Introduction 1 2 Here we report our clinical experience in three patients with the ectopic GHRH-syndrome derived from a population of over 200 acromegalic patients, diagnosed and treated at the Leiden University Medical Center from 1976 till 2002. Long-term follow-up studies in these patients and results of medical therapy with somatostatin analogs are scarce. The purpose of this report is to expand our knowledge of this rare clinical entity. In addition, we report detailed results of diurnal GH secretion, before and after removal of the GHRH-producing source in order to investigate whether pulsatile and basal GH secretion due to GHRH overproduction differs from that of a primary pituitary somatotropinoma. Methods Basal concentrations of hormones, including prolactin, free thyroxin, triiodothyronine, cortisol, testosterone, estradiol, progesterone, IGF-I, and IGFBP3 were measured. In addition the following tests were performed: Oral glucose loading test (75 g), TRH test (200 μg i.v. ), and a GHRH test (50 μg i.v. ) and the following hormones were measured: glucose tolerance test: GH, insulin and glucose at 0, 30, 60, 90 and 120 min, TRH test: TSH, prolactin and GH at −15, 0, 15, 20, 30 45, 60, 90 and 120 min; GHRH test: GH and prolactin at 0, 20, 30, 45, 60, and 90 min. For the 24-h GH secretion profile the patients were hospitalized, and an indwelling i.v. cannula was inserted in a forearm vein, and blood samples were withdrawn at 10-min intervals. The patients were free to move around, but not to sleep during daytime. Meals were served at 0800, 1230 and 1730 h. Lights were turned off between 2200 and 2400 h. Assays Plasma GH was measured with a sensitive time-resolved fluoro-immunoassay (Wallac Oy, Turku, Finland). The assay is specific for the 22 kDa GH. The standard was biosynthetic recombinant human GH (Genotropin, Pharmacia & Upjohn, Uppsala, Sweden), and was calibrated against the WHO First International Reference Preparation 80/505 (to convert μg/l to mU/l multiply by 2.6). The limit of detection of this assay (defined as the value 2 SD above the mean value of the zero standard) was 0.01 mU/l (0.0038 ng/ml). The intraassay coefficient of variation varied between 1.6% and 8.4% in the range from 0.01 μg/l to 18 μg/l and interassay coefficient of variation was 2.0–9.0% in the same range. Total IGF-I was determined by RIA (Incstar, Stillwater, MN) after extraction and purification on ODS-silica columns. The intraassay coefficient of variation was less than 11%. The detection limit was 1.5 nmol/l. Age-related normal data were determined in the same laboratory. The measurement of IGFBP3 was performed by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). The limit of detection of this assay was 0.08 mg/l, and the interassay coefficient variation was below 6.8%. Deconvolution analysis 3 4 Approximate entropy 5 m r r 6 7 9 r m Copulsatility 10 11 Clinical findings at diagnosis and initial treatment Case 1 1 1 Fig. 1 Upper panel shows the abdominal CT at the level of the pancreas and the lower panel the selective arteriography of the superior mesenterial artery  The patient was the index case of a large kindred affected by MEN-I syndrome. Later, a gene mutation was revealed in exon 2 of chromosome 11q13. During long-term follow-up GH and IGF-I concentrations remained normal. Six years later, in 1988, the patient developed diabetes mellitus, initially treated with oral hypoglycemic drugs and with insulin from 1992 onwards. The patient also had mild bilateral nodular adrenal hyperplasia since 1988, with no evidence of growth during the last recent 10 years. No excess of adrenal (cortex and medulla) hormones or precursors was demonstrable during follow-up. During the last 3 years the plasma level of pancreatic polypeptide (PP) increased to 350 nmol/l (normal < 100 nmol/l). Repeat CT and MRI scanning of the upper abdomen failed to reveal the presence of a pancreatic tumor thus far. 2 Fig. 2 Follow-up of GH and IGF-I concentration in patient 1 after removal of the pancreatic source of GHRH. Fasting serum GH concentrations are depicted as circles, and IGF-I as triangles  Case 2 3 4 5 111 6 Fig. 3 Dynamic GH (circles) and PRL (triangles) tests in two patients with a GHRH-secreting lung carcinoid before (closed symbols) and after surgery (open symbols). Note the GH increase after TRH (200 μg) administration in both patients, the decrease in serum GH concentration in patient 2 after GHRH (50 μg), and the moderate GH decrease after i.v. octreotide (50 μg)  Fig. 4 MRI scans of the pituitary gland of patient 2 (left panel) and of patient 3 (right panel) before treatment  Fig. 5 Chest X-rays of patient 2 (left panel) and patient 3 (right panel), showing the large bronchial carcinoid  Fig. 6 Octreoscans of the patients 2 and 3. In the female patient (left panel) the tumor is seen in the right lower lobe of the lung and also the positive staining of the pituitary gland. The male patient (right panel) had a large tumor in the left lung, without pituitary staining  7 Fig. 7 before and triangles circles  3 Case 3 3 3 AE 12 13 4 5 111 6 . 7 Histopathological studies Pituitary gland The removed part of the anterior pituitary gland of patient 1 consisted of hyperplastic cells, immunostaining positively for GH. The removed tissue of the third patient consisted of a mixture of hyperplasia and adenoma formation. The cells stained positively for both GH and PRL. GHRH-producing tumors Patient 1 Patient 2 Patient 3 3 Somatostatin analog therapy 9 8 9 Fig. 8 MRI of the pituitary gland of patient 2 during therapy with octreotide. These pictures were taken after 12 months treatment with 300 μg octreotide given as a continuous subcutaneous infusion  Fig. 9 GH (circles) and IGF-I (triangles) concentrations during long-term treatment with octreotide. Patient 3 received only the long-acting repeatable form, but patient 2 was treated initially with chronic sc octreotide infusion. The time of change into the slow-release formulation is indicated by the arrow. Normal values for IGF-I for this age: <32 nmol/l. Normal value for random GH < 5 mU/l  9 111 131 9 with octreotide and CT Fig. 10 Serum GH concentrations obtained by 10 min blood sampling for 24 h. Patient 2 was studied before therapy and after surgical removal of the lung tumor. Note that GH concentration decreased more than 10-fold and that the secretion pattern became more regular, but basal GH concentration remained slightly elevated. The left lower panel represent the profile of patient 3 after pituitary surgery, but before removal of the carcinoid tumor. Nadir values were clearly increased. After thoracic surgery and under octreotide treatment GH secretion pattern visually normalized  GH secretory profiles 10 1 Table 1 Deconvolution of the 24 hour serum GH profiles in patients with ectopic GHRH syndrome and controls  Patient 2 before surg. Patient 2 after surg. Female controls Patient 3 before surg.  Patient 3 after surg.  Male controls Jaffe’s patient Vance’s patient Pulse frequency (no/24 h) 30 16 17 (14–21) 30 24 12 (7–14) 30 21 Half-life (min) 15.5 15.9 12.9 (12.0–15.5) 15.6 15.7 17.5 (15.2–19.7) 24.6 16.2 Pulse half-duration (min) 20.9 32.9 27.7 (22.7–29.8) 25.3 23.2 25.3 (19.5–34.4) 30.5 41.3 Pulse height (mU/l /min) 4.92 0.442 0.387 (0.198–0.881) 0.227 0.160 0.141 (0.087–0.422) 6.0 1.87 Pulse mass (mU/l) 101 14.4 10.2 (7.3–18.5) 5.66 3.68 4.44 (2.42–9.55) 181 76.5 Basal secretion(mU/l /24 h) 2,261 30.2 9.1 (5.5–17.6) 18.0 14.9 3.4(1.4–5.3) 3,900 4,60 Pulsatile secretion (mU/l /24 h) 3,047 230 173 (122–312) 170 88 43.5 (17.8–103) 5,400 1,600 Total secretion(mU/l /24 h) 5,309 260 182 (132–325) 188 103 47 (19.7–107) 9,300 2,060 Blood samples were taken at 10-min intervals for 24-h and analyzed by multiparameter deconvolution. The female patient (no. 2) was studied before surgical removal of the GHRH-secreting bronchus carcinoid and repeat sampling study was done after thoracic surgery under octreotide LAR. The male patient (no. 3) was studied first after adenomectomy of the pituitary tumor, but before thoracic surgery. The second sampling study was performed after removal of the bronchial carcinoid during octreotide-LAR treatment. The serum profiles of the patients reported in literature were digitized and deconvoluted with the assay precision according to the authors. The GH data were subsequently transformed from μg-mass units into mU using the conversion factor 2.0. Reference values were obtained in nine males and 10 females healthy controls. Values shown are medians and 95% confidence intervals between brackets 2 14 15 1 P Table 2 Approximate entropy of GH secretion in ectopic GHRH syndrome Patient Before removal of the ectopic GHRH source After surgery and during octreotide treatment Reference values, Median and 95% CI No. 2 (female) 1.256 0.686 0.400 (0.300–0.440) No. 3 (male) 0.842 0.561 0.240 (0.166–0.350) Jaffe’s patient 1.533  0.240 (0.166–0.350) Vance’s patient 1.248  0.240 (0.166–0.350) Calculations were performed on GH data series consisting of 145 samples obtained at 10 min intervals during 24 h. Normal values were derived from nine males and 10 females healthy controls Discussion 1 16 17 18 19 32 2 33 33 34 17 35 34 36 37 38 39 40 18 17 33 35 31 44 41 43 44 8 45 8 7 9 7 46 47 48 49 50 Therapy 51 48 52 18 2 19 21 24 25 15 53 27 54 55 57 22 58 59 63