The presence of somatostatin receptors in malignant neuroendocrine tumor tissue predicts responsiveness to octreotide.

In 77 percent of patients suffering from a malignant carcinoid syndrome, administration of the somatostatin analog, octreotide (SMS 201-995, Sandostatin) induced clinical improvement coupled with a decrease in 24-hour urinary 5-hydroxyindole acetic acid (5-HIAA). This finding prompted an evaluation to determine the correlation between the presence of somatostatin receptors in tumor tissue and the response to octreotide in patients with advanced, metastatic, neuroendocrine tumors. In tissues of 31 tumors (20 carcinoid, eight islet-cell carcinoma, three medullary thyroid carcinomas), the presence of somatostatin receptors was analyzed by binding of the somatostatin analog 125I-Tyr3-SMS 201-995 and autoradiography. Receptors were detected in 16 of 20 samples of carcinoid tissues; all but one patient with receptor-positive tumors improved clinically after treatment with octreotide, and the urine 5-HIAA level was reduced a median of 63 percent (range, 39-94 percent) compared to values before treatment. Of the receptor-negative carcinoid patients, only one showed clinical improvement, which was minimal, and there was a negligible reduction in 5-HIAA after octreotide therapy. All eight patients with metastatic islet-cell carcinomas were positive for somatostatin receptors. Symptomatic improvement and a > 50 percent decrease in the level of at least one of the pathologically elevated marker hormones was seen in all eight. None of the three patients with medullary carcinoma of the thyroid had a decrease in calcitonin, and all three were initially somatostatin receptor-negative. We conclude that the presence of somatostatin receptors in malignant neuroendocrine tumor tissue appears to correlate with the response to octreotide therapy. Analysis of somatostatin receptors in malignant neuroendocrine carcinoma tissue should be included in future prospective clinical trials of this synthetic peptide.     

Gastrointestinal carcinoid tumours. Histogenetic, histochemical, immunohistochemical, clinical and therapeutic aspects

The increased knowledge of the pathobiology of gastrointestinal carcinoid (neuroendocrine) tumours and the improved therapeutic possibilities have brought a demand for more precise diagnosis. Although the carcinoid tumours can often be tentatively recognized in routinely processed microscopic slides, their more accurate identification requires additional diagnostic procedures. General neuroendocrine markers such as the argyrophil reaction of Grimelius and immunohistochemistry with application of antibodies against chromogranin A and of neuron-specific enolase are discriminatory staining methods which are used to reveal the neuroendocrine origin of almost all highly differentiated carcinoid tumours of the gastrointestinal tract. Mid-gut carcinoids, which predominate among these tumours almost unexceptionally contain serotonin. This biogenic amine can be demonstrated by the argentaffin reaction of Masson, serotonin immunoreactively or by formalin-induced fluorescence. The characteristic staining pattern of mid-gut carcinoids is almost invariably preserved in the metastatic deposits and consequently the staining methods for identifying serotonin can also be used on metastases to reveal a primary mid-gut carcinoid. The enterochromaffin-like (ECL) cell carcinoids of the body and fundic area of the stomach often seen in association with pernicious anaemia are argyrophil with the Sevier-Munger silver stain. Other neuroendocrine tumours, viz. antral, duodenal and rectal carcinoids should be studied by a battery of relevant peptide hormone antisera for adequate diagnosis. During the last decade new peptide hormones have been found in circulation in patients with carcinoid tumours, but serotonin and urinary 5-HIAA are still the most important markers for carcinoids of the mid-gut origin. Other clinically useful tumour markers are chromogranin A + B, pancreatic polypeptide, human chorionic gonadotropin alpha and beta subunits. For localizing procedures, angiography is the most reliable investigative method for primary tumours in the gut, whereas CT-scan and ultrasound investigations are good for detection of liver metastases. During the last five years, the therapy for malignant carcinoid tumours has been considerably improved. Chemotherapy has only revealed objective response rates in about 10-30% of the patients giving median survivals from start of therapy of about 10 months. Recently treatment with alpha interferons and the new somatostatin analogue octreotide have given objective responses in 50-75% of patients with malignant mid-gut carcinoid tumours. These patients have now a median survival from start of therapy of 70 months when treated with alpha interferons. In the future new therapies will come into use such as monoclonal antibodies and perhaps also agents blocking different growth factors.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Interferon levels in human pulmonary tumors are lower than plasma levels

It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.     

Biological activity of human alpha-interferons studied using specific antisera

Preparation of highly active rabbit antisera (AS) to human recombinant alpha 2-interferon and their use for studying biological properties of natural and plasmid alpha-interferons are described. By exhaustion of AS by alpha 3-interferon there were prepared practically monospecific AS not reacting with antigenic determinants of alpha 3-interferon. It was found that alpha 3-interferon represented a significant portion of human lymphoblastoid interferons and was included in PH-labile alpha-interferon from serum of patients with Kaposi carcinoma. AS to alpha 2-interferon completely neutralized antiviral and antiproliferative activity of the homologous subtype alpha-interferon and stimulation of cytotoxicity of human natural killer cells induced by it. It neutralized also the same effects of the heterologous subtypes (alpha 3 and alpha F/D) and leukocytic interferon, but the neutralization level was lower. The results of the study confirmed the polyfunctional nature of the interferon molecule.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

Serotonin in cytologic samples of liver metastases of carcinoid tumors. An immunocytochemical study with a monoclonal antibody

Cytologic samples of malignant carcinoid tumors were examined with regard to the presence of serotonin by immunocytochemical staining with a monoclonal antibody. Serotonin immunoreactivity occurred in tumor cells derived from carcinoids of the small intestine while bronchial carcinoid tumor cells were nonreactive. Acetone-alcohol fixation of the cells was a prerequisite for an adequate staining. The serotonin-immunoreactive tumors were also argentaffin positive. The results indicate that cytologic specimens of neuroendocrine tumors, such as carcinoid, can be successfully assayed for the presence of serotonin by an immunocytochemical procedure.     

Additive and synergistic antitumor effects with toremifene and interferons

MFC-7 cells were exposed to toremifene, human alpha and gamma interferons and combinations of them in vitro. Growth of the cells was followed by ATP bioluminescence method. Rats bearing DMBA-induced tumors were treated with toremifene, rat gamma interferon and their combination daily for five weeks. The growth of the tumors was followed by palpation weekly. Toremifene and interferons inhibited the growth of MCF-7 cells. Interferons alpha and gamma were additive; toremifene and interferons were additive or at the best synergistic. Toremifene inhibited the growth of DMBA-induced tumors. Rat gamma interferon alone had no clear effect on the tumor growth. Combination of toremifene and gamma interferone was the most effective treatment and did not show any detectable toxicity. Toremifene and interferons have interesting interactions. Clinical studies using the combination might be warranted.     

New diagnostic method in pulmonary carcinoid

In clinical oncology tumor markers could be helpful in diagnostics, and may play a complementary role beside the imaging systems in the follow up of patients with malignant disease. Chromogranin A can be considered as a marker in neuroendocrine malignancies such as pulmonary carcinoids. Based on recommendations adopted from the literature, the authors measured chromogranin A levels in sera of patients with carcinoid tumors of different stages. Although the patient number is low, our data suggest that in cases where carcinoid metastasis can be detected, chromogranin A levels are elevated.     

The biological relevance of gastric neuroendocrine tumors.

Gastric neuroendocrine tumors were originally thought to have a low incidence (three percent). Since endoscopic diagnostic procedures have become clinical routine, they are now found more frequently (relative incidence up to 41 percent). In recent years, classifications have been developed that attempt to consider the biological relevance of these tumors. Four types of gastric neuroendocrine tumor may be distinguished: Type 1 gastric neuroendocrine tumor is most common. It is associated with chronic atrophic fundus gastritis, hypergastrinemia and often with pernicious anemia. Usually it is multicentric and smaller than one cm, does not produce any symptoms and has an excellent prognosis. Type 2 gastric neuroendocrine tumor is second in frequency. It has no association with other diseases, is solitary and has no predilection for a particular localization. It may be larger than 1 cm, produce a carcinoid syndrome or Zollinger-Ellison syndrome and have a metastasis rate of up to 30 percent. Type 3 gastric neuroendocrine tumor is rare and always associated with Zollinger-Ellison syndrome and multiple endocrine neoplasia type I. It occurs as multiple lesions in the gastric body fundus and has a lower metastatic rate than type 2 gastric neuroendocrine tumor. Type 4 gastric neuroendocrine tumor corresponds to a small-cell carcinoma.     

The incidence and clinical significance of antibodies to interferon-a in patients with solid tumors

It is well known that natural and recombinant proteins can cause antibody formation in the host. We have studied the incidence of binding and neutralizing antibodies in carcinoid patients (n=327). All together 204 patients received interferon-α 2b (Intron-A), median does 15 MU range 9–35 MU/week subcutaneously and 51% of the patients developed binding antibodies by immunoassay and 17% showed positive neutralization assay but high titer antibodies (>800 NU/ml) were only found in 4% of the patients. The median time until the development of binding antibodies was 26 months and neutralizing antibodies 25 months. Twenty-nine patients received interferon-α 2a (Roferon), median does 18 MU/week subcutaneously and 45% developed binding antibodies, 38% had positive neutralization assay and 28% presented high titer antibodies. Binding and neutralizing antibodies occurred at the same time after median six months of treatment. Patients treated with Wellferon (n=45) and leukocyte interferon (n=48), median dose of 15 MU/week subcutaneously did not develop any neutralizing antibodies. The majority of the interferon-α 2 antibodies were of the IgG isotype. The clinical relevance of the development of high titer neutralizing antibodies was evaluated in the patients. All together 17 patients developed high titer neutralizing antibodies and of these 12 patients showed loss of antitumor response measured as increased level of tumor markers and of tumor progression. In nine of these patients a switch to human leukocyte interferon reinstituted an antitumor response. Neutralizing antibodies against recombinant interferon-α 2a and 2b might occur in patients with carcinoid tumors. The incidence of high titer neutralizing antibodies is significantly higher in patients treated with interferon-α 2a compared to interferon-α 2b. A significant number of patients lost the antitumor effect during development of neutralizing antibodies at high titers, but human leukocyte interferon can be used as rescue treatment.     

Overexpression of wild-type PKD2 leads to increased proliferation and invasion of BON endocrine cells

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2WT) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.     

Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest.

Alpha interferon is a potent growth inhibitor of Daudi Burkitt's lymphoma cells. We show here that alpha-interferon signaling interacted simultaneously with several components of the basic cell cycle machinery, causing cells to enter into a state that had many features characteristic of the G0 state. Within a few hours after alpha-interferon treatment, cyclin D3 mRNA and protein levels dropped to undetectable levels and, in parallel, the activities of cyclin A- and cyclin E-associated kinases were significantly reduced. The latter resulted from the rapid alpha-interferon-mediated elimination of cdc25A, a phosphatase that is required for antagonism of negative tyrosine phosphorylation of cdk2 in cyclin-cdk complexes. This regulation represents a novel mechanism through which an external inhibitory cytokine interacts with the cell cycle machinery. At later time points after alpha-interferon treatment, the levels of the 55-kDa slowly migrating hyperphosphorylated form of cyclin E and of cyclin A were also reduced. The antiproliferative effects were reversible, and cultures from which alpha interferon was removed reentered S phase after a lag that typically corresponded to approximately two doubling times. During this lag period, the expression of cyclin D3 and cyclin A, as well as of the cdc25A phosphatase, continued to be switched off, in spite of the removal of alpha interferon from the cell surface. In contrast, c-myc, which represents another downstream target gene that is subjected to negative regulation by alpha interferon, was relieved from suppression much earlier, concomitant with the decay in early signaling of the cytokine. The delayed pattern of cyclin reexpression provides evidence that alpha-interferon signaling imposes a G0-like state on this system.     

Isolated carcinoid tumor metastatic to the thyroid gland: report of a case initially diagnosed by fine needle aspiration cytology

BACKGROUND: Neuroendocrine tumor metastatic to the thyroid gland is rare and may be difficult to differentiate from primary thyroid neuroendocrine tumors, such as medullary thyroid carcinoma (M/ITC). This report describes an unusual case of bronchial carcinoid metastatic to the thyroid diagnosed by fine needle aspiration cytology (FNAC). CASE: A 42-year-old woman with an undiagnosed bronchial carcinoid tumor presented to our clinic with a solitary nodule in the thyroid gland. FNAC of the nodule showed loosely cohesive groups of cuboidal tumor cells with scant, slightly granular cytoplasm; centrally located nuclei with a coarsely granular, salt-and-pepper chromatin pattern and inconspicuous nucleoli. Immunocytochemically the tumor cells were positive for neuron-specific enolase, chromogranin and synaptophysin and negative for thyroglobulin, calcitonin and carcinoembryonic antigen. The cytologic diagnosis of a metastatic neuroendocrine carcinoma was confirmed histologically. CONCLUSION: Metastasis to the thyroid gland may pose a diagnostic problem, particularly with tumors of neuroendocrine origin, as these have similar cytologic features in various organs. The correct preoperative cytologic diagnosis of metastatic carcinoid tumor in patients without a prior history of cancer and differential diagnosis with MTC are crucial because prognosis, workup and treatment are different in each.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors.

The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with 35S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Traitement médical des tumeurs endocrines digestives

Gastroenteropancreatic neuroendocrine tumors constitute a highly heterogeneous group of tumors with very different prognoses. It is important to distinguish between the well-differentiated neuroendocrine tumors (carcinoid tumors affecting the gastrointestinal tract and pancreatic endocrine tumors), which generally progress slowly, and the poorly differentiated endocrine tumors, which are characterized by being aggressive and of rapid progression. The treatment of the poorly differentiated forms is essentially based on chemotherapy, although prognosis remains poor. The well-differentiated forms require a more complex approach – depending on the site of the primary tumor, staging, the resectability of the lesions, and disease spread – and should therefore be defined within a multidisciplinary setting. The treatment of choice remains surgical excision of the primary tumor and metastases. No additional treatment has been demonstrated to be effective. For functional tumors, symptomatic treatment with a somatostatin analog for carcinoid tumors or a protein pump inhibitor for gastrinoma must be started rapidly to reduce the complications related to hormone secretion. For metastatic disease or for tumors that are not amenable to complete resection, several options can be proposed: careful monitoring, chemoembolization of liver metastases, systemic chemotherapy, or enrollment in therapeutic protocols offering targeted therapies. Stepwise introduction of these various therapies prolongs survival, even in metastatic disease.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors

Summary The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with ³⁵S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Does vinblastine add to the potency of alpha interferon in the treatment of renal cell carcinoma?

The combination of alpha interferon and vinblastine has been reported to yield a response rate of 30-40% in previously untreated patients with metastatic renal cell carcinoma. This combination was given to nine patients with advanced metastatic renal carcinoma after they failed or relapsed on alpha-interferon alone, to attempt to evaluate the role of vinblastine in this combination. Neither complete nor partial response was observed. Two patients had disease stabilization for two and seven months. Our preliminary results suggest that vinblastine did not add to the efficacy of interferon in this group of patients.     

Neuroendocrine tumors of the small intestine and the appendix - management guidelines (recommended by The Polish Network of Neuroendocrine Tumors)

Polish recommendations regarding management of patients suffering from neuroendocrine tumors of small intestine and appendix are presented. Small intestine, especially ileum represent most common origin of these tumors. Majority of them are well differentiated and grow slowly. Rarely, they are less differentiated with fast growth and poor prognosis. Symptoms are atypical, diagnosis could be often accidental. In 4-10% of patients typical symptoms of carcinoid syndrome are present. Chromogranin A is useful in the laboratory diagnostics, and urinary excretion of 5-hydroxyindoloacetic acid is helpful for the diagnostics and monitoring of the disease. Histopathological diagnostics was extensively described. Ultrasound, colonoscopy, capsule endoscopy, baloon enteroscopy, computed tomography, magnetic resonance and somatostatin analogs scintigraphy could be used for the visualization. The treatment of choice in the neuroendocrine tumors of small intestine and appendix is radical or palliative surgery, if possible using endoscopy. Pharmacotherapy consists of biotherapy and chemotherapy. The crucial in biotherapy is somatostatin analogs application, possible in symptomatic treatment of hormonally functioning tumors. This is treatment of choice in carcinoid crisis. Interferon alfa could be applied because of the same indications as somatostatin analogs, except for carcinoid crisis. Chemotherapy is less successful in disseminated or locally advanced intestinal neuroendocrine tumors, so radioisotope therapy should be considered in each case of unresectable tumor.     

Adrenomedullin is a novel marker of tumor progression in neuroendocrine carcinomas.

Adrenomedullin is a multi-functional polypeptide hormone. Its involvement in angiogenesis and vasodilator action support the hypothesis that adrenomedullin may be a secretory product of neuroendocrine tumors and contribute to tumor progression. Plasma levels of adrenomedullin were measured by radioimmunoassay in 46 patients with neuroendocrine carcinomas of the gastroenteropancreatic and bronchial system. Tissue expression of adrenomedullin was studied using monoclonal antibodies on pretreated paraffin embedded tissues in a group of 31 patients. Adrenomedullin plasma levels were significantly elevated in patients compared to healthy age-matched controls (p < 0.001). The highest plasma levels were found in patients with neuroendocrine carcinomas of bronchial, midgut and unknown origin. Patients with progressive disease had higher plasma levels than patients with stable disease (p < 0.001). Of the examined tumor samples, 55 % showed cytoplasmic staining for adrenomedullin > 5 % of the total tumor area. Plasma levels and tissue expression of adrenomedullin did not correlate with functional activity of the tumors or presence of the carcinoid syndrome, but did with tumor progression (p < 0.001 and p < 0.014). In conclusion, plasma and tissue expression of the angiogenic peptide adrenomedullin are predictive of tumor progression in patients with neuroendocrine carcinomas. Adrenomedullin might represent a useful prognostic marker in patients with neuroendocrine carcinomas.