A meta-analysis of the accuracy of a neuroendocrine tumor mRNA genomic biomarker (NETest) in blood.

BACKGROUND: The lack of an accurate blood biomarker in neuroendocrine tumor (NET) disease has hindered management. The advance of genomic medicine and the development of molecular biomarkers has provided a strategy-liquid biopsy-to facilitate real-time management. We reviewed the role of a blood mRNA-based NET biomarker, the NETest, as an in vitro diagnostic (IVD). PATIENTS AND METHODS: A systematic review of the literature using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was undertaken. The methodological quality was evaluated using the QUADAS-2 tool. We identified ten original scientific papers that met the inclusion criteria. These were assessed by qualitative analysis and thereafter meta-analysis. Data were pooled and a median [95% confidence interval (CI)] diagnostic odds ratio (DOR), positive likelihood ratio (+LR), and negative likelihood ratio (-LR) were calculated. For the meta-analysis, a generic inverse variance method was undertaken using the accuracy and area under the curve (AUC) data. RESULTS: The ten studies exhibited moderate to high methodological quality. They evaluated NETest usage both as a diagnostic and as a monitoring tool. The meta-analysis identified the diagnostic accuracy of the NETest to be 95%-96% with a mean DOR of 5 853, +LR of 195, and -LR of 0.06. The NETest was 84.5%-85.5% accurate in differentiating stable disease from progressive disease. As a marker of natural history, the accuracy was 91.5%-97.8%. As an interventional/response biomarker, the accuracy was 93.7%-97.4%. The pooled AUC for the NETest was 0.954 ± 0.005, with a z-statistic of 175.06 (P < 0.001). CONCLUSIONS: The NETest is an accurate biomarker suitable for clinical use in NET disease management. The meta-analysis supports the utility of the NETest as an IVD to establish a diagnosis and monitor therapeutic efficacy. The use of this as a biomarker provides information relevant to NET management consistent with observations regarding utility of liquid biopsies in other oncological disciplines.

Rab25 as a tumour suppressor in colon carcinogenesis.

Recent investigations have increasingly focussed attention on the roles of intracellular vesicle trafficking in the regulation of epithelial polarity and transformation. Rab25, an epithelial-specific member of the Rab family of small GTPases, has been associated with several epithelial cancers. Whereas Rab25 overexpression is associated with ovarian cancer aggressive behaviour, Rab25 expression is decreased in human colon cancers independent of stage. Recent studies of mouse models of intestinal and colonic neoplasia have demonstrated that Rab25 deficiency markedly promotes the development of neoplasia. Some of these effects appear related to alterations in ß1-integrin trafficking to the cell surface. These findings all suggest that Rab25 is a tumour suppressor for colonic neoplasia.

Targeted disruption of the Kvlqt1 gene causes deafness and gastric hyperplasia in mice.

The KvLQT1 gene encodes a voltage-gated potassium channel. Mutations in KvLQT1 underlie the dominantly transmitted Ward-Romano long QT syndrome, which causes cardiac arrhythmia, and the recessively transmitted Jervell and Lange-Nielsen syndrome, which causes both cardiac arrhythmia and congenital deafness. KvLQT1 is also disrupted by balanced germline chromosomal rearrangements in patients with Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth and cancer. Because of the diverse human disorders and organ systems affected by this gene, we developed an animal model by inactivating the murine Kvlqt1. No electrocardiographic abnormalities were observed. However, homozygous mice exhibited complete deafness, as well as circular movement and repetitive falling, suggesting imbalance. Histochemical study revealed severe anatomic disruption of the cochlear and vestibular end organs, suggesting that Kvlqt1 is essential for normal development of the inner ear. Surprisingly, homozygous mice also displayed threefold enlargement by weight of the stomach resulting from mucous neck cell hyperplasia. Finally, there were no features of BWS, suggesting that Kvlqt1 is not responsible for BWS.

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells.

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

Myosin vb is associated with plasma membrane recycling systems.

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Distribution of A-kinase anchoring proteins in parietal cells.

Recent investigations have suggested that subcellular compartmentalization of second messenger responsive enzyme systems may be responsible for specific patterns of cellular activation. The type II cAMP-dependent kinase (A-kinase) is localized to particular subcellular domains through the binding of the regulatory subunit (RII) dimer to A-kinase anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have investigated the presence of AKAPs throughout the gastrointestinal tract, with specific emphasis focused on the gastric parietal cell. All gastrointestinal tissues contained at least one detectable AKAP (60 kDa), with five AKAPs (50-140 kDa) in fundic and antral mucosa. Isolated gastric glands contained four AKAPs. Two AKAPs (50 and 78 kDa) were detected in purified parietal cells, with the 78 kDa AKAP (AKAP78) specific to parietal cell enriched populations. RII-binding to all of these AKAPs was abolished by preincubation of [32P]RII with a synthetic peptide representing the RII-binding region of the AKAP, HT-31. AKAP78 was distributed throughout all membrane fractions of subfractionated parietal cells, with the largest amount of RII-binding detected in the light membrane fraction. Identification of A-kinase regulatory subunits by photoaffinity labeling with 8-azido-[32P]cAMP demonstrated that RII segregated into the same parietal cell subfractions as AKAP78. A majority (approximately 60%) of AKAP78 was detected in the Triton X-100-insoluble fraction, suggesting that this protein resides in a cytoskeletal domain. AKAP78 may be involved in localizing the type II A-kinase to specific intracellular locations in the parietal cell.

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

Distribution and immunocytochemical colocalization of peptide YY and enteroglucagon in endocrine cells of the rabbit colon.

Peptide YY (PYY) is 36 amino acid peptide hormone present in high concentrations in the colon where it is colocalized with enteroglucagon in L cells. A selective release of PYY and enteroglucagon from the rabbit colon has been described, raising the question of the exact localization of the two hormones in the rabbit colon. We have therefore examined the distribution of PYY and enteroglucagon as well as somatostatin in the rabbit colon using RIA and electron microscopic immunocytochemistry. PYY and enteroglucagon were present in high concentrations in the colorectal mucosa with peak concentrations in the left colon (PYY 544 +/- 87 pmol/g, enteroglucagon 152 +/- 10 pmol/g). Electron microscopic examination of the colonic mucosa demonstrated a large population (65%) of EC cells, a moderate population (30%) of L cells, and a small population (5%) of D cells. By immunogold labeling serotonin was localized to EC cells, PYY and enteroglucagon to L cells, and somatostatin to the D cell. Double immunogold labeling revealed PYY and enteroglucagon in all L cells examined (93 cells). A majority of the secretory granules (83%) were labeled by both PYY and glucagon antibodies, whereas a significant portion of granules (15%) was labeled by the PYY antibodies alone. The results demonstrate that L cells are the sole source of PYY and enteroglucagon in the rabbit colon and that L cells contain different populations of secretory granules. The existence of different secretory granules in L cells may explain the selective release of PYY and enteroglucagon observed in the rabbit colon.

Identification of somatostatin and gastrin receptors on enterochromaffin-like cells from Mastomys gastric tumors.

Histamine-secreting enterochromaffin-like (ECL) cells of the gastric fundus of the Mastomys can develop into solid ECL cell tumors, either spontaneously or after induction by acid inhibition. We used this tumor tissue to perform in vitro receptor autoradiography for somatostatin (SS), gastrin, and substance-P, using, respectively, [125I]Tyr3-octreotide, [125I]gastrin-17, and [125I]Bolton-Hunter-labeled substance-P as radioligands. A high density of SS receptors was found in the nontumor fundic mucosa, where gastrin receptors were only barely detectable. However, in the group of spontaneously developing ECL cell tumors, a high density of SS and gastrin receptors was observed, homogeneously distributed in the tumor tissue. In addition, the loxtidine-induced ECL cell tumors expressed a high density of SS and gastrin receptors. The receptors were specific for the respective peptide and of high affinity, with a dissociation constant (Kd) of 0.90 nM for SS receptor and 0.87 nM for gastrin receptors. No substance-P receptors were detected on the ECL cell tumors, although they were present in the muscle layers of the Mastomys gastric fundus. These results demonstrate that ECL-derived tumors express receptors for both SS and gastrin. This observation is consistent with the proposal that there is substantial regulation of the histamine-producing ECL cell by SS and gastrin. The presence of gastrin receptors is compatible with a role for gastrin as a trophic factor in ECL cell hyperplasia and neoplasia. The expression of SS receptors may be of diagnostic and therapeutic relevance in the regulation of ECL function and neoplastic transformation.

Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

Association of calcium/calmodulin-dependent kinase with cytoskeletal preparations: phosphorylation of tubulin, neurofilament, and microtubule-associated proteins.

Calcium and calmodulin have been implicated in the regulation of cytoskeletal function. In this report, we demonstrate that microtubule preparations from rat brain contain a calcium/calmodulin-dependent protein kinase that phosphorylates endogenous MAP-2, tubulin, synapsin I, and neurofilament proteins. This cytoskeletal-associated kinase has been biochemically characterized and shown to be identical to Type II calcium/calmodulin-dependent protein kinase (CaM kinase II). The subunits of CaM kinase II represented major calmodulin-binding proteins in cytoskeletal preparations. A monoclonal antibody against the 52000 Da subunit of CaM kinase II specifically labeled cytoskeletal elements in cortical neurons. These results indicate that CaM kinase II is associated with the neuronal cytoskeleton and may play a role in mediating some of the effects of calcium on cytoskeletal function.

Gastrin-dependent inhibitory effects of octreotide on the genesis of gastric ECLomas.

BACKGROUND: The efficacy of octreotide in the regulation of endocrine tumor secretion and symptomatology has been well documented. Its effects on neuroendocrine tumor generation and cell proliferation are less well understood. The purpose of this study was to determine if blockade of somatostatin receptors by octreotide would alter gastrin levels and influence enterochromaffin-like (ECL) cell proliferation. METHODS: The well-established gastric ECLoma model of the rodent, mastomys, was used. Animals received loxtidine (1 mg/kg/day), an irreversible H2 blocker, and subcutaneous slow release, octreotide pellet implants (150 or 300 micrograms/kg/day) or placebo pellets for a 4-month period. RESULTS: Control parameters for gastric mucosal thickness, plasma gastrin level, ECL cell density, and bromodeoxyuridine-positive cells were 517 +/- 20 microns, 46.1 +/- 11.4 pmol/L, 7.4 +/- 0.9 cells/visual field, and 13.8 +/- 2.6 cells/visual field, respectively. After loxtidine-placebo treatment all values were significantly increased (p < 0.05; 883 +/- 70 microns, 192.8 +/- 10.6 pmol/L, 97 +/- 16.2 cells/visual field, and 51.7 +/- 19.2 cells/visual field, respectively). High dose octreotide significantly inhibited all parameters (668 +/- 3.5 microns, 66.2 +/- 20.5 pmol/L, 37.0 +/- 8.0 cells/visual field, and 10.9 +/- 2.2 cells/visual field; p < 0.05). Low dose octreotide failed to significantly inhibit ECL cell density mucosal thickness, or cell proliferation. CONCLUSIONS: Irreversible H2 receptor blockade results in hypergastrinemia and ECL cell tumor generation. Hypergastrinemia, ECL cell hyperplasia, and cell proliferation are significantly inhibited by in vivo blockade of somatostatin receptors by administration of octreotide.

Inhibition of parietal cell H+ secretion by transforming growth factor alpha: a possible autocrine regulatory mechanism.

Transforming growth factor-alpha (TGF alpha) and TGF alpha/epidermal growth factor receptor messenger ribonucleic acid have recently been demonstrated in isolated parietal cells. The aim of this study was to investigate the effects of TGF alpha on basal and stimulated secretion in vitro with the isolated rabbit parietal cell model. Acid secretion was assessed indirectly with cell uptake of carbon 14-labeled aminopyrine [( 14C]AP). TGF alpha (10(-11) to 10(-7) mol/L) had no effect on unstimulated [14C]AP uptake. TGF alpha dose dependently inhibited histamine (10(-5) to 10(-6) mol/L)-stimulated but not forskolin (10(-5) to 10(-7) mol/L)-stimulated [14C]AP uptake. This effect on histamine-stimulated activation was reversed by pertussis toxin (200 ng/ml) before incubation. TGF alpha had no effect on carbachol (10(-5) to 10(-6) mol/L)-stimulated [14C]AP uptake. Specific HCO3-buffer studies demonstrated that these observations were independent of extracellular buffer and possible TGF alpha effects on intracellular pH. Our data indicate that TGF alpha inhibits acid secretion by specifically uncoupling histamine/cyclic adenosine monophosphate transduction at the guanosine triphosphate-binding protein. TGF alpha, unlike epidermal growth factor, has no effect on carbachol stimulation, which suggests a qualitative difference between the biologic actions of TGF alpha and epidermal growth factor. Possible autocrine-paracrine modulation of histamine stimulation by TGF alpha invokes a novel regulatory mechanism of parietal cell secretion.

A manganese-dependent protein kinase in gastric gland cytosol.

A protein kinase activity completely dependent on Mn2+ was studied in the cytosolic fraction from rabbit-isolated gastric glands. The manganese-dependent protein kinase (MNPK) activity phosphorylated major 33 kd (pp33) and minor 140 kd (pp140) endogenous proteins. The MNPK activity displayed a Kact for Mn2+ of 7.5 mmol/L. MNPK showed no preference for adenosine triphosphate or guanosine triphosphate with a Kact of 10 mumol/L for both. The kinase was differentiated from other known kinases since calmodulin, cyclic adenosine monophosphate, and phospholipids failed to stimulate pp33 phosphorylation. Furthermore, the protein kinase inhibitors trifluoperazine, the Walsh inhibitor protein, and heparin, as well as the phosphatase inhibitor p-nitrophenylphosphate failed to alter MNPK activity. The results indicate that novel MNPK activity is present in gastric gland cytosol. Elucidation of intracellular protein kinase activities may provide insights into the regulation of gastric secretory process.

A somatodendritic distribution of Rab11 in rabbit brain neurons.

Comparisons of subcellular targeting in neurons and epithelial cells have led to suggestions that apical epithelial antigens localize to axons and nerve terminals. We have studied the distribution in brain of the small GTP-binding protein, Rab11, which is localized to apical vesicular populations in epithelial cells. Sections of rabbit brain were examined by immunohistochemistry using a monoclonal antibody against rabbit Rab11. Rab11 immunoreactivity was present exclusively in neurons. In all regions examined, including forebrain, cerebellum, thalamus and brainstem, Rab11 immunoreactivity was observed in cell bodies and dendrites. No staining was observed in hippocampal neurons. These results indicate that the distribution of Rab11 does not support previous suggestions that apical epithelial markers should localize to axons and synaptic endings.

Intrahepatic cholangiojejunostomy as a palliative procedure in primary sclerosing cholangitis.

Primary sclerosing cholangitis is an idiopathic disease characterized by progressive diffuse stricture of extrahepatic and intrahepatic bile ducts. Eighteen patients with end-stage symptoms of primary sclerosing cholangitis were evaluated during a 10-year period from 1976 to 1986. Nine patients presented with disease amenable to intrahepatic cholangiojejunostomy. All patients presented with elevated liver function test results, and six of nine patients had a history of ulcerative colitis. The mean survival after intrahepatic cholangiojejunostomy was 3.9 years (range, 4 months to 10 years). Two of three of the patients with biliary cirrhosis died within 1 year after surgery. Four of nine patients remain alive today, with a mean survival of 4.7 years. For patients with end-stage primary sclerosing cholangitis, intrahepatic cholangiojejunostomy provides effective surgical palliation in those without secondary biliary cirrhosis.

Neuropeptide Y inhibition of vasoactive intestinal polypeptide-stimulated ion transport in the rabbit distal colon.

We have studied the effect of neuropeptide Y on basal and vasoactive intestinal polypeptide-stimulated changes in the short-circuit current of strips of colonic mucosa from New Zealand white rabbits mounted in Ussing chambers. When administered to the basolateral surface, neuropeptide Y is found to decrease basal short-circuit current. Neuropeptide Y inhibits vasoactive intestinal peptide-stimulated increases in short-circuit current in a concentration-dependent fashion by a tetrodotoxin-insensitive mechanism. Also, neuropeptide Y inhibited increases in short-circuit current produced by direct stimulation of adenylate cyclase with forskolin. Furthermore, neuropeptide Y prevents vasoactive intestinal peptide-stimulated increases in tissue cyclic adenosine monophosphate levels. These results indicate that neuropeptide Y administered to the basolateral membrane inhibits vasoactive intestinal peptide-stimulated short-circuit current changes by a tetrodotoxin-insensitive mechanism that decreases tissue levels of cyclic adenosine 3',5'-monophosphate.

Transforming growth factor-alpha (TGF alpha) inhibition of parietal cell secretion: structural requirements for activity.

Parietal cells of the gastric fundus produce transforming growth factor-alpha (TGF alpha) which functions as a potent inhibitor of acid secretion. We have previously demonstrated that TGF alpha can inhibit aminopyrine uptake in isolated rabbit parietal cells. In this study, we have evaluated the components of TGF alpha structure which determine its ability to inhibit parietal cell function. Both human and rat TGF alpha inhibited histamine stimulation by increasing the EC50 for agonist stimulation. Three fragments containing the third loop domain of TGF alpha (rat TGF alpha 34-43, human TGF alpha 34-43 and human TGF alpha 34-50) all inhibited histamine stimulation with IC50 values 20, 33 and 4-fold higher, respectively, than that of the native molecule. Rat TGF alpha inhibited carbachol stimulation throughout an agonist dose response. Human TGF alpha was only effective in inhibiting carbachol if incubations were performed in the presence of air rather than 100% O2. In air incubation, all three of the TGF alpha fragments inhibited carbachol stimulation but, in contrast to the effects on histamine, the peptides all were virtually equipotent with the native molecule. The human sequence fragments, like the native human TGF alpha, elicited no inhibition when incubations were performed in the presence of 100% O2. The results suggest that there are pharmacological differences in the response of isolated parietal cells to TGF alpha-mediated inhibition of histamine and carbachol. In addition, in contrast with previous investigations on the mitogenic action of TGF alpha, third loop fragments of TGF alpha retain the capacity to inhibit aminopyrine accumulation.