Clinical Endocrinology (1992) 37,460-467

Case of the Month

Cushing’s syndrome associated with ectopic production of corticotrophin-releasing hormone, corticotrophin and vasopressin by a phaeochromocytoma

Timothy O’Brlen’, William F. Young, Jr*, David G. Davllat, Bernd W. ScheIthauerS, Kalman KovacsQ, Eva Horvaths, Wylle Valell and Jon A. van Heerdenll ‘Division of Endocrinology, Metabolism and Internal Medicine, tDivision of Thoracic Diseases and Internal Medicine, *Section of Surgical Pathology and VDepartment of Surgery, Mayo Clinic and Mayo Foundation, Rochester, MN, and §Department of Pathology, St Michaels Hospital, University of Toronto, Toronto, Ontario, Canada, and (IClayton Foundation Laboratories for Peptide Biology, the Salk Institute, USA

(Received 12 December 1991; returned for revision 3 February 1992; finally revised 9 March 1992; accepted 2 April 1992)

Summary

We describe a case of Cushlng’s syndrome caused by a phaeochromocytoma secretlng cortlcotrophln-releasing hormone (CRH) and cortlcotrophln (ACTH). A 49-year-old white woman presented with a 1-month history of lower limb oedema, polydlpsla and polyurla. Physical examln- ation revealed a patlent with plethorlc facles, lanugo-type facial hair, central obesity, red abdominal striae, lower limb oedema, and blood pressure of 210/115 mmHg. Laboratory studies showed high plasma ACTH and mar- kedly elevated urinary cortisol excretion that suppressed more than 50% with hlgh-dose dexamethasone admlnls- tratlon. Computed tomographic scan of the abdomen showed a 4 t m left adrenal tumour. Catecholamlnes and metabolites were markedly increased In a 24-hour urlne collectlon. Results of venous catheterization studies showed that CRH and ACTH were secreted by the tumour. In addition, with ovine CRH admlnlstratlon, Inferior petros- a1 sinus sampllng showed pituitary secretlon of ACTH. Left adrenalectomy resulted In complete remlsslon of Cushlng’s syndrome. Light mlcroscoplc and Immunohls- tochemlcal studles revealed a phaeochromocytoma that produced CRH, ACTH and vasopressln. RNA studies showed that this tumour, In contrast to normal adrenal and

Correspondence: Dr W. F. Young, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905, USA.

other reported phaeochromocytomas, transcribed a lone pituitary-slzed (1200 nucleotlde) pro-oplomelanocortln mRNA. This is the second reported case of a CRH- secreting phaeochromocytoma.

Pituitary corticotrophin (ACTH) excess is the most frequent cause of ACTH-dependent Cushing’s syndrome; however, approximately 20% of cases are caused by ectopic secretion of ACTH (Howlett et al., 1986), most often due to carcinoid tumours and small cell carcinoma of the lung and less often to medullary thyroid cancer, pancreatic islet cell tumours, and various other carcinomas, e.g. ovarian, prostatic, col- onic, pulmonary and oesophageal carcinomas (Jex ef a[., 1985). Phaeochromocytoma is a rare cause of this syndrome and the first such case was reported by Bourgoignie et al. (1964). Only 21 well documented cases have been reported (Beaser et al., 1986; Berenyi et al., 1977; Bertagna et al., 1982; Bourgoignie et al., 1964; Bruining et al., 1985; Forman ef al., 1979; Hoffman et af., 1980; Interlandi et al., 1985; Jessop et al., 1987; Kakudo et al., 1984; Mendonca et al., 1988; Schroeder et al., 1984; Van Brummelen et al., 1982). Besides ACTH, phaeochromocytomas have been reported to secrete other substances, including parathyroid hormone-related peptide (Kimura et al., 1990) and growth hormone-releasing hormone (GHRH) (Roth et al., 1986).

Pro-opiomelanocortin (POMC) is the precursor peptide for ACTH, p-endorphin, 8- and y-lipotrophins, and a-, p- and y-melanocyte-stimulating hormones. These peptide pro- ducts have been demonstrated immunohistochemically in several extrapituitary, non-tumorous tissues (Tanaka et al., 1982; Nicholson et al., 1987; Bruni et al., 1979; Emson et al., 1984; Liotta et al., 1977; Texier et al., 1991). Various POMC transcript sizes have been identified at the RNA level. The mechanism for the generation of these different size tran- scripts is an alternative transcription start site usage both within the 5’ untranslated region (de Keyzer et al., 1989a) and in the third exon (Lacaze-Masmonteil et al., 1987). In contrast to normal and tumorous pituitary, in which the expected 1200 nucleotide (nt) transcript is found (de Keyzer et al., 1985), normal adrenal tissue transcribes both 800- and 1200-nt sized mRNAs (DeBold et al., 1988). However, adrenal tumours, such as phaeochromocytomas, have been

460

Clinical Endocrinology (1992) 36 CRH and ACTH secretion by phaeochrornocytorna 461

found to produce 800-, 1300-, and 2300-nt-size and larger POMC transcripts (de Keyzer et al., 1989b; DeBold et al., 1988). We report a patient with corticotrophin-releasing hormone (CRH)-secreting, ACTH-secreting, and vasopres- sin (ADH)-producing phaeochromocytoma that in contrast to normal adrenal and other reported phaeochromocytomas expressed a lone 1200-nt POMC mRNA.

Methods

Hormone levels were measured by standard radioimmu- noassay technique. Plasma was extracted for CRH measure- ment as described by Plotsky et al. (1990). CRH radioimmu- noassay was performed as previously described (Vale et al., 1983).

The protocol for the use of the ovine CRH (oCRH) stimulation test (Young et al., 1990) was approved under an investigational exemption for a new drug by the United States Food and Drug Administration, the United States Public Health Service and by the Mayo Clinic Institutional Review Board. Informed written consent was obtained from the patient before the test was administered.

Routinely processed tissues were sectioned and stained by the haematoxylin-eosin (H & E), periodic acid-Schiff (PAS), and Gordon-Sweet methods. Immunostaining was per- formed according to the technique of Hsu et al. (198 I), using antisera to growth hormone, prolactin, ACTH, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone and a-subunit, as well as to CRH (donated by Dr T. Sano, Department of Pathology, University Medical School of Tokashima, Tokashima, Japan), dilution 1 : 500; and ADH (Serotec LTD, Oxford, England), dilution 1 : 1000. Normal pituitary served as positive control, whereas substi- tution of specific antisera with normal rabbit serum served as negative control.

For POMC mRNA demonstration, an oligonucleotide probe (NEP-20/4, Dupont Canada Inc.) of 30 nucleotides

GTAI-3’) was used. The probes were labelled by the 3’-end method with (-’%)dATP-alpha S and terminal deoxynucleo- tidy1 transferase using a kit (NEP-100, Dupont Canada Inc.) and purified with NENSORBTMZO cartridge included in the kit. In-situ hybridization was carried out on paraffin sections applying 3 x lo5 c.p.m. of POMC probe and 1 x lo6 p(dT) probe as described previously (Kovacs et al., 1989; Lloyd et a/., 1990). For in-situ hybridization combined with immuno- cytochemistry, the ABC method for ACTH was applied after 2 x SSC washings. Anti-hACTH (NIDDKD, Bethesda, Maryland) diluted 1:2000 was used. The specificity of hybridization was checked by predigestion of tissue sections with 100 pg/l RNase A (Sigma) and by competition study

(5’-d[CTT-GCC-CCA-GCG-GAA-GTG-CTC-CAT-GGA-

with tenfold excess of unlabelled probe; human kidney was used as negative control.

Total RNA was extracted from normal pituitaries obtained at autopsy and from the phaeochromocytoma at surgery by homogenization and centrifugation through a caesium chloride cushion (Chirgwin et al., 1979). Synthetic POMC transcripts were made from the T7 orientation of the pGEM7Zf+ plasmid containing the 1100 base pair 3’ genomic fragment of the POMC gene (Whitfield et al., 1982). RNA samples were glyoxylated, fractionated on 1 ‘Yo agarose gels, transferred to nitrocellulose filters and hybridized overnight with the nick-translated POMC-containing vector as described above (Thomas, 1980). Filters were subse- quently washed in 1 xSSC, 0.1% SDS for 20 minutes, followed by 3 washes of 20 minutes each at 68°C in 0.2 x SSC, 0.1 % SDS before autoradiography (Sambrook et al., 1989).

Small tissue samples were routinely fixed and processed for electron microscopy and studied with a Philips 410-LS electron microscope.

Case report

A 49-year-old white woman presented with a I-month history of lower limb oedema, polydipsia and polyuria. She had a 6-year history of mild untreated hypertension and a mildly increased level of fasting blood glucose. She denied weight gain or easy bruising. Physical examination revealed a p,atient with plethoric facies, lanugo-type facial hair, central obesity, red abdominal striae, lower limb oedema, and blood pressure of 210/115 mmHg.

Laboratory evaluation demonstrated blood glucose of 17.3 mmol/l (normal, 3.9-5.6 mmol/l), serum sodium of 139 mmol/l (normal, 135-145 mmol/l), and serum potassium of 3.6 mmol/l (normal, 3.6-4.8 mmol/l). The osmolality of a morning voided urine sample was 758 mmolfkg (normal, 300-800 mmol/kg). Baseline hypercortisolism was con- firmed with markedly increased urinary free cortisol of 34374 nmol per 24 hours (normal, < 298 nmol) (Table I). Plasma ACTH concentration was elevated at 121 pmol/l (normal, 0- I3 pmol/l). Urinary cortisol excretion suppressed more than 50% from baseline with the 2-day high-dose (8- mg) dexamethasone test (Table 1). Although the serum cortisol level did not decrease, the urinary ketogenic steroids and 17-ketosteroids did suppress with dexamethasone ad- ministration.

Computed tomographic scan of the abdomen revealed a 4- cm mass in the left adrenal gland and a normal appearing right adrenal gland (Fig. 1). A 24-hour urine collection was obtained and the following values were determined: meta- nephrines, 28.4 pmol (normal, < 7. l pmol); noradrenaline,

462 T. O'Brien eta / . Clinical Endocrinology (1992) 36

Table 1 Results of preoperative evaluation of Cushing's syndrome

24-h urine Plasma values

ACTH Cortisol Cortisol (pmol/l) (nmol/l) (nmo1/24 h)

Normal values < I 3 193-690 66298 Baseline 121 2042 34 374 Dexamethasone (8 mg/day) 2207 10791

ACTH, adrenocorticotrophic hormone; KGS, ketogenic steroids; 17-KTS, 17-ketosteroids.

Fig. 1 Computed tomographic scan of abdomen showing a 4 x 3- cm mass (arrows) of left adrenal gland.

2814 nmol (normal, 89-473 nmol); adrenaline, 6129 nmol (normal, 0-109 nmol); and dopamine, 1823 nmol (normal, <2183 nmol).

To confirm that the left adrenal phaeochromocytoma was a causal factor of the hypercortisolism, venous sampling studies for ACTH and CRH were completed. Simulta- neously obtained blood samples from right and left renal veins, left adrenal vein, and peripheral veins were assayed for ACTH and CRH. The peripheral vein CRH levels were 2 ng/l prior to adrenal vein catheterization and 11 ng/l simultaneous with adrenal vein catheterization. The baseline peripheral vein CRH level was similar to that found in normal controls (1.9f1.0, n=4) and in 10 patients with pituitary-dependent Cushing's disease (1.9 0.8). The left adrenal gland was found to be the source of ACTH and CRH

Table 2 Results of venous sampling for ACTH and CRH

Left adrenal vein 308

Left renal vein* 31 Right renal vein 31

Peripheral vein 35

Peripheral vein 37

83 11 4 4 2

ACTH, adrenocorticotrophic hormone; CRH, corticotrophin releasing hormone. * Sample obtained distal to left adrenal vein.

v 60

40

t i O -'5 b I Id ' 2b I 3b I 410 ' 5b ' 6'0

Time (rnin)

Flg. 2 Plasma ACTH levels in blood samples from 0, right and 0, left inferior petrosal sinuses and A, a peripheral vein before and after intravenous administration of oCRH ( I p/kg) (arrow).

hypersecretion (Table 2). Blood samples from the interior petrosal sinuses (IPS) were obtained before and after administration of oCRH (Fig. 2). At baseline, there was no gradient between the levels of ACTH measured centrally and peripherally. However, oCRH administration resulted in 1.5 (left side) and 1.9 (right side) central (IPS) to peripheral (P) ACTH gradient (IPS: P-ACTH). The peripheral ACTH and cortisol levels increased 30 and 1 % after oCRH administra- tion, respectively. The results suggested that the left adrenal mass was secreting ACTH and CRH and that the inferior petrosal sinus ACTH gradient after oCRH administration was due to ectopic CRH 'priming' by the left adrenal phaeochromocytoma.

The hyperglycaemia and hypertension were controlled with 80 units of insulin per day and combined initial a- (phenoxybenzamine) and subsequent p-adrenergic (propra-

Clinical Endocrinology (1992) 36 CRH and ACTH secretion by phaeochromocytoma 463

Fig. 3 a, Typical histologic features of phaeochromocytoma of adrenal gland include increased vascularity, Zellballen formation, and monomorphism of tumour cells. Adrenal cortex is visible in upper right corner. H&E. x 40. b, The chromogranin reaction was strongly positive. Immunostain. x 63.

nolol) blockade. A left adrenalectomy was performed via the anterior route. The post-operative course was uneventful. The left adrenal gland showed cortical hyperplasia and contained a typical solitary phaeochromocytoma (3-4 x 3.3 x 3 cm). Microscopic examination showed spindle and polygonal cells with amphophilic granular cytoplasm (Fig. 3a); the latter was variably PAS-positive and immuno- reactive for neuron-specific enolase, chromogranin, and synaptophysin (Fig. 3b). Hormones immunohistochemically demonstrated included ACTH, /I-endorphin, CRH, and

operatively. This atypically rapid recovery of the HPA axis was consistent with the findings on the IPS study and probably due to chronic CRH ‘priming’ of the anterior pituitary corticotrophs, thus preventing corticotroph atro- phy and the more typical, prolonged recovery post-operati- vely. Post-operative 24-hour urinary metanephrine and fractionated urinary catecholamine values were normal. The patient remains normotensive and euglycaemic 1 year after surgery.

ADH (Fig. 4). Ultrastructural study showed features typical of phaeochromocytoma.

Dlscussion

In-situ hybridization preparations documented the pres- ence of POMC mRNA. Further characterization of the transcript showed it was a single mRNA species of 1200 nts (Fig. 5).

Post-operatively, the signs and symptoms of Cushing’s syndrome resolved. The patient became normotensive, and antihypertensive agents were discontinued. Hyperglycaemia resolved and insulin therapy was discontinued. The hypotha- lamic-pituitary-adrenal (HPA) axis recovered promptly and exogenous glucocorticoids were discontinued 1 month post-

In this report, we describe a patient with Cushing’s syndrome due to a phaeochromocytoma secreting both ACTH and CRH. The findings on dynamic endocrine testing in patients with hypercortisolism caused by concomitant ectopic ACTH and CRH secretion may be difficult to distinguish from those in patients with hypercortisolism due to eutopic (pituitary) ACTH secretion. In both instances, urinary cortisol excre- tion may suppress with high-dose dexamethasone adminis- tration and plasma ACTH levels may increase in response to oCRH administration (Schteingart et al., 1986; Nieman et

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Clinical Endocrinology (1992) 36 CRH and ACTH secretion by phaeochromocytorna 465

al., 1986). Our patient showed a 68% suppression in urinary cortisol excretion with high-dose dexamethasone testing and a 30% increase in peripheral plasma ACTH levels after oCRH administration. Although both of these responses are positive and consistent with pituitary-dependent Cushing’s syndrome, the following criteria are more specific for an ACTH-secreting pituitary tumour: ( I ) suppression in uri- nary cortisol excretion with dexamethasone administration (e.g. > 90% decrease from baseline) (Flack et al., 1992), and (2) stimulation in plasma ACTH with oCRH administration (e.g. > 50% from baseline) (Kaye & Crapo, 1990). Elevated plasma CRH levels and an abnormality on computerized imaging, as found in our case, may help distinguish between ectopic ACTH-CRH and eutopic ACTH-dependent hyper- cortisolism.

IPS sampling has been used to distinguish between ectopic and eutopic sources of ACTH (Oldfield et al., 1991). A catheter is passed to the IPS bilaterally and blood samples are drawn simultaneously. Basal 1PS:P-ACTH ratio > 2 or peak ratio > 3 after oCRH administration are consistent with a diagnosis of pituitary-dependent disease, whereas lower ratios are consistent with a diagnosis of ectopic ACTH secretion (Oldfield et al., 1991). An unusual feature of our case was the increase in IPS ACTH levels with a peak IPS: P- ACTH ratio of 1.9 after oCRH administration. We propose that this was due to production of CRH by the phaeochro- mocytoma, thus preventing suppression of the pituitary- adrenal axis as would be seen in other causes of ectopic ACTH secretion. A case of ectopic secretion of CRH from a bronchial carcinoid has been reported in which there was a basal 1PS:P-ACTH ratio > 2 (Case records, 1987). In the only other reported case of CRH-secreting phaeochromo- cytoma, IPS studies were not reported (Jessop et of., 1987).

CRH-like immunoreactivity has also been found in nor- mal human tissues and in certain tumours (Wakabayashi et al., 1985). In a recently reported case, immunostaining was used to identify CRH in phaeochromocytoma associated with ectopic ACTH syndrome(Jess0p er al., 1987). Immuno- staining was positive for ACTH, B-endorphin, CRH and ADH in our case. Plasma ADH levels were not measured in our patient and we do not know if it was secreted by the tumour. CRH and ADH may have acted in a paracrine fashion to cause tumoral secretion of ACTH.

Another interesting aspect of our case was the size of the POMC gene transcript. As stated above, variably sized POMC transcripts have been demonstrated in non-pituitary tissues. An 800-nt-sized transcript is often found in normal liver, lung and thyroid (DeBold et al., 1988) tissue. In normal adrenal glands, both 800- and 1200-nt mRNAs are usually detected (DeBold ef al., 1988). S1 nuclease studies have shown that the shorter 800-nt transcript results from

alternative transcription start site initiation within the third exon (Lacaze-Masmonteil et d., 1987). In contrast, non- pituitary tumours, such as phaeochromocytoma (de Keyzer et al., 1989b; DeBold et al., 1988) and thymic carcinoids (de Keyzer et al., 1985; Tsukada et af., 1981), transcribe 1200-nt- and 1450-nt-sized mRNAs and longer species. SI nuclease studies of these tumours have shown that the start site for initiation of transcription for the 1450-nt mRNA is in the 5’ untranslated region of the POMC gene (de Keyzer et al., 1989a). As shown in Fig. 5, the phaeochromocytoma in our patient was different from both normal and tumorous adrenal tissue in that only a 1200-nt species was detected. There is much speculation about significance and mechan- isms of the alternative modes of POMC gene transcription in

1 2 3

1100 nt

Pit T7 Pheo, FIg. 5 Northern blot of POMC RNA. Lane 1, 10 pg of normal pituitary total RNA. Lane 2, 5 pg of T7 synthetic POMC mRNA from 1100 base pair genomic insert. Lane 3, 15 pg total RNA from phaeochromocytoma.

466 T. O‘Brien et a/. Clinical Endocrinology (1992) 36

non-pituitary tissues. The variable start site usage is thought possibly to reflect the different states of differentiation of the tissues studied, that is, normal tissue versus tumorous tissue (DeBold et al., 1988). A second possibility is that there may be tissue-specific differences in the distribution of transacting factors for transcription initiation between pituitary and non-pituitary tissues. Although we did not perform tran- scription start site studies, it appears that a lone start site for POMC transcription was preferred in the non-pituitary tumour in our case and resulted in the exclusive production of a pituitary-sized transcript.

Acknowledgements

Supported in part by grant MT-6349 awarded by the Medical Research Council of Canada and by the generous contributions of Mr and Mrs Francis A Wittern, Sr and Mr and Mrs Francis A. Wittern, Jr.

We thank Mr Steve Sutton and Ms Joan Vaughan for performing the CRH assays, Mrs Nancy Ryan for her expertise in the performance of immunocytologic studies, Dr Eric D. Wieben for advice and guidance on POMC mes- senger analyses, and Dr Lucia Stefaneanu for supervising the in-situ hybridization studies. We gratefully acknowledge Dr Daniel Linkie of Ferring Pharmaceuticals for the formula- tion and supply of oCRH.

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