Immunohistochemical characterization of a steroid-secreting oncocytic adrenal carcinoma responsible for paraneoplastic hyperparathyroidism
Magalie Haissaguerre, 1, *. +@D Estelle Louiset,2,1 Christofer C. Juhlin, 3,4 Adam Stenman, 5,6[D Christophe Laurent,7 Helene Trouette,8 Herve Lefebvre,2,9 and Antoine Tabarin1D
1Endocrinology and Endocrine Oncology, Bordeaux University Hospital, Bordeaux 33000, France
2UNIROUEN, INSERM, NorDiC, Rouen, Normandie University, 76000, France
3Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
4Department of Pathology and Cancer Diagnostics, Karolinska University Hospital, Stockholm, Sweden 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
6Department of Breast, Endocrine Tumors and Sarcoma, Karolinska University Hospital, Stockholm, Sweden
“Digestive Surgery, Bordeaux University Hospital, Bordeaux, France
8 Anatomopathology, Bordeaux University Hospital, Bordeaux, France
9Department of Endocrinology, Diabetes and Metabolic Diseases, Rouen University Hospital, Rouen, France
*Corresponding author: Department of Endocrinology, Haut Leveque Hospital, University of Bordeaux, 1 avenue de Magellan, 33604 PESSAC, France. Email: magalie.haissaguerre@chu-bordeaux.fr
Abstract
We report a unique case of a 44-year-old man with paraneoplastic hyperparathyroidism due to an oncocytic adrenocortical carcinoma (stage pT3NOROMO, ENSAT 2 with a 4% Ki-67). Paraneoplastic hyperparathyroidism was associated with mild adrenocorticotropic hormone (ACTH)- independent hypercortisolism and increased estradiol secretion responsible for gynecomastia and hypogonadism. Biological investigations performed in blood samples from peripheral and adrenal veins revealed that the tumor secreted parathyroid hormone (PTH) and estradiol. Ectopic PTH secretion was confirmed by abnormally high expression of PTH mRNA and clusters of PTH immunoreactive cells in the tumor tissue. Double-immunochemistry studies and analysis of contiguous slides for the expression of PTH and steroidogenic markers (scavenger receptor class B type 1 [SRB1], 3ß-hydroxysteroid dehydrogenase [3ß-HSD], and aromatase) were performed. The results suggested the presence of two tumor cells subtypes with large cells with voluminous nuclei producing only PTH and that were distinct from steroid- producing cells.
Keywords: adrenocortical carcinoma, hypercalcemia, hyperparathyroidism, ectopic PTH secretion
Significance
Ectopic parathyroid hormone (PTH) secretion responsible for paraneoplastic hyperparathyroidism has been reported very rarely. Here, we report the first case of paraneoplastic hyperparathyroidism due to an oncocytic adrenocortical carcinoma. Mild cortisol excess and increased estradiol secretion responsible for gynecomastia and hypogonadism preceded PTH-induced hypercalcemia. Ectopic PTH secretion was confirmed by biochemical analysis in blood from adrenal and per- ipheral veins and PTH expression in tumoral tissue using quantitative RT-PCR and immunohistochemistry. Immunohistochemical studies revealed the presence of two distinct tumor cell subtypes secreting either PTH or steroids, a finding suggesting the secondary occurrence of a PTH-secreting subclone.
Introduction
Primary hyperparathyroidism (PHPT) results from parathy- roid disease, including adenoma, hyperplasia, and rarely car- cinoma. While paraneoplastic hypercalcemia usually results from parathyroid hormone-related protein (PTHrp) secre- tion, ectopic secretion of parathyroid hormone (PTH) has rarely been reported.1 We describe a unique case of paraneo- plastic hypercalcemia due to an oncocytic adrenocortical
carcinoma (ACC) with immunocytochemical demonstration of a double tumoral component responsible for steroid and parathyroid hormone (PTH) secretion.
Case description
A 42-year-old man was admitted for severe hypercalcemia (4.0 mmol/L) associated with vomiting, hypophosphatemia (0.33 mmol/L, N=0.80-1.40), and 25-OH-D vitamin defi- ciency (21 ng/ml, N> 30). Medical history included untreated
+ The two authors have contributed equally to this work.
A
1000
Adrenalectomy
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Estradiol (pmol/L)
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Adrenalectomy
800
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400
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200
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0
3 yr
1
3 month
3 yr
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3 month
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20
Adrenalectomy
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6
Adrenalectomy
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15
LH (U/L)
4
10
5
2
0
0
3 yr
1
3 month
3 yr
1 3 month
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Adrenalectomy
F
800
Adrenalectomy
Calcium (mM)
4
PTH (pg/mL)
600
3
400
2
200
0
3 yr
0.5 yr
1
3 month
3 yr
1 3 month
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Adrenalectomy
H
15
Adrenalectomy
Cortisol (nM)
600
ACTH (pmol/mL)
10
400
5
200
0
3 yr
1
3 month
3 yr
1
3 month
gynecomastia and impotence lasting for 3 years. Calcemia meas- ured 6 months before admission was normal (Figure 1). Primary hyperparathyroidism was diagnosed in the face of inappropriate serum PTH concentrations and increased urinary calcium excre- tion (10.44 mmol/24 h, N = 2.40-7.20). Circulating PTHrp concentration was normal. 123] 99mTc-sestamibi parathyroid scintigraphy did not show abnormal uptake. Cervical ultrason- ography only revealed an 8 mm left-sided thyroid nodule. Meticulous exploratory cervicotomy did not find abnormal parathyroid tissue. Resection of three of the four grossly normal parathyroid glands with subtotal thyroidectomy was performed. No pathological parathyroid glands were found at histopatho- logical analysis, while PHPT persisted post-operatively. First,
intravenous hyperhydration was performed in the emergency department. The patient was treated initially with IV hyperhy- dratation followed by IV infusion of zoledronic acid adminis- trated every 3 weeks and cinacalcet 60 mg twice a day. This regimen maintained calcemia around 3.00 mmol/L until adrenal surgery.
Hormonal workup of gynecomastia revealed hypogonado- tropic hypogonadism (serum testosterone = 0.25 ng/ml, N = 3-11 ng/ml) associated with increased circulating estra- diol levels (Figure 1). No symptoms of Cushing’s syndrome were observed. However, mild adrenocorticotropic hormone (ACTH)-independent hypercortisolism was diagnosed based on normal 24 h urinary free cortisol, impaired 1 mg overnight
A
C
B
dexamethasone test (cortisol = 150 nmol/L), and consistently suppressed plasma ACTH levels. In contrast with ACTH lev- els, serum dehydroepiandrosterone sulfate (DHEA-S) concen- tration was normal (5.3 umol/L). Serum 11-deoxycortisol and 17-hydroxy-progesterone concentrations were normal. The computed tomography scan revealed a heterogeneous 10 cm left adrenal tumor exhibiting intense uptake during 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18FDG PET) scanning (Figure 2).
Adrenal-to-peripheral vein gradients were found for PTH, estradiol, and cortisol following simultaneous left adrenal and antecubital vein sampling performed during adrenalec- tomy (27.6, 4.1, and 2.4, respectively). Plasma PTH concen- trations were 10-fold higher in the left adrenal vein than those in the antecubital vein. Pathological examination diag- nosed an oncocytic ACC (Figure 3A, B) with a Lin-Weiss- Bisceglia score including 1 major criteria (venous invasion) and 3 minor criteria (size >10 cm, necrosis, capsular invasion) and a Helsinki score of 10.2 The reticulin algorithm confirmed malignancy including disruption of the reticulin network and the presence of necrosis and venous invasion. The stage of the ACC was pT3N0R0M0, ENSAT 2 with a Ki-67 of 4%.
One day after surgery, hypocalcemia (2.0 mmol/L) occurred with low plasma PTH levels (7 pg/mL) requiring alfacalcidol and calcium supplementation. One month following surgery, serum estradiol decreased from 806 pmol/L to 89 pmol/L and gonadotropic function evaluation was normal (serum tes- tosterone = 5.42 ng/ml). The pituitary-adrenal axis function was normalized (Figure 1). Vitamin D and calcium supple- mentation was progressively stopped after 9 months while maintaining normocalcemia. Three years later, the patient was still in remission.
Methods
The adrenal tumor from the patient and three additional de- identified ACCs without associated PTH hypersecretion, as well as five de-identified parathyroid adenomas (PA) with es- tablished preoperative PTH elevation, were collected at sur- gery and immediately dissected by the pathologists. The expression of PTH was analyzed using qRT-PCR on an ABI 7900HT Real-Time PCR System (Applied Biosystems). Parathyroid hormone expression levels were calculated from threshold cycle values, normalized to B2M values (ACt), and
expressed as 2(-ACt). Results are illustrated as median ± IQR. Data were analyzed using IBM SPSS Statistics 23 (IBM SPSS, Armonk, NY, USA).
Immunohistological studies were performed on deparaffi- nized tissue sections with antibodies targeting PTH (clone MRQ-31) using an automated Ventana Benchmark Ultra sys- tem (Ventana Medical Systems, Tucson, USA). De-identified control tissues were mounted onto the same slide as the target tissue and served as negative and positive controls. Other sec- tions were incubated with antibodies against PTH, scavenger receptor class B type 1 (SRB1), 3ß-hydroxysteroid dehydrogen- ase (3ß-HSD; 15516-1-AP), and aromatase (MCA20775). Immunoreactivities were detected by using anti- immunoglobulin coupled to peroxidase (EnVision mouse or rabbit IgG/HRP) and diaminobenzidine or fluorescent- conjugated anti-immunoglobulin (Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse) antibodies. The tissue sec- tions were examined on an Axoscope 7 microscope or on a TCS SP8 MP confocal microscope at the Cell Imaging Platform of Normandie (PRIMACEN).
Results
Quantitative RT-PCR analyses revealed that the patient’s ACC overexpressed PTH mRNA, as compared to control ACC albeit at a lower level than that of parathyroid adenomas (additional data). Immunohistochemical studies showed a het- erogeneous distribution of clusters of PTH-immunoreactive ACC cells (Figure 3C). At high magnification, PTH immunos- taining was mainly detected in large cells with voluminous nu- clei. Parathyroid hormone immunoreactivity appeared granular and accumulated close to nucleus or plasma mem- brane, suggesting a localization of PTH in organelles. To fur- ther characterize the phenotype of tumor cells, we looked for the presence of steroidogenic cell markers (Figure 3E-G). The tumor contained numerous immunopositive cells for SRB1, the high-density lipoprotein (HDL) cholesterol receptor known to be expressed by steroidogenic cells. In accordance with tumoral estradiol secretion, numerous 3ß-HSD and aro- matase immunoreactive cells were also detected in the tumor. Interestingly, aromatase immunoreactivity was localized in small tumor cells but not in large cells with voluminous nuclei. Indeed, double immunofluorescence experiments revealed the presence of two distinct cell populations immunoreactive for either 3ß-HSD or PTH, which were intermingled in some tis- sue areas (Figure 3H). Similarly, analysis of immunostainings performed on contiguous slices showed distinct distributions of PTH- and aromatase-positive cells in some tumor areas (Figure 3I, J). In addition, some regions containing numerous aromatase-positive cells were devoid of PTH immunostaining.
Discussion
We report herein a unique case of paraneoplastic hyperpara- thyroidism due to an oncocytic ACC, an entity that has not been, to the best of knowledge, previously reported. Ectopic se- cretion of PTH has rarely been associated with various carcin- omas (bladder, stomach, liver, lung, ovarian), neuroendocrine pancreatic tumor, and pheochromocytoma.1 However, de- tailed histopathological and molecular information concerning these tumors are lacking in most cases or are ambiguous as illus- trated by the absence of PTH expression in tumoral cells or as- sociation with a stronger tumoral expression of PTHrp.3,4
The adrenal tumor described herein was classified as onco- cytic and malignant based on the Lin-Weiss-Bisceglia score. Oncocytic tumors are characterized by granular eosinophilic cytoplasm due to accumulation of mitochondria. Most onco- cytic adrenal tumors are benign. Common histological charac- teristics include cytoplasmic granularity; diffuse immunochemical positivity for vimentin, Melan A, synapto- physin, and alpha inhibin; and particular reticuline frame- work. Majority of oncocytic adrenal tumors develops on the left adrenal, as observed in our patient. Approximately 20% of oncocytic adrenal tumors are malignant. The diagnosis of malignancy relies on the histological scores of Lin-Weiss- Bisceglia rather than the Weiss score and the reticulin and Helsinki scores. Owing to their rarity, the characteristics of oncocytic ACCs are poorly known. Analysis of the literature suggests that 80% of oncocytic ACCs are non-secreting and are associated with a better prognosis than non-oncocytic ACC. In our case report, the Ki-67 proliferation index was low (4%), an unusual observation in ACC, that may explain, together with an ENSAT stage of 3 and oncocytic nature, the lack of tumor relapse more than 4 years following surgery.”
As observed in our case, ACCs are often associated with steroidogenesis dysregulation responsible for the secretion, at variable intensity, of multiple steroid hormones, mainly cor- tisol and androgens.4-8 Estrogen-producing ACCs are rarer (1%-2% of ACCs).6,9,10 A disorganized steroidogenesis with immunohistochemical expression of aromatase and lack of en- zyme converting estradiol to estrone has been shown in an es- tradiol and testosterone-co-secreting ACC.8 Moreover, heterogeneous expression of steroidogenic enzymes involved in cortisol and testosterone secretion and adrenal expression of ACTH receptor have been detected in an adrenal oncocyto- ma from a patient with hirsutism.11 In our patient, both gyne- comastia and gonadotropic insufficiency were resolutive after tumor surgery. Given the mild intensity of cortisol and DHEA-S secretion, we hypothesize that the hypogonadotro- phic hypogonadism in our patient was secondary to estradiol secretion by the ACC. Aromatase expression by tumoral cells suggests that estradiol synthesis could have resulted partly from intratumoral conversion of androgens although alterna- tive steroidogenic pathways cannot be excluded.5,6,12
Secretion of peptide hormones (endothelin, calcitonin) has been shown in exceptional cases of ACC.7,13 In our case, PTH tumoral secretion was demonstrated by detection of (i) a high left adrenal vein to peripheral vein PTH gradient, (ii) absence of parathyroid adenoma and hyperplasia, (iii) dra- matic decrease in plasma PTH concentration after adrenalec- tomy resulting in transient hypoparathyroidism, (iv) high expression level of PTH mRNA in the tumoral tissue, and (v) detection of clusters of PTH immunoreactive cells in the tu- mor tissue. The fact that a minority of tumoral cells expressed PTH is consistent with the quantitatively modest expression of PTH mRNA as compared to parathyroid adenomas although it was unequivocally greater than in control ACCs. The micro- scopical appearance of PTH immunoreactivity in the cyto- plasm of tumor cells suggests that PTH was stored in secretory granules, as classically observed for peptide hor- mone release through exocytosis. Parathyroid hormone- producing tumor cells, characterized by large size and voluminous nuclei, exhibited a distinct morphology from that of aromatase-positive cells. Double-immunochemistry studies and analysis of contiguous slides revealed that PTH-producing cells were immunonegative for 3ß-HSD and
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aromatase, indicating that they were not steroidogenic cells. These data suggest the existence of at least two distinct tumor cell subtypes secreting either steroids or PTH.
Hypercalcemia occurred 3 years following the occurrence of gynecomastia. We therefore hypothesize that the development of the PTH-secreting cell component was a second event dur- ing the ACC evolution. Despite the absence of detailed mo- lecular studies, the hypothesis of the secondary occurrence of a PTH-secreting subclone is consistent with evidence for multiple somatic mutational events in the pathogenesis of ACC.14,15
Funding
This work was supported by Institut National de la Santé et de la Recherche Médicale Unité 1239, the Conseil Régional de Normandie, and the European Regional Development Fund.
Conflicts of interest: No conflict of interest for all the authors.
Patient consent
Written informed consent was obtained from the patient for publication of the submitted article.
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