ASO Author Reflections: Indolent Growth and Small Bowel Neuroendocrine Tumor Management.

Surgical treatment is central to management of small bowel neuroendocrine tumors (SBNETs). Current controversies include whether to resect asymptomatic primary tumors in the setting of unresectable metastases, the role of minimally invasive surgery, and how best to incorporate/sequence medical treatments. Low SBNET incidence, long event-times, and variability in disease burden, surgical technique, and institutional treatment preferences remain obstacles to conducting randomized surgical trials for SBNETs. With increasing referral of these patients to high-volume centers, cooperation between experienced SBNET clinicians should allow design of high-quality randomized trials to test new treatments and answer key questions.

The joint IAEA, EANM, and SNMMI practical guidance on peptide receptor radionuclide therapy (PRRNT) in neuroendocrine tumours.

Peptide receptor radionuclide therapy (PRRNT) is a molecularly targeted radiation therapy involving the systemic administration of a radiolabelled peptide designed to target with high affinity and specificity receptors overexpressed on tumours. PRRNT employing the radiotagged somatostatin receptor agonists (90)Y-DOTATOC ([(90)Y-DOTA(0),Tyr(3)]-octreotide) or (177)Lu-DOTATATE ([(177)Lu-DOTA(0),Tyr(3),Thr(8)]-octreotide or [(177)Lu-DOTA(0),Tyr(3)]-octreotate) have been successfully used for the past 15 years to target metastatic or inoperable neuroendocrine tumours expressing the somatostatin receptor subtype 2. Accumulated evidence from clinical experience indicates that these tumours can be subjected to a high absorbed dose which leads to partial or complete objective responses in up to 30 % of treated patients. Survival analyses indicate that patients presenting with high tumour receptor expression at study entry and receiving (177)Lu-DOTATATE or (90)Y-DOTATOC treatment show significantly higher objective responses, leading to longer survival and improved quality of life. Side effects of PRRNT are typically seen in the kidneys and bone marrow. These, however, are usually mild provided adequate protective measures are undertaken. Despite the large body of evidence regarding efficacy and clinical safety, PRRNT is still considered an investigational treatment and its implementation must comply with national legislation, and ethical guidelines concerning human therapeutic investigations. This guidance was formulated based on recent literature and leading experts' opinions. It covers the rationale, indications and contraindications for PRRNT, assessment of treatment response and patient follow-up. This document is aimed at guiding nuclear medicine specialists in selecting likely candidates to receive PRRNT and to deliver the treatment in a safe and effective manner. This document is largely based on the book published through a joint international effort under the auspices of the Nuclear Medicine Section of the International Atomic Energy Agency.

Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis.

Juvenile polyposis (JP; OMIM 174900) is an autosomal dominant gastrointestinal hamartomatous polyposis syndrome in which patients are at risk for developing gastrointestinal cancers. Previous studies have demonstrated a locus for JP mapping to 18q21.1 (ref. 3) and germline mutations in the homolog of the gene for mothers against decapentaplegic, Drosophila, (MADH4, also known as SMAD4) in several JP families. However, mutations in MADH4 are only present in a subset of JP cases, and although mutations in the gene for phosphatase and tensin homolog (PTEN) have been described in a few families, undefined genetic heterogeneity remains. Using a genome-wide screen in four JP kindreds without germline mutations in MADH4 or PTEN, we identified linkage with markers from chromosome 10q22-23 (maximum lod score of 4.74, straight theta=0.00). We found no recombinants using markers developed from the vicinity of the gene for bone morphogenetic protein receptor 1A (BMPR1A), a serine-threonine kinase type I receptor involved in bone morphogenetic protein (BMP) signaling. Genomic sequencing of BMPR1A in each of these JP kindreds disclosed germline nonsense mutations in all affected kindred members but not in normal control individuals. These findings indicate involvement of an additional gene in the transforming growth factor-beta (TGF-beta) superfamily in the genesis of JP, and document an unanticipated function for BMP in colonic epithelial growth control.

Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22-23.

Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome that carries a significant risk for the development of colorectal cancer. Microdeletions of one of the two predisposing genes to JP, BMPR1A, have been associated with a severe form of JP called juvenile polyposis of infancy. Many of these deletions have also been found to contiguously include PTEN, which is the gene responsible for the development of Cowden syndrome. The advent of molecular techniques that localize genomic copy number variations and others that target specific genes such as multiplex-ligation probe analysis has allowed researchers to explore this area further for deletions. Here, we review the literature for microdeletions described on chromosome 10q22-23 in patients with JP and other intestinal polyposis syndromes.

The rate of germline mutations and large deletions of SMAD4 and BMPR1A in juvenile polyposis.

Juvenile polyposis (JPS) is an autosomal dominant syndrome that predisposes individuals to develop gastrointestinal polyps and cancer. Germline point mutations in SMAD4 and BMPR1A have been identified as causing JPS in approximately 40-60% of patients, but few studies have looked at the rate of large deletions. In this study, we determined the overall prevalence of genetic changes of SMAD4 and BMPR1A by sequencing and by screening for larger deletions. DNA was extracted from 102 JPS probands, and each exon and intron-exon boundary of SMAD4 and BMPR1A were sequenced. Coding and non-coding exons of SMAD4 and BMPR1A were screened for deletions with multiplex ligation-dependent probe amplification (MLPA). By sequencing, 20 probands had point mutations of SMAD4 and 22 of BMPR1A. By MLPA, one proband had deletion of most of SMAD4, one of both BMPR1A and PTEN, one of the 5' end of BMPR1A, and another of the 5' end of SMAD4. The overall prevalence of SMAD4 and BMPR1A point mutations and deletions in JPS was 45% in the largest series of patients to date. Large deletions are less frequent in JPS patients, but represent other heritable causes of JPS, which should be screened for in pre-symptomatic genetic testing.

Mutations in the SMAD4/DPC4 gene in juvenile polyposis.

Familial juvenile polyposis is an autosomal dominant disease characterized by a predisposition to hamartomatous polyps and gastrointestinal cancer. Here it is shown that a subset of juvenile polyposis families carry germ line mutations in the gene SMAD4 (also known as DPC4), located on chromosome 18q21.1, that encodes a critical cytoplasmic mediator in the transforming growth factor-beta signaling pathway. The mutant SMAD4 proteins are predicted to be truncated at the carboxyl-terminus and lack sequences required for normal function. These results confirm an important role for SMAD4 in the development of gastrointestinal tumors.

The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function.

Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing.

Concentration-dependent substate behavior of native AMPA receptors.

AMPA-type glutamate receptors mediate most excitatory postsynaptic currents (EPSCs) at central synapses, and their conductance determines in part the size of EPSCs. The conductance of a recombinant AMPA receptor depends on the number of agonist molecules bound to the channel. Here we tested whether native AMPA and kainate receptors show this behavior in outside-out patches from neurons in situ by measuring conductance levels of single channels over a wide range of agonist concentrations. We found that the conductance of AMPA, but not kainate, receptors depended strongly on agonist concentration. Our results suggest that alterations in the glutamate concentration in the synaptic cleft may change the apparent unitary conductance of postsynaptic AMPA receptors.

Regions of alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein.

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.

Heterogeneous conductance levels of native AMPA receptors.

The single-channel properties of AMPA receptors can affect information processing in neurons by influencing the amplitude and kinetics of synaptic currents, yet little is known about the unitary properties of native AMPA receptors in situ. Using whole-cell and outside-out patch-clamp recordings from granule cells in acute cerebellar slices, we found that migrating granule cells begin to express AMPA receptors before they arrive in the internal granule cell layer and receive synaptic input. At saturating agonist concentrations, the open probability of channels in outside-out patches from migrating cells was very high, allowing us to identify patches that contained only one or two active channels. Analysis of the single-channel activity in these patches showed that individual AMPA receptors exhibit as many as four distinguishable conductance levels. The conductance levels observed varied substantially for different channels, although on average the values fell within the range of unitary conductances estimated previously for synaptic AMPA receptors. In contrast to patches from migrating granule cells, we rarely observed directly resolvable single-channel currents in patches excised from the somata of granule cells in the internal granular layer, even though these cells gave large AMPA receptor whole-cell currents. We did, however, detect AMPA receptors with apparent unitary conductances of <1 pS in patches from both migrating and mature granule cells. Our results suggest that granule cells express a heterogeneous population of AMPA receptors, a subset of which are segregated to postsynaptic sites after synaptogenesis.

Subunit interactions and AMPA receptor desensitization.

Most AMPA-type glutamate receptors (GluRs) exhibit rapid and virtually complete desensitization when activated by glutamate, and at some central synapses it is largely desensitization that determines the decay of EPSCs. However, the mechanisms underlying the conformation change that results in desensitization are not fully understood. AMPA receptor subunits that contain a single amino acid substitution have been shown to form homomeric channels that show markedly reduced desensitization. We show here that the coexpression of wild-type GluR1 with one such mutant, GluR1(L497Y), results in heteromeric channels that show desensitization behavior that is intermediate between wild-type and mutant homomers. The relative amplitudes of the multiple exponential components present in the decay of glutamate-evoked currents depended on the relative abundance of wild-type and mutant subunits and were described by the combinatorial distribution of the two types of subunits into tetrameric, but not pentameric, assemblies. Our results are consistent with recent structural data suggesting that AMPA receptors are tetrameric assemblies composed of two dimers.

K-ras mutation in adenomas and carcinomas of the ampulla of vater.

The role of K-ras mutations in the progression of tumors of the ampulla of Vater is not well understood. To study the frequency and timing of K-ras mutations in ampullary tumors, areas of invasive carcinoma and adjacent adenomas were microdissected from paraffin blocks from 96 resected tumors. DNA was extracted, PCR amplification of K-ras exon 1 was performed, and PCR products were sequenced. Statistical analysis of K-ras mutations with respect to patient survival and clinicopathological factors was performed using the chi2 test, log-rank test, and Cox proportional hazard model. Thirty-four of 92 ampullary carcinomas (37.0%) and 25 of 46 adenomas (54.3%) had mutations in K-ras exon 1. Twenty-two of 23 (95.7%) adenomas adjacent to carcinomas with K-ras mutations also had K-ras mutations. The only clinicopathological factor significantly associated with K-ras mutation was tumor size >2 cm (P = = 0.035). Patient survival did not correlate with the K-ras mutation status (P = 0.31). We conclude that K-ras mutations are frequent in both adenomas and carcinomas of the ampulla of Vater and appear to occur as an early genetic event. The spectrum of mutations is similar to that observed in colorectal neoplasms, and these do not significantly correlate with patient survival.

The prevalence of MADH4 and BMPR1A mutations in juvenile polyposis and absence of BMPR2, BMPR1B, and ACVR1 mutations.

BACKGROUND: Juvenile polyposis (JP) is an autosomal dominant syndrome predisposing to colorectal and gastric cancer. We have identified mutations in two genes causing JP, MADH4 and bone morphogenetic protein receptor 1A (BMPR1A): both are involved in bone morphogenetic protein (BMP) mediated signalling and are members of the TGF-beta superfamily. This study determined the prevalence of mutations in MADH4 and BMPR1A, as well as three other BMP/activin pathway candidate genes in a large number of JP patients. METHODS: DNA was extracted from the blood of JP patients and used for PCR amplification of each exon of these five genes, using primers flanking each intron-exon boundary. Mutations were determined by comparison to wild type sequences using sequence analysis software. A total of 77 JP cases were sequenced for mutations in the MADH4, BMPR1A, BMPR1B, BMPR2, and/or ACVR1 (activin A receptor) genes. The latter three genes were analysed when MADH4 and BMPR1A sequencing found no mutations. RESULTS: Germline MADH4 mutations were found in 14 cases (18.2%) and BMPR1A mutations in 16 cases (20.8%). No mutations were found in BMPR1B, BMPR2, or ACVR1 in 32 MADH4 and BMPR1A mutation negative cases. DISCUSSION: In the largest series of JP patients reported to date, the prevalence of germline MADH4 and BMPR1A mutations is approximately 20% for each gene. Since mutations were not found in more than half the JP patients, either additional genes predisposing to JP remain to be discovered, or alternate means of inactivation of the two known genes are responsible for these JP cases.

Cloning of the cDNA encoding the sodium channel beta 1 subunit from rabbit.

Here, we report the nucleotide (nt) sequence of the cDNA encoding the sodium channel beta 1 subunit from rabbit (o beta 1). Cloning of the o beta 1 cDNA was accomplished by reverse transcription-polymerase chain reaction using rabbit brain RNA as a template for cDNA synthesis. The nt sequence of the o beta 1 cDNA predicts a 218-amino-acid polypeptide which is 96.3 and 97.3% identical to the beta 1 from human (h beta 1) and rat (r beta 1), respectively.

Responses of rat dorsal horn neurons to natural stimulation and to iontophoretically applied norepinephrine.

Extracellular recordings were obtained of 177 neurons throughout the lumbar spinal dorsal horn of urethane- or halothane-anesthetized rats. These neurons all responded to iontophoretically applied L-glutamate and their responses to natural stimulation of the ipsilateral hindlimb were characterized. Iontophoretically applied norepinephrine was tested on 94 of these neurons. Fifty-one neurons were inhibited and 22 were excited. Norepinephrine produced a biphasic inhibitory/excitatory effect on nine neurons. Norepinephrine was exclusively inhibitory on superficial dorsal horn neurons that responded only to innocuous brush and touch and on neurons in the nucleus proprius that responded to brush, touch, and noxious skin pinch. Norepinephrine excited some superficial brush/touch/pinch neurons and produced short inhibitions that were followed by prolonged excitations of some nucleus proprius neurons that responded only to noxious skin pinch. Neurons in the base of the dorsal horn that responded to low-threshold proprioceptive stimulation were excited by norepinephrine. Both the inhibitory and excitatory effects of norepinephrine were stereoselective, but they were not blocked by receptor subtype-selective antagonists. Desensitization to norepinephrine occurred for 30% of the neurons. This study demonstrates that the inhibitory effects of norepinephrine on rat dorsal horn neurons are not restricted to neurons that are responsive to noxious stimuli and that some of these neurons are primarily excited by norepinephrine. The excitatory effects of norepinephrine on low-threshold proprioceptive neurons may contribute to norepinephrine's known enhancement of spinal flexor reflex activity.

Midbrain deafness following head injury.

A patient is described who became deaf following a head injury. Postmortem examination revealed bilateral lesions of the lateral lemnisci and inferior colliculi. The clinical pattern of midbrain deafness is examined and compared with syndromes of cortical and peripheral auditory impairment.

The effect of unilateral dorsal root ganglionectomies or ventral rhizotomies on alpha 2-adrenoceptor binding to, and the substance P, enkephalin, and neurotensin content of, the cat lumbar spinal cord.

The density of alpha 2-adrenoceptor binding sites and the content of substance P, enkephalins, and neurotensin were determined in quadrants of the lumbar spinal enlargement of control cats and of cats upon which either unilateral dorsal root ganglionectomies or unilateral ventral rhizotomies had been performed. The performance of unilateral dorsal root ganglionectomies resulted in a significant decrease (45-55%) of substance P content in the ipsilateral dorsal horn 7 and 21 days postoperatively. The concentration of alpha 2-adrenoceptor binding sites ([3H]rauwolscine Bmax) in the ipsilateral dorsal horn was consistently and significantly decreased at these same postganglionectomy times (20% reduced relative to the contralateral dorsal horn). Enkephalin content 7 and 21 days after ganglionectomies was not significantly different from control, whereas the neurotensin content of the ipsilateral dorsal horn was significantly increased in the 21-day survival cats. The performance of unilateral ventral rhizotomies did not produce any statistically significant changes in the density of alpha 2-adrenoceptor binding sites or in the substance P or enkephalin content of any spinal quadrant. The neurotensin content of both the ipsilateral dorsal and ipsilateral ventral quadrants of the ventral rhizotomized cats was significantly increased. The significant decrease of alpha 2-adrenoceptor binding site concentration in the ipsilateral dorsal horn after unilateral dorsal root ganglionectomies suggests that approximately 20% of the alpha 2-adrenoceptors present within the cat lumbar spinal dorsal gray are located on the axons or terminals of primary sensory afferents. Consistent with this interpretation of the ganglionectomy results, we found significant levels of saturable [3H]rauwolscine binding to homogenates of the cat L4-Sl spinal dorsal root ganglia. Because alpha 2-adrenoceptor binding sites in the ipsilateral ventral lumbar spinal gray were not significantly reduced after unilateral ventral rhizotomies, our results provide no evidence for the location of alpha 2-adrenoceptor on lumbar spinal motoneurons.

Intrathecal 6-hydroxydopamine or cervical spinal hemisection reduces norepinephrine content, but not the density of alpha 2-adrenoceptors, in the cat lumbar spinal enlargement.

The effect of the intrathecal administration of the catecholaminergic neurotoxin 6-hydroxydopamine, or of hemisection of the spinal cord at the Cl level, on the density of alpha 2-adrenoceptors and on the norepinephrine, dopamine, and serotonin content in the cat lumbar spinal enlargement was determined 2, 7 or 21 days after performance of each type of lesion. The intrathecal administration of 6-hydroxydopamine produced a time-dependent reduction of norepinephrine content in the cat lumbar spinal enlargement (95% reduction at 21 days) without significantly altering the serotonin content in this same tissue of the same cats. The dopamine content of the dorsal horn was not changed significantly, whereas ventral horn dopamine content was depleted after intrathecal 6-hydroxydopamine. alpha 2-Adrenoceptor binding site density was not significantly different from control either 2 or 21 days after 6-hydroxydopamine, but was increased significantly (50%) over the control density 7 days after 6-hydroxydopamine. Hemisection of the cervical spinal cord produced a bilateral 40-60% reduction of norepinephrine content in both the dorsal and ventral horns of the cat lumbar spinal enlargement 7 and 21 days later. Cervical hemisection did not significantly alter the alpha 2-adrenoceptor binding site density in these same cats either 2, 7, or 21 days after performance of the lesion. It is concluded that alpha 2-adrenoceptors located on the terminals of descending noradrenergic or other spinopetal fibers do not represent a significant fraction of the total population of alpha 2-adrenoceptors present in the dorsal or ventral cat lumbar enlargement.

Introduction of the glutamate receptor subunit 1 into motor neurons in vitro and in vivo using a recombinant herpes simplex virus.

We developed and characterized a recombinant herpes simplex virus vector and used it to introduce the complementary DNA encoding glutamate receptor subunit 1 flip into postmitotic motor neurons. Infection of purified motor neurons in vitro with this vector resulted in selective, high-level expression of glutamate receptor subunit 1 immunoreactivity in nearly 100% of the neurons. Patch-clamp experiments demonstrated that the protein product of the glutamate receptor subunit 1 flip transgene assembles into functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels. Herpes simplex virus-glutamate receptor subunit 1 flip was introduced into spinal cord cells by direct injection into the ventral horn and selectively into motor neurons by sciatic nerve injection. High levels of expression were sustained for at least one week and were accompanied by changes in the ionic permeability of AMPA receptors in transgene-expressing neurons. Throughout the first week of infection, there was little evidence for toxicity. Herpes simplex virus provides a versatile tool for manipulating the glutamate receptor phenotype of postmitotic neurons and will permit study of the role of individual glutamate receptor subunits in neuronal physiology and pathophysiology.

Expression and heteromeric interactions of non-N-methyl-D-aspartate glutamate receptor subunits in the developing and adult cerebellum.

The localization and expression of ionotropic non-N-methyl-D-aspartate glutamate receptors (GluR) were investigated in the developing and adult rat cerebellum using subunit-specific polyclonal antibodies for immunocytochemical, immunoblot and immunoprecipitation studies. In P7 animals, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor immunoreactivity was detected in all layers of the cerebellar cortex with the exception of the external granule cell layer. Antibodies against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR4 gave strong immunoreactive staining of Bergmann glia in both young and adult animals, and both antibodies showed prominent staining of the molecular layer in the adult cerebellum. Dense immunoreactive staining of Purkinje cell somata and dendrites was obtained with anti-GluR2/3/4c in both the developing and adult cerebellum. Whereas each of the three alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antibodies stained the internal, but not the external, granule cell layer, immunostaining for the kainate-type subunits GluR6/7 and KA2 was detected in both the external and internal granule cell layers. as well as in the molecular layer in both P7 and adult cerebellum. Immunoblot analysis of total cerebellar protein indicated that the level of GluR4 expression increased 15-fold from P1 to P18, whereas the expression of the KA2 subunit protein was nine-fold lower in adult cerebellum than it was at P1. The expression of GluR1 increased moderately (two-fold) from P1 to adult. Subunit interactions between GluR1 and GluR4, as well as between GluR6/7 and KA2, were demonstrated in immunoprecipitation experiments; and the GluR4 and KA2 subunits appear to be present exclusively in heteromeric assemblies with GluR1 and GluR6/7, respectively. The results show that the various alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type subunits are differentially expressed during cerebellar development and further define the possible subunit composition of non-N-methyl-D-aspartate receptors in the major cerebellar cell types.