Prognostic factors for survival after surgery for adrenal metastasis.

AIM: To better define the indications for adrenalectomy for adrenal metastasis we have analysed factors predicting survival in our institutional series. METHODS: A consecutive series of 30 patients undergoing adrenalectomy for metastasis (1996-2007), excluding patients with simultaneous ipsilateral renal cell carcinoma (RCC), was studied. Metastases were regarded as synchronous (<6 mo), or metachronous (>6 mo), depending on the interval after primary surgery. Survival was calculated from time of adrenalectomy and factors influencing survival were identified. RESULTS: The tumour diagnoses were RCC n = 9, malignant melanoma n = 5, non-small-cell lung cancer n = 5, colorectal carcinoma n = 4, foregut carcinoid n = 2, adrenocortical carcinoma, breast cancer, hepatocellular carcinoma, urothelial carcinoma, and liposarcoma (one each); nine adrenal metastases were synchronous and 21 metachronous. Ten patients had undergone previous surgery for extra-adrenal metastases. Out of 30 adrenalectomies 10 were laparoscopic (LAdx) and 20 open (OAdx) procedures without surgical complications. The local recurrence rate was low: LAdx 1/10, OAdx 1/20, and the median survival was 23 months. Independent prognosticators of favourable survival were adrenalectomy for potential cure (p = 0.01), no previous metastasis surgery (p = 0.02), and tumour type (p = 0.043), with better prognosis for patients with adrenal metastasis from colorectal carcinoma and RCC and worse prognosis in non-small-cell lung cancer and malignant melanoma. CONCLUSIONS: Surgery for adrenal metastasis is safe and the indication for this procedure in an individual patient can be supported by several prognostic factors. The survival benefit in patients with adrenalectomy for potential cure indicates a therapeutic value of adrenalectomy in selected patients.

Prolonged survival after hepatic artery embolization in patients with midgut carcinoid syndrome.

BACKGROUND: Hepatic artery embolization (HAE) is a palliative treatment for patients with liver metastases from neuroendocrine tumours. HAE reduces hormonal symptoms, but its impact on survival has been questioned. METHODS: Biochemical responses and survival in consecutive patients with disseminated liver metastases from midgut carcinoid tumours were studied after HAE. Repeat HAE was performed in selected patients with radiological and biochemical signs of progression. RESULTS: Of 107 patients who had HAE, the median survival from the first procedure was 56 (range 1-204) months. Prolonged survival showed a strong correlation with reduction of urinary 5-hydroxyindoleacetic acid (P = 0.003) and plasma chromogranin A (P = 0.001) levels. The biochemical response to repeat HAE was similar to that for the first procedure (P = 0.002). The complication rate was low (7.5 per cent), as was the mortality rate (1.9 per cent) within 1 month of HAE. CONCLUSION: HAE is safe, provides good control of hormonal symptoms, and prolongs survival in biochemically responsive patients. It is a valuable palliative option for patients with midgut carcinoid syndrome due to liver metastases and can be repeated in patients with a favourable response to the first procedure.

Successful receptor-mediated radiation therapy of xenografted human midgut carcinoid tumour.

Somatostatin receptor (sstr)-mediated radiation therapy is a new therapeutic modality for neuroendocrine (NE) tumours. High expression of sstr in NE tumours leads to tumour-specific uptake of radiolabelled somatostatin analogues and high absorbed doses. In this study, we present the first optimised radiation therapy via sstr using [(177)Lu-DOTA(0)-Tyr(3)]-octreotate given to nude mice xenografted with the human midgut carcinoid GOT1. The tumours in 22 out of 23 animals given therapeutic amounts showed dose-dependent, rapid complete remission. The diagnostic amount (0.5 MBq [(177)Lu-DOTA(0)-Tyr(3)]-octreotate) did not influence tumour growth and was rapidly excreted. In contrast, the therapeutic amount (30 MBq [(177)Lu-DOTA(0)-Tyr(3)]-octreotate) induced rapid tumour regression and entrapment of (177)Lu so that the activity concentration of (177)Lu remained high, 7 and 13 days after injection. The entrapment phenomenon increased the absorbed dose to tumours from 1.6 to 4.0 Gy MBq(-1) and the tumours in animals treated with 30 MBq received 120 Gy. Therapeutic amounts of [(177)Lu-DOTA(0)-Tyr(3)]-octreotate rapidly induced apoptosis and gradual development of fibrosis in grafted tumours. In conclusion, human midgut carcinoid xenografts can be cured by receptor-mediated radiation therapy by optimising the uptake of radioligand and taking advantage of the favourable change in biokinetics induced by entrapment of radionuclide in the tumours.

Uptake of meta-iodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters.

The radio-iodinated noradrenaline analogue meta-iodobenzylguanidine (MIBG) can be used for scintigraphy and radiation therapy of neuroendocrine (NE). The aim of the present study was to study the importance of vesicular monoamine transporters (VMATs) for the uptake of (123)I-MIBG in NE tumours. In nude mice, bearing the human transplantable midgut carcinoid GOT1, all organs and xenografted tumours accumulated (123)I after i.v. injection of (123)I-MIBG. A high concentration of (123)I was maintained in GOT1 tumours and adrenals, which expressed VMATs, but rapidly decreased in all other tissues. In the VMAT-expressing NE tumour cell lines GOT1 and BON and in VMAT-expressing primary NE tumour cell cultures (carcinoids, n=4 and pheochromocytomas, n=4), reserpine significantly reduced the uptake of (123)I-MIBG. The membrane pump inhibitor clomipramine had no effect on the uptake of (123)I-MIBG in GOT1 and BON cells, but inhibited the uptake in one out of four primary carcinoid cell cultures and three out of four primary pheochromocytoma cell cultures. In conclusion, VMATs and secretory granules are of importance for the uptake and retention of (123)I-MIBG in NE tumours. Information about the type and degree of expression of VMATs in NE tumours may be helpful in future to select patients suitable for radiation therapy with radio-iodinated MIBG.

Dosimetric comparison of radionuclides for therapy of somatostatin receptor-expressing tumors.

PURPOSE: Therapy of tumors expressing somatostatin receptors, sstr, has recently been clinically tested using somatostatin analogues labeled with (111)In and (90)Y. Several other radionuclides, i.e., (131)I, (161)Tb, (64)Cu, (188)Re, (177)Lu, and (67)Ga, have also been proposed for this type of therapy. The aim of this work was to investigate the usefulness of the above-mentioned radionuclides bound to somatostatin analogues for tumor therapy. METHODS: Biokinetic data of (111)In-labeled octreotide in mice and man were used, primarily from our studies but sometimes from the literature. Dosimetric calculations were performed with the assumption that biokinetics were similar for all radionuclides bound to somatostatin analogues. The cumulated tumor:normal-tissue activity concentration, TNC was calculated for the various physical half-lives of the radionuclides. Using mathematical models, the tumor:normal-tissue mean absorbed dose rate ratio, TN D and tumor:normal-tissue mean absorbed dose ratio, TND, were calculated for various tumor sizes in mice and humans. RESULTS: TNC of radionuclide-labeled octreotide increased with physical half-life for most organs, both in mice and in humans. TN D showed that radionuclides emitting electrons with too high energy are not suitable for therapy of small tumors. Furthermore, radionuclides with a higher frequency of photon emissions relative to electron emissions will yield lower TN D and are thus less suitable for therapy than radionuclides with a lower frequency of photon emissions. The TND was highest for (161)Tb in both mice and humans. CONCLUSIONS: The results demonstrate that long-lived radionuclides, which emit electrons with rather low energy and which have low frequency of photon emissions, should be the preferred therapy for disseminated small sstr-expressing tumors.

Biokinetics of 111In-DTPA-D-Phe(1)-octreotide in nude mice transplanted with a human carcinoid tumor.

The long time biokinetics of the radiolabeled somatostatin analogues 111In-DTPA-D-Phe(1)-octreotide was studied in nude mice transplanted with the human carcinoid tumor, GOT1. The results were compared with those from the patient with the original tumor. This patient has been diagnosed and later treated with 111In-DTPA-D-Phe(1)-octreotide. The animals received about 2 MBq 111In-DTPA-D-Phe(1)-octreotide (0.1 microg) by injection into a tail vein. The animals were killed 0.5 h-14 d after injection of the radiopharmaceutical. Tumor tissue and normal tissues were collected and weighed and measured for 111In activity. The 111In uptake in the tumor was higher than in all normal tissues except the kidneys. The tumor-to-normal-tissue activity concentration, TNC, increased with time for all normal tissues studied. These data were similar to those observed for the original tumor in the patient. The similar biokinetics for 111In-DTPA-D-Phe(1)-octreotide in the tumor-bearing mice and the patient makes this animal model suitable as a model for evaluation of therapy of somatostatin receptor (sstr) expressing tumors with radiolabeled somatostatin analogues. Furthermore, the increase with time of TNC both in mice and the patient indicates that long-lived radionuclides are preferred for therapy with radiolabeled somatostatin analogues.

A transplantable human carcinoid as model for somatostatin receptor-mediated and amine transporter-mediated radionuclide uptake.

A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue 111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to beta-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms.

[Liver transplantation in neuroendocrine tumors prolongs symptom-free period, might also be a cure]. / Levertransplantation vid neuroendokrina tumörer. Ger längre tids symtomfrihet, kanske även bot.

Several neuroendocrine tumours, such as carcinoids and pancreatic endocrine tumours, may manifest relatively slow tumour growth. The patients may suffer from severe hormonal symptoms, largely due to liver metastases which sometimes are amenable to cytoreductive surgery. If residual tumour after primary tumour resection is multilobar, liver transplantation may be one way to treat hormonal symptoms and possibly prolonging survival. Early long-term outcome of liver transplantation in patients with neuroendocrine tumours suggests prognosis to be more favourable for carcinoids than for endocrine pancreatic tumours. It is suggested that liver transplantation may be appropriate for patients with isolated hepatic tumour disease in the following situations: 1, tumour recurrence after liver surgery for cure; 2, non-resectable liver disease, especially in cases of severe hormonal symptoms; and 3, disease progression after hepatic arterial embolisation and medical therapy. These indications are discussed in the light of three case reports.

Comparison of survival between malignant neuroendocrine tumours of midgut and pancreatic origin.

The survival of 64 consecutive patients with disseminated midgut carcinoid tumours was compared in a retrospective study with that of 25 consecutive patients with sporadic malignant endocrine pancreatic tumours treated according to similar surgical principles. The presence of hepatic metastases implied a worse prognosis in neuroendocrine tumours of pancreatic rather than midgut origin. This infers that these tumour types must be separated when treatments are evaluated.

Somatostatin Receptors in the Diagnosis and Therapy of Neuroendocrine Tumor.

The expression of somatostatin receptors in neuroendocrine tumors has facilitated the diagnosis and surgical treatment of patients with these tumors. After injection of a radiolabeled long-acting somatostatin analog, (111)In-octreotide, scintigraphic tumor imaging can ben performed as well as intraoperative tumor localization. During localization studies very high (111)In concentration values were found in tumor tissues versus normal tissues, especially in carcinoid tumors and endocrine pancreatic tumors. Studies on such tumors in cell culture further indicated internalization of (111)In into tumor cells, which is a prerequisite for a radiobiological effect from short range Auger and conversion electrons. Attempts to systemic radionuclide therapy via somatostatin receptors in patients with neuroendocrine tumors have been initiated.

Internalization of indium-111 into human neuroendocrine tumor cells after incubation with indium-111-DTPA-D-Phe1-octreotide.

UNLABELLED: Neuroendocrine tumor cells frequently overexpress somatostatin receptors at their cell surfaces. To evaluate the possibility of using the somatostatin analog 111In-DTPA-D-Phe1-octreotide for radiation therapy, we studied the binding and subsequent internalization of 111In into three types of cultured human neuroendocrine tumor cells. METHODS: Primary cultures of gastric carcinoid, midgut carcinoid and glucagonoma cells were incubated with 111In-DTPA-D-Phe1-octreotide and cell-surface bound, internalized and released 111In activity was measured. Electron microscopic autoradiography was also performed. RESULTS: All three cell types specifically (80%-95%) bound 111In-DTPA-D-Phe1-octreotide and internalized 111In. After 1 hr pulse incubation with 111In-DTPA-D-Phe1-octreotide, there was an initial decrease in intracellular 111In to about 50% during the subsequent 6-hr incubation. Almost no further release was observed during the remaining 18-42 hr studied. Autoradiography showed that the internalized 111In was found in the cytoplasm and nucleus in the midgut carcinoid cells. CONCLUSION: Indium-111 DTPA-D-Phe1-octreotide might be useful for radiation therapy of patients with surgically incurable tumors having high somatostatin receptor densities.

A method for selective infusion in the thyroid artery of the rat and mouse.

A technique for in vivo infusion in the superior thyroid artery in rats and mice was developed and evaluated. The influx catheter is inserted in retrograde direction into the superior carotid artery. The infusate mixed with blood is directed exclusively to the thyroid lobe via the superior thyroid artery. The thyroid isthmus is divided and the other lobe serves as a control. Thyroid ultrastructure was unaltered after infusion for at least 4 h and the follicle cells displayed a normal morphological response to TSH. Electron microscopical autoradiography (125I, [3H]leucine) was performed using 20-80 times less label as compared with iv administration. Infusion of forskolin, a stimulator of adenylate cyclase, increased the intrathyroidal cyclic AMP levels about 10-fold. Infusion of the ionophore monensin yielded typical dilations of Golgi cisternae as well as reduced secretion of newly synthesized protein into the follicle lumen. The arterial infusion technique developed is useful when in vitro methods or systemic administration of substances are unsuitable. The technique permits selective administration of small amounts of experimental substances to the thyroid in high concentrations.

Forskolin-induced elevation of cyclic AMP does not cause exocytosis and endocytosis in rat thyroid follicle cells in vivo.

Using a newly developed infusion technique, the in vivo effects of forskolin and dibuturyl cyclic AMP (dbcAMP) on exocytosis and endocytosis in thyroid follicle cells were studied in thyroxine-treated rats and mice. Reactants were selectively infused via the superior thyroid artery to one thyroid lobe. The contralateral lobe served as a control. In the rat, a supramaximal i.v. dose of thyrotropin (TSH, 500 mU) induced a slight increase in thyroidal tissue levels of cyclic adenosine monophosphate (cAMP) while TSH 50 mU i.v. had no effect on cAMP levels. On the other hand both doses of TSH stimulated exocytosis, signified by a decrease in the number of exocytotic vesicles and endocytosis, signified by the appearance of pseudopods and colloid droplets. Selective thyroid infusion of dbcAMP (5 mM) or forskolin (25 microM), which induced a 10-fold increase in thyroid cAMP levels, did not induce any morphological sign of exocytosis or endocytosis in the follicle cells. The morphological response to TSH given i.v. was quantitatively unaltered by simultaneous infusion of forskolin. In contrast to the findings in rats, infusion of forskolin and dbcAMP in mice induced endocytosis. In conclusion, our findings in the mouse are in agreement with earlier studies in this and other species, indicating that cAMP mediates the effects of TSH on endocytosis and probably also on exocytosis. In contrast, our observations in the rat thyroid in vivo lead to the conclusion that cAMP is not the main intracellular mediator of exocytosis and endocytosis in this species. This conclusion is at variance with previous reports, mostly from in vitro studies.(ABSTRACT TRUNCATED AT 250 WORDS)

A technique for superfusion of isolated granulosa cells. Dynamics of cAMP production and steroidogenesis in response to gonadotropins.

A superfusion model for isolated ovarian cells was developed and characterized in detail. Granulosa cells isolated from pre-ovulatory rat ovarian follicles were placed in superfusion (perifusion) chambers with a volume of 125 microliters. Culture medium was pumped through the chambers, collected in 20-min fractions of 600 microliters and analysed for cAMP and steroids. Viability was confirmed by morphological examination. The use of polycarbonate membranes to retain the cells in the chambers was abandoned since the membranes caused severe cell damage. The temporal relationships between gonadotropic stimuli and the release of cyclic 3':5'-adenosine monophosphate (cAMP) and steroids was investigated. Within 10 min FSH elicited transient increase in the release of cAMP and progesterone but had no effect on testosterone or estradiol-17 beta release. Amplitude and duration of the response in cAMP and progesterone release were correlated to concentration and length of the FSH pulse when these parameters were varied within the ranges 1-100 micrograms/l and 30-270 min, respectively. Compared with the cAMP response, the progesterone response peaked up to 30 min later and lasted 1 to 2 h longer but could not be extended to more than approximately 6 h, not even with longer FSH pulses. These results could indicate a development of desensitization.

Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland.

We have developed a post-embedding immunogold technique for electron microscopic localization and quantitation of thyroglobulin (TG), thyroxine (T4), and triiodothyronine (T3) in rat thyroid. Labeling for TG was located on rough endoplasmic reticulum, Golgi apparatus, exocytotic vesicles, luminal colloid, colloid droplets, and lysosomes, whereas labeling for thyroid hormones was located on luminal colloid, colloid droplets, and lysosomes. We tested different procedures of fixation, dehydration, embedding, polymerization, and immunoincubation to optimize ultrastructural preservation and immunolabeling. Fixation with glutaraldehyde and osmium was possible with retained antigenicity. Dehydration temperature and the choice of embedding resin were the two crucial factors for good immunolabeling. Low-temperature dehydration greatly improved immunolabeling and could be combined with embedding in the methacrylate LR White or the epoxide Agar 100 (equivalent of Epon 812) polymerized at 40-60 degrees C, as the temperature during subsequent embedding and polymerization was of little importance for the immunoreactivity. Labeling on LR White sections was always higher than on Agar 100 sections. Various etching procedures were tested without improved specific labeling. Etching with hydrochloric acid gave nonspecific labeling of certain cell compartments.

The effect of monensin on thyroglobulin secretion. Studies with isolated follicles from pig thyroids.

The effect of monensin on the secretion of thyroglobulin was studied in open follicles isolated from pig thyroid tissue; in this system, thyroglobulin is secreted into the incubation medium. When monensin was present during a 4-h chase incubation after pulse-labelling with 3H-leucine, the secretion of labelled thyroglobulin was reduced by about 85%; in electron-microscopic autoradiographs of rat thyroid lobes labelled and chase-incubated under similar conditions the relative number of grains over follicle lumina was strongly reduced when monensin was present during the chase. These observations are in agreement with the consensus that monensin arrests transport of secretory proteins in the Golgi complex. In other experiments, pulse-labelled follicles were chase-incubated for 1.5 h whereby labelled thyroglobulin was transported from the RER to exocytic vesicles. Monensin present during a subsequent chase of 0.5 h caused only a moderate decrease of labelled thyroglobulin secretion. TSH present during the second chase-stimulated secretion in both control and monensin-exposed follicles. TSH also caused a drastic reduction of exocytic vesicles in rat thyroid lobes, and the number of vesicles remaining in the cells was the same in controls and lobes exposed to the ionophore. The observations are interpreted to show that monensin does not inhibit the basal or TSH-stimulated transport of thyroglobulin from the site of monensin-induced arrest in the Golgi complex to the apical cell surface or the exocytosis of thyroglobulin.

Effect of a second dose of thyrotropin on exocytosis and endocytosis in the rat thyroid gland.

The effect of TSH on exocytosis and endocytosis in thyroid follicle cells was studied with electron microscopic stereology. Groups of rats, pretreated with T4 for 2 d, were injected iv with TSH (500 mU) 20 min, 2, 4, 6, and 8 h before perfusion fixation. Parallel groups were given a second dose of TSH 2, 4, 6 and 8 h after the first one and the thyroids were fixed after 20 min. In controls, injected with saline, the membrane surface area (msa) of exocytotic vesicles was about the same as that of the apical plasma membrane. Pseudopods and colloid droplets were not present. TSH induced exocytosis. The msa of exocytotic vesicles was reduced by about 85% already after 20 min and was even more reduced after 2 h. At later times after injection of TSH the msa of exocytotic vesicles increased gradually to reach control levels after 8 h. TSH also induced endocytosis. After 20 min the increase in msa of endocytotic structures (pseudopods, colloid droplets and micropinocytotic vesicles) corresponded to the decrease in msa of exocytotic vesicles during the same time interval. The msa of endocytotic structures remained high at 2 h but decreased gradually at later time intervals and after 8 h no pseudopods and colloid droplets remained in the apical cell region. A second injection of TSH given 2 h after the first one when few exocytotic vesicles were present did not influence the msa of endocytotic structures. At later time intervals a second dose of TSH stimulated exocytosis as well as endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Turnover of apical plasma membrane in thyroid follicle cells of normal and thyroxine-treated rats.

In thyroid follicle cells exocytotic vesicles transfer newly synthesized thyroglobulin to the follicle lumen and new membrane to the apical plasma membrane. In a previous study data obtained by quantitative electron microscopy were used to estimate the turnover of the pool of exocytotic vesicles in follicle cells of normal and thyroxine-treated (2 days) rats. In the present study, these kinetic data were combined with stereological measurements to calculate the amount of membrane added to the apical plasma membrane by exocytosis and, indirectly, to estimate the turnover of this membrane. In follicle cells of normal rats the area of the membrane added was about 240 micron2/h (180 micron2/h after correction for stereological overestimation) and in thyroxine-treated rats about 105 micron2/h (corrected 80 micron2/h). These areas corresponded to the addition of 1.2% and 0.7% respectively, of the apical plasma membrane per minute. In each cell the total volume of the exocytotic vesicles emptying their content into the follicle lumen in normal rats was 6.2 micron3/h (corrected 4.7 micron3/h) and in thyroxine-treated rats 3.0 micron3/h (corrected 2.3 micron3/h). Considerations based on the findings in the present study suggest that in normal rats macropinocytosis (formation of colloid droplets) is the most important endocytotic pathway for internalization of colloid, while micropinocytosis is the major pathway of membrane internalization. In thyroxine-treated rats macropinocytosis is inhibited. The low volume capacity of the micropinocytotic pathway probably explains the accumulation of colloid protein, mainly thyroglobulin, observed in such rats.