitotane treatment. The clinical course of the disease was followed during and after treatment. The effects of mitotane on progesterone production and cell proliferation were studied in cultured human ovar- ian granulosa cells.
Results: Computed tomography and vaginal ultrasonography during mitotane treat- ment repeatedly demonstrated ovarian cysts of varying size without solid intralocu- lar structures. Two women became amenorrheic during the treatment period. After mitotane cessation, the ovarian cysts disappeared and normal menstrual cycles re- sumed. One woman had an uncomplicated pregnancy two years after mitotane treat- ment. In one woman, who underwent salpingo-oophorectomy, histological analysis demonstrated benign ovarian cysts. Mitotane impeded the synthesis of progester- one, reduced the stimulatory effect of gonadotropins on progesterone formation, and reduced labeling with [3H]thymidine in cultured granulosa cells.
Conclusions: Therapeutic concentrations of mitotane are associated with the for- mation of benign ovarian cysts and amenorrhea. Mitotane-induced suppression of ovarian steroidogenesis and impediment of the proliferative capacity of steroid-pro- ducing cells are suggested potential pathogenetic mechanisms underlying mitotane- induced ovarian dysfunction and cyst development. Mitotane treatment does not compromise future ovarian function.
KEYWORDS
adrenocortical carcinoma, gonadotropins, granulosa cells, mitotane, ovarian cysts, steroidogenesis
Abbreviations: ACC, adrenocortical carcinoma; CT, computed tomography; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone.
@ 2020 Nordic Federation of Societies of Obstetrics and Gynecology
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AOGS Acta Obstetricia et Gynecologica Scandinavica
Adrenocortical carcinoma (ACC) is a rare and aggressive tumor (an- nual incidence 0.7-2.0 per million) with a slight female preponderance (55%-60%). The age distribution is bimodal with a peak in childhood and a second larger peak between 40 and 50 years of age.1,2 About one half of affected patients present with hormonal symptoms, so may be diagnosed at an early stage.3,4 Some cases of ACC are detected as the result of local compression symptoms, while about 10% of the cases are detected incidentally.5 In neoplasia, enzymatic deficiencies in steroid synthesis result in abnormal steroid intermediaries and in- dividual urinary steroid profiles are used to detect ACC recurrence.6,7
The adrenolytic drug mitotane interacts with mitochondrial func- tion, thereby blocking the production of corticosteroids and inducing mitochondrial fragmentation and cell death.8-10 Stage I ACC tumors have a 5-year survival rate of 60%-80%, whereas the corresponding survival rate for stage II tumors is 35%-50%.11 Late-stage ACC tumors (stage III-IV) have poor prognosis, but active surgical management and monitored mitotane treatment seem to offer survival advantages.7,8,11-15
The development of ovarian cysts in women treated with mito- tane was reported in a recent study on premenopausal women.16 However, little is known about the histopathological characteristics of this cyst type and the mechanisms underlying their formation.
The present study reports on five women with stage III-IV ACC, all of whom repeatedly developed ovarian cysts during mitotane treatment. To elucidate whether cyst development could be as- cribed to a direct effect of mitotane on the ovary, the production of progesterone and the labeling with tritiated thymidine were studied in cultures of human granulosa cells exposed to mitotane in vitro.
2 MATERIAL AND METHODS |
2.1 Clinical study
The clinical course involving symptomatology, diagnosis, primary and secondary operative procedures, and adjuvant treatment with mitotane was followed in five women of reproductive age who were diagnosed with stage III-IV ACC. The women were admitted to the Section of Endocrine and Sarcoma Surgery, Department of Surgery, Sahlgrenska University Hospital, between 1998 and 2005. After ini- tial surgery they were followed up according to protocol.
2.2 Experimental procedures
Granulosa cells were obtained from follicular fluid samples aspirated from volunteers in connection with in vitro fertilization. Follicular fluid was pooled from 3-5 women in each experiment. After the ad- dition of an equal volume of culture medium containing 10% fetal bovine serum to the follicular fluid, the granulosa cells were resus- pended and stored at 37℃ until completion of the aspiration proce- dure. The granulosa cells were prepared by repeated sedimentation
Key Message
The use of mitotane as an adjuvant treatment for adreno- cortical carcinoma seems to induce the development of be- nign ovarian cysts without compromising fertility. In vitro, mitotane suppresses progesterone formation and cell pro- liferation of cultured granulosa cells.
and gently resuspended in culture medium in a conical test tube (15 mL) at room temperature. The culture medium was M-199 with Earle’s salts, L-glutamine, and sodium bicarbonate (Sigma-Aldrich) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% fetal bovine serum (Life Technologies Ltd). The cells were cultured in 0.5 mL of medium for 4-18 days in 96-well microtiter plastic plates (Falcon®, Becton, Dickinson and Company) under 5% CO2 in humidified air in an incubator at 37℃. The culture medium was changed every other day, and cell morphology was checked daily until the granulosa cells had achieved 30%-60% confluence. Culture proceeded in the corresponding medium with 1% fetal bovine serum for 24 h, when the agents to be tested were added. After the initial experiments, comparing various culture times and concentrations of substances, granulosa cells were cultured at standardized con- centrations of the added agents: mitotane (1-(2-chlorophenyl)-1-(4- chlorophenyl)-2,2-dichloroethane, 10-5 M; Sigma-Aldrich); human chorionic gonadotropin (hCG) (Pregnyl®, 10 mU/ml; Organon AB, Gothenburg, Sweden); and follicle-stimulating hormone (FSH) (Puregon®, 1 U/ml; Organon AB). All agents were prepared in stock solutions, kept frozen at -20℃, and diluted in culture medium to ap- propriate concentrations before each experiment.
In experiments aiming to evaluate progesterone formation, the media were collected after 24 h, immediately frozen, and stored at -20℃ until analysis. Progesterone was measured in aliquots from each well using a time-resolved fluoroimmunoassay (Wallac Oy). In another series of experiments aiming to estimate cell prolifera- tion, cell culture occurred in the presence of [3H]thymidine (Sigma- Aldrich, 2 uCi/mL). In the main series of experiments, incubation was stopped after 24 h when the culture media were removed. After 3 rinses with plain M-199, the cells were precipitated with 10% ice- cold trichloroacetic acid and stored at 4℃ overnight. After further rinsing in 5% trichloroacetic acid, the cells were dissolved in 0.1 M NaOH at 40℃, and aliquots were removed for the determination of radioactivity using liquid scintillation counting (Packard). In all ex- periments, an equal volume of plain or radioactive culture medium was added to non-stimulated control cells. All experiments were performed in 6 wells for each tested agent.
2.3 3 | Statistical analyses
The mean values of progesterone (ng/ml) and radiolabeling in six wells were measured in each experiment. Total number of granulosa
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AOGS Acta Obstetricia et Gynecologica Scandinavica
| Patient no. | Age (years) | Stage | Surgery | Hormonal markers before surgery | Amenorrhea/ normalized menstruations | Cyst formation/ Diameter | Duration of mitotane treatment |
|---|---|---|---|---|---|---|---|
| 1 | 38 | III | 1) Adrenalectomy Caval thrombectomy 2) Pulmonary lobe resection | Cortisolª DHEAS | During mitotane/ after treatment | Repeatedly during treatment/30-100 mm | 60 mo |
| 2 | 20 | III | 1) Nephroadrenalectomy Caval thrombectomy Splenectomy | Cortisolª DHEASb Testosterone | Before mitotane/ last year of treatment | After 12 months of treatment/70 mm | 44 mo |
| 3 | 45 | III | 1) Adrenalectomy 2) Salpingo- oophorectomy Supravaginal hysterectomy | Cortisolª DHEASb Testosterone Androstenedione | Before, during and after treatment | Repeatedly during treatment/20-60 mm | 34 mo |
| 4 | 34 | III | 1) Adrenalectomy | No | No amenorrhea | Repeatedly during treatment/25-103 mm | 46 mo |
| 5 | 20 | IV | 1) Adrenalectomy 2) Kidney resection | Cortisolª | During mitotane/ after treatment | After 14 months of treatment/50 and 70 mm | 38 mo |
Note: 1) Primary surgery.
2) Repeat surgery.
Abbreviation: DHEAS, dehydroepiandrosterone sulphate.
ªCushingoid habitus.
bVirilism.
cells varied between single experiments, so the progesterone con- centrations varied accordingly. Therefore, compiling the results, the formation of progesterone and the incorporation of [3H]thymidine were expressed as percent of control values, and the statistics were based on the mean percentage of each experimental group. Student’s paired t test was used to evaluate the differences between control and treated groups. Values of P < . 05 were considered significant.
2.4 Ethical approval
The study was performed in accordance with The Code of Ethics of the Declaration of Helsinki and approved by the Regional Ethics Committees at the University of Gothenburg, Sweden (ref. no. 439-04, 2005). All women provided written informed consent after receiving thorough oral and written information about the study.
3 RESULTS
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3.1 Clinical data
The clinical characteristics of the five women are presented in Table 1. The women with stage III-IV ACC were aged 20-45 years. Two women (patients 1 and 4) were para-2, while the other three were nulliparous at the time of diagnosis. None of the women had
a history of gynecological disease. At the diagnosis of ACC, all women had normal ovaries, as confirmed on computed tomogra- phy (CT).
In one woman (patient 1), the adrenal tumor (a right-sided tumor of 7 x 10 cm) was incidentally diagnosed after CT in connection with a pulmonary embolism. One woman (patient 2) booked an appoint- ment with her gynecologist because of amenorrhea. Subsequent hormonal investigation revealed elevated serum levels of androgens and increased urinary secretion of cortisol. Another woman (patient 4) self-diagnosed a subcostal tumor mass shortly after a 6 weeks preterm delivery, and subsequent CT revealed an adrenal tumor. Four women presented with cushingoid habitus; two of them also had virilizing symptoms including acne (patient 2), hirsutism, an en- larged clitoris, and a low-pitched voice (patient 3). In all women, pre- operative tests for hormonal markers showed that serum and urine cortisol levels were elevated. Dehydroepiandrosterone sulfate was markedly elevated in three women, two of them presenting with vi- rilization, one of them having elevated testosterone while the other had elevated testosterone and androstenedione. At presentation, FSH, luteinizing hormone (LH), and steroid hormone-binding globu- lin were within the normal range in all five women.
The women underwent radical operations, including adrenalec- tomy, splenectomy, and caval thrombectomy (Table 1). One woman (patient 3), who had enlarged para-aortal lymph nodes and ovar- ian cyst progression, underwent a secondary laparotomy including salpingo-oophorectomy. She died of metastatic disease 6 months later. Another woman (patient 5) presented with cushingoid habitus
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AOGS Acta Obstetricia et Gynecologica Scandinavica
before admittance. She underwent laparoscopic adrenalectomy for a 4 x 6 cm tumor. Five months later, she had a kidney resection for local recurrence. She was healthy at follow up 58 months after surgery. Another woman (patient 4) had disease recurrence (pulmo- nary metastases) 62 months after the primary surgery. The other two women (patients 1 and 2) remained disease-free at 129 and 156 months after surgery, respectively.
After the primary surgery, mitotane treatment was initiated combined with hydrocortisone because of the suppressive action of mitotane on steroidogenesis. The mitotane dose was successively increased to the therapeutic interval (14-20 µg/mL). The mitotane treatment continued for up to five years. The urinary profile of ste- roid excretion, an indicator of disease recurrence, was repeatedly controlled during and after treatment. As a result of the possible teratogenic effects of mitotane, the women were recommended to prevent pregnancy during treatment. In the present study, a copper intrauterine contraceptive device was chosen to avoid hormonal inter- ference with monitoring of mitotane therapy. According to the clinical guidelines for mitotane treatment, contraceptives containing estro- gen might facilitate progression of ACC tumors expressing estrogen receptors.11
Two women (patients 2 and 3) were amenorrheic at presentation (Table 1), while two others became amenorrheic during treatment. In 1 of them (patient 1), the FSH and LH levels became slightly elevated; in the other (patient 5), they remained within the normal range. Normal menstrual cycles resumed after treatment. One woman (pa- tient 2) experienced the start of regular cycles during the last year of treatment. Eight months after mitotane cessation, the drug was undetectable in the serum. She became spontaneously pregnant and had an uncomplicated delivery 2 years later. Another woman (pa- tient 4) continued to have normal menstrual cycles throughout the treatment period.
Ovarian cysts were detected at routine follow up by CT, mag- netic resonance imaging, and/or vaginal ultrasonography (Figure 1), demonstrating unilocular or multilocular cysts on one or both sides. The cysts were thin-walled, varied in size from 20 to 103 mm, and did not express any typical ultrasonographical signs of malignancy.17 The cysts spontaneously disappeared during the treatment period, but they often recurred (Table 1). A dramatic change in cyst size could occur within a couple of weeks. Thus, in one woman (patient 4), the ovaries appeared normal until 9 months after the initiation
of mitotane treatment, when a 66-mm cyst was found in the right ovary (Figure 1A) and a 30-mm cyst was found in the left ovary. At follow up 10 weeks later, the cyst on the right side had disappeared, whereas the cyst on the left side had progressed to a trilocular cyst measuring 103 mm in diameter (Figure 1B). At this stage, the patient felt an increased urge to urinate. Otherwise, cyst development was asymptomatic. The levels of marker CA-125, occasionally controlled, remained normal in all women. Ovarian cyst formation was not cor- related with serum levels of mitotane or gonadotropins. After ces- sation of treatment, the ovaries were normal as judged by CT and vaginal ultrasound. In the woman who underwent salpingo-oopho- rectomy (patient 3), the preoperatively diagnosed bilateral ovarian cysts (70 mm and 40 mm in diameter, respectively) appeared mac- roscopically benign, and subsequent histological analysis confirmed their benign status.
3.2 Experimental data
During 24 h of culture, mitotane significantly (P < . 05) reduced pro- gesterone release into the culture medium (Figure 2). In addition, the stimulatory effects of FSH and hCG on progesterone secretion were significantly (P < . 05) reduced by mitotane treatment. After 24 h of culture in the presence of [3H]thymidine, mitotane and hCG signifi- cantly reduced radiolabeling (P < . 05) (Figure 3). After culture in the presence of both mitotane and hCG, this reduction was even more pronounced. In 4 experiments proceeding for 72 h, hCG slightly stim- ulated radiolabeling, which was substantially reduced by mitotane.
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4 DISCUSSION
Clinically, we observed five women who developed ovarian cysts under adjuvant mitotane treatment. After cessation of the mito- tane regimen, complete regression of the ovarian cysts occurred. This finding is in accordance with previous observations.16,18 One woman underwent salpingo-oophorectomy. A histopathological ex- amination showed benign results. This fact in addition to the benign picture of the cysts during ultrasonography and consistently normal CA-125 levels support a conservative attitude when ovarian cysts are diagnosed in mitotane-treated women.
(A)
NT
R12 666-09
(B)
103M
SEL ch
AOGS Acta Obstetricia et Gynecologica Scandinavica
450
400
*(vs C)
Progesterone, percent of control
350
300
250
*(vs hCG)
*(vs C)
200
*(vs FSH)
150
100
*(vs C)
50
0
1-13
11- 13
11- 10
11- 10
11-8
n=7
C
M
hCG
hCG+M
FSH
FSH+M
160,0
140,0
Radioactivity, percent of control
120,0
100,0
*(vsC)
80,0
*(vsC)
60,0
*(vs hCG)
40,0
20,0
0,0
n=6
n=6
n=6
n =6
n =4
n =4
n =4
C, 24 h
M
hCG
hCG+M
C, 72 h
hCG
hCG+M
Menstrual disorders have been observed in many women treated with mitotane,16,18 indicating mitotane-induced ovarian dysfunction. In the present study, 2 of 5 women became amenorrheic during mi- totane treatment and normal cycles reappeared after therapy. One woman who was amenorrhoeic before the start of mitotane treat- ment experienced normal menstrual cycles during therapy. She be- came pregnant spontaneously and had an uneventful pregnancy and uncomplicated delivery 2 years after treatment. Hence, mitotane treatment does not appear to be associated with permanent damage to vital reproductive functions.
The question arises whether the observed ovarian cyst forma- tion and development of amenorrhea were caused by mitotane and, if so, by what mechanisms? One way to clinically elucidate a possible causative effect would be to follow serum mitotane concentrations in relation to the ultrasound-monitored cyst formation and regres- sion. However, when examined, we did not detect any dose-depen- dent relationship between mitotane and cyst appearance.
Mitotane exerts its adrenotoxic effect by blocking the mitochondrial enzymes responsible for the biosynthesis of pregnenolone, the common precursor of adrenal and gonadal steroids.9,10 A suppressive effect of mitotane on steroid synthesis was observed in cultured adrenocortical cells established from human ACC.9,19 It appears likely that mitotane
affects adrenal steroid formation but may interfere with ovarian ste- roidogenesis by the same mechanism. The observations in the present study that mitotane suppresses steroid production in cultured granulosa cells support this notion. A recent study in mice20 showing that mitotane induces a decrease in antral follicles, decreases the levels of FSH recep- tors, and interferes with ovulation adds further support to this concept.
An impediment of ovarian steroidogenesis in the in vivo situation could alter the ovarian-pituitary axis, leading to ovarian cyst develop- ment. For instance, a change in the relationship between serum LH and FSH levels is associated with functional cyst formation in women with polycystic ovary syndrome.21 Further, a decline in ovarian steroid formation in vivo could lead to an increased pituitary secretion of FSH, which in turn could enhance the proliferation of granulosa cells and secretion of follicular fluid and subsequent cyst formation. However, it does not appear likely that the ovarian-hypophyseal axis is circumstan- tially disturbed during mitotane treatment because serum FSH and LH levels were only slightly elevated in 1 woman but remained within the normal physiological range in the other women. The fact that 1 woman continued to have regular menstrual cycles throughout the treatment period supports this concept. Our observations are in contrast to the findings in another study showing that the levels of gonadotropins as well as sex hormone-binding protein were elevated during mitotane treatment.16 On the other hand, subtle disturbances in the delicate balance between FSH and LH may lead to ovulatory disturbances, as shown in women with secondary amenorrhea.22 Another hypothetical explanation for the development of ovarian cysts could be a distur- bance in gonadotropin secretion due to increased androgen produc- tion by the ACC tumor cells.21 However, this hypothesis seems less likely as all 5 women in the present study had a long period of tumor remission during mitotane treatment but still developed ovarian cysts. Yet, another possibility is that mitotane stimulates cyst formation in the ovary by acting on local ovarian factors, for example cytokines, leading to a modified response to gonadotropins.23,24
As mentioned, the peak of ACC incidence comprises premeno- pausal women 40-50 years of age. In the present report as well as the study by Salenave et al,16 only premenopausal women were in- cluded. Whether postmenopausal women respond differently to mi- totane remains to be determined.
Mitotane administration destroys the zona fasciculata and zona reticularis of the adrenal cortex in dogs.25 Similar gross and ultra- structural changes were observed in cells from human adrenal corti- cal cell lines exposed to mitotane in vitro.10,19 The demonstration in this study that mitotane reduces radiolabeling with tritiated thymi- dine indicates that mitotane has an apoptotic effect on steroid-pro- ducing cells of the ovary.
The use in this study of pooled granulosa cells from follicles of dif- ferent women may make the results more general regarding the effects of mitotane. On the other hand, granulosa cells exposed to high con- centrations of gonadotropins in vivo may show a different response to gonadotropins in vitro compared with granulosa cells obtained from natural cycles. A preculture period in 1% fetal bovine serum, as ap- plied in the present study, before addition of the tested compounds may have rendered the cells more responsive. Desensitization and
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poor response of the granulosa cells to LH after long-term cell culture have been demonstrated.24 Such a desensitization might underlie the inhibitory effect of hCG on radiolabeling during 24-h culture, which appeared to be overcome after another 48-h exposure to hCG.
When diagnosing oncological diseases of the adrenal cortex, it is important to apply an interdisciplinary approach because of common features in adrenal and ovarian steroid production and in trophic hormones along the hypothalamic-pituitary-adrenal-ovarian axis. The results of the present study indicate that the ovarian cysts associated with mitotane treatment are benign functional cysts.
5 CONCLUSION
Adjuvant treatment with mitotane at therapeutic concentrations seems to be associated with benign ovarian cyst formation and transient amen- orrhea development without any persistent harmful effects on ovarian function. Hence, women treated with mitotane can conceive naturally after discontinuing treatment. Mitotane-induced suppression of ovar- ian steroid formation and the apoptotic effects of the drug on ovarian cell growth, as demonstrated here, under in vivo conditions might lead to a deranged ovarian-pituitary feedback, an altered ovarian sensitivity to gonadotropins, and subsequent ovarian cyst development.
CONFLICTS OF INTEREST
None.
ORCID
Erling Ekerhovd İD https://orcid.org/0000-0001-7234-8557
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How to cite this article: Abrahamsson G, Ekerhovd E, Janson PO, et al. Ovarian cyst formation in women of reproductive age receiving mitotane as part of the treatment of adrenocortical carcinoma: Clinical and experimental observations. Acta Obstet Gynecol Scand. 2020;00:1-6. https://doi.org/10.1111/aogs.13869