Mitotane treatment in patients with metastatic testicular Leydig cell tumor associated with severe androgen excess

Mitotane (o,p′DDD) is established in the adjuvant and advanced-stage treatment of adrenocortical carcinoma and counteracts both tumor growth and tumor-related steroid production. Both the adrenal glands and the gonads are steroidogenically active organs and share a common embryogenic origin. Here, we describe the effects of mitotane in two patients with metastatic Leydig cell tumor (LCT) of the testes and associated severe androgen excess (serum testosterone 93 and 88 nmol/L, respectively; male reference range 7–27 nmol/L). Both men suffered from severe restlessness, insomnia and irritability, which they described as intolerable and disrupting normal life activities. Urinary steroid profiling by gas chromatography–mass spectrometry (GC–MS) confirmed excess androgen production and revealed concurrent overproduction of glucocorticoids and glucocorticoid precursors, which under physiological conditions are produced only by the adrenal glands but not by the gonads. In a palliative approach, they were commenced on mitotane, which achieved swift control of the hormone excess and the debilitating clinical symptoms, restoring normal quality of life. GC–MS demonstrated normalization of steroid production and decreased 5α-reductase activity, resulting in decreased androgen activation, and imaging demonstrated disease stabilization for 4–10 months. In conclusion, mitotane can be highly effective in controlling steroid excess in metastatic LCTs, with anti-tumor activity in some cases.


Introduction
Testicular Leydig cell tumors (LCTs) are rare stromal tumors, comprising 1-3% of all testicular neoplasms (1,2).LCTs result in precocious puberty in 10% of affected children due to excess androgen secretion (3).Affected adult men most commonly present with a painless testicular mass and significant androgen excess (4) and can also have tumor-related estrogen excess, manifesting with gynaecomastia in 10-30% of cases (4,5,6).An estimated 10-15% of testicular LCTs are malignant (3,7), although the true proportion remains debated (6,8).The primary approach to malignant LCTs is surgical, usually involving orchidectomy, retroperitoneal lymph node dissection and lifelong surveillance (9).LCT metastases are rare and are detected on average 10 years after primary surgery (7), but therapeutic options are very limited, with no known role for radiotherapy and lack of efficacy of cytotoxic chemotherapy (7,9).Therefore, prognosis for this rare endocrine cancer is poor, with an approximate median survival of two years (3,4,10).
During human foetal development, gonads and adrenal glands both derive from the urogenital ridge and after separation they develop distinct steroidogenic features, with gonadal sex steroid production and adrenal production of glucocorticoids, mineralocorticoids and adrenal androgen precursors.Mitotane (o,p′DDD) is routinely used in the treatment of adrenocortical cancer, where it has been shown to control adrenal steroid excess and, to a degree, tumor proliferation (11).Mitotane also diminishes androgen action by inhibiting 5α-reductase (12) and hence activation of testosterone to 5α-dihydrotestosterone.Thus, we considered mitotane as a potentially useful drug in patients with metastatic Leydig cell tumor, in particular, in patients with tumor-associated androgen excess.Here, we describe the effects of mitotane treatment in two patients with metastatic LCT, leading to a significant biochemical and clinical amelioration of the signs and symptoms of tumor-related steroid excess, and also to temporary radiological stabilization of previously rapid disease progression.

Methods
Urinary steroid metabolome profiling at baseline and during mitotane treatment was carried out by gas chromatography-mass spectrometry, utilizing selected-ion-monitoring analysis for identification and quantification of 32 distinct steroid metabolites reflective of 24-h net steroid output, as previously described (13).Serum steroid measurements were carried out in the routine clinical biochemistry setting, using established and validated tandem mass spectrometry (androstenedione, testosterone) and immunoassays (DHEAS, 17β-oestradiol), respectively.

Case 1
A 51-year-old patient presented with severe restlessness, insomnia, impaired concentration, increased aggressiveness, redness of the face and body hair growth, all gradually developing over the last six months.Fifteen years previously, he had undergone an orchidectomy for LCT, and thirteen years later, excision of a retroperitoneal mass, confirmed on histology as LCT metastasis.Imaging revealed multiple lesions consistent with liver, lung and retroperitoneal metastases.Immunohistochemistry of a tissue biopsy confirmed vimentin-positive, inhibinnegative metastatic LCT.Serum testosterone was very high at 93 nmol/L (normal male reference range 7-27 nmol/L).Urinary steroid profiling by gas chromatography-mass spectrometry (GC-MS) showed increased androgen metabolite excretion (sum of androsterone and etiocholanolone 101,476 µg/24 h; adult male reference range <8000 µg/24 h) as well as increased excretion of DHEA, metabolites of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and cortisol (230 µg/24 h; normal <130) (Fig. 1A).Prognosis was assessed as poor and the patient declined chemotherapy.However, he agreed to the initiation of mitotane treatment in an attempt to improve the clinical signs and symptoms of tumor-related androgen excess that were significantly limiting his quality of life.Mitotane dose was gradually titrated to 3 g per day, with concurrent hydrocortisone replacement (20 mg tid).Within a few weeks, androgen excretion decreased from 101,476 to 12,827 µg/24 h, with evidence of significant inhibition of 5α-reductase activity and normalization of other steroids that were increased at baseline (Table 1).Plasma mitotane concentrations considered therapeutic (anti-proliferative) in the context of adrenocortical carcinoma (14-20 mg/L) (16) were reached after 5 months of treatment (Supplementary Table 1, see section on supplementary data given at the end of this article).Follow-up imaging still showed progressive disease at two months, but stable disease according to RECIST 1.1 criteria after six months of mitotane treatment (Supplementary Fig. 1).Alongside the decrease in androgens, the patient reported a significant improvement of his previously debilitating clinical signs and symptoms.He returned to full-time work and enjoyed good quality of life.After 10 months of mitotane treatment, he died suddenly of a suspected myocardial infarction; no post-mortem examination was carried out.

Case 2
A previously fit-and-well 59-year-old man presented with a right testicular mass and underwent orchidectomy; histopathology revealed malignant LCT.Three years later, he presented with lower back pain, and imaging showed a large retroperitoneal mass, confirmed as disease recurrence by transcutaneous biopsy.He underwent laparoscopic removal of the mass together with retroperitoneal lymph node dissection.One year later, follow-up imaging revealed disseminated metastases, including liver, kidney and peritoneal deposits.He was unwell, with agitation, anxiety and insomnia.Biochemical work-up showed increased serum testosterone (88.5 nmol/L, norma:l 7-27), oestradiol (744 pmol/L, normal <156), androstenedione (7.0 nmol/L, normal: 0.8-3.1)and DHEAS (>27 µmol/L, normal: 0.91-6.76).GC-MS profiling showed increased steroid excretion including androgen metabolites (69,108 µg/24 h, normal <8000) and cortisol (414 µg/24 h, normal <130) (Fig. 1A).He rejected chemotherapy and agreed to palliative mitotane treatment with concurrent hydrocortisone replacement; mitotane was administered employing the high-dose saturation regimen (day 1 500 mg tds, day 2 1000 mg tds, and from day 3 onwards 1500 mg tds; therapeutic plasma mitotane levels were reached after 4 months (Supplementary Table 1)).Mitotane decreased serum androgen production within four weeks.Six months after treatment initiation, plasma testosterone had decreased to 29.1 nmol/L and oestradiol to 177 pmol/L, while androstenedione and DHEAS had normalized.Urinary steroid profiling 4 months after initiation of miotane showed a decline in all previously raised steroid metabolites and decreased 5α-reductase activity.This was paralleled by significant clinical improvement in signs and symptoms, specifically reduced restlessness, aggressiveness and insomnia.Imaging four months after initiation of mitotane revealed a mixed response, with regression of some previous lesions, but emergence of new metastatic deposits in lung and abdomen.The patient passed away 12 months after his second recurrence, i.e. six months after the start of mitotane treatment.

Discussion
Here, we used mitotane, an established drug in adrenocortical carcinoma, in two patients with metastatic testicular LCT associated with severe androgen excess, clinically manifesting with severe restlessness, insomnia, irritability and impaired concentration.Both patients experienced significant improvement in signs and symptoms with mitotane therapy, swift normalization of steroid excess and some stabilization of radiologically quantified tumor load.
In a comprehensive PubMed search (search terms: Leydig cell tumor, malignant Leydig cell tumor, metastatic Leydig cell tumor, mitotane, lysodren, and o,p′DDD), we identified eight cases of LCT treated with mitotane (Table 2).Four patients received mitotane as second-or third-line treatment for metastatic LCT for a very short time only (3 days-8 weeks); none of them showed a biochemical, clinical or radiological response.The remaining four cases received mitotane as first-line treatment for metastatic LCT, with treatment duration varying between 10 weeks and 33 months (Table 2).All four patients experienced significant radiological tumor response and reduction in steroid excess during mitotane  The male reference range is derived from the 24-h urine steroid excretion observed in 24 healthy men aged 40-60 years.The numbers of the steroid metabolites relate to the numbers in Fig. 1A.The total glucocorticoid metabolites were calculated as the sum of metabolites 20, 22-25 and 27-30.

Figure 1 (
Figure 1 (Panel A) Steroid synthesis in the two patients with metastatic testicular Leydig cell tumor as assessed by mass spectrometrybased 24-h urinary steroid profiling before initiation of mitotane treatment (log scale; closed circles, patient 1; open triangles, patient 2).Box plots represent medians and interquartile ranges from a group of 24 healthy male volunteers (age: 40-60 years); whiskers represent the full range.(Panel B) Immunohistochemical staining for sterol-Oacyltransferase 1 (SOAT1) using formalin-fixed paraffinembedded tissue from the recurrent tumor of patient 2, demonstrating high (60% of cells) to moderate (30% of cells) expression of SOAT1 in the tumor tissue.

Table 2
Previously reported cases of patients with widespread metastases from testicular Leydig cell carcinoma treated with mitotane, presented in the order of duration of treatment.