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.

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.

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.

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.

Germline SMAD4 or BMPR1A mutations and phenotype of juvenile polyposis.

BACKGROUND: Juvenile polyposis (JP) is an inherited condition predisposing to upper gastrointestinal (UGI) polyps and colorectal cancer. Two genes are known to predispose to JP, SMAD4 and bone morphogenetic protein receptor type 1A (BMPR1A). The object of this study was to determine the differences in phenotype of patients with SMAD4 or BMPR1A mutations (MUT+) compared with those without (MUT-). METHODS: DNA was extracted from 54 JP probands and used for polymerase chain reaction of all exons of SMAD4 and BMPR1A. Products were then sequenced and analyzed for mutations. Medical record data were used to create a JP database, and statistical analysis was performed using Fisher's exact and unpaired t-tests. RESULTS: Nine of 54 patients had germline SMAD4 mutations, 13 had BMPR1A mutations, and 32 had neither. There were no significant differences between SMAD4+ and BMPR1A+ cases in terms of clinical factors examined, except for a family history of UGI involvement (P <.01). There was a higher prevalence of familial cases in MUT+ patients (P =.09), >10 lower gastrointestinal polyps (P =.06), and frequency of family history of gastrointestinal cancer compared with MUT- patients (P =.01). CONCLUSIONS: Patients with germline SMAD4 or BMPR1A mutations have a more prominent JP phenotype than those without, and SMAD4 mutations predispose to UGI polyposis.

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.

Small bowel sarcoma: analysis of survival from the National Cancer Data Base.

BACKGROUND: Small bowel sarcomas (SBS) are rare, accounting for 10% of small bowel cancers. As a result, few studies of SBS have had enough patients to accurately define their natural history and to determine the factors that have an impact on patient survival. The objective of this study was to examine patient and tumor factors in SBS and to determine prognostic factors for disease-specific survival (DSS) using the National Cancer Data Base. METHODS: Data from the National Cancer Data Base for patients diagnosed with primary SBS between 1985 and 1995 were analyzed. The chi2 statistic was used to determine significant differences between groups of patient, tumor, and treatment factors. DSS was calculated for patients diagnosed between 1985 and 1990. Significant differences in survival were determined using the Wilcoxon statistic for univariate analyses and by Cox regression in multivariate analyses. RESULTS: Of 14,253 small bowel tumors diagnosed between 1985 and 1995, sarcomas represented 10.1%. Overall, 5-year DSS was 38.9%, with a median survival of 34.1 months (n = 590). By univariate analysis, patient age, sex, tumor size, tumor grade, histologic type, general summary stage, nodal status, and whether cancer-directed surgery was performed were significantly correlated with DSS. In multivariate analysis, tumor size <5 cm, leiomyosarcoma histology, and localized disease were found to be significant favorable prognostic factors for DSS. CONCLUSIONS: SBS are rare tumors that are challenging in terms of their histopathologic classification, grading, and staging. Patients with SBS were treated predominantly by surgery, with a minority receiving adjuvant therapy. Tumor size, histologic type, and general summary stage were independent prognostic factors for 5-year DSS in patients with SBS, which is improved relative to 5-year DSS seen in patients with small bowel adenocarcinoma.

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.

Minimally invasive parathyroid surgery.

More surgeons are performing unilateral exploration for primary hyperparathyroidism (HPT) than ever before. This article reviews the factors that have led to the trend toward less invasive surgery. Discussion includes the history of unilateral exploration for HPT, the advent of magnetic resonance sestamibi imaging, and the development of intraoperative assays for parathyroid hormone. Results of minimally invasive techniques, including radio-guided parathyroidectomy, endoscopic parathyroidectomy, and outpatient parathyroidectomy, also are presented.

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.

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.

Development of glutamatergic synaptic activity in cultured spinal neurons.

The development of glutamatergic synapses involves a sequence of events that are still not well understood. We have studied the time course of the development of glutamatergic synapses in cultured spinal neurons by characterizing spontaneous synaptic currents recorded from cells maintained in vitro for different times. At short times in culture (2 days in vitro; DIV2), spontaneous synaptic activity consisted almost solely of N-methyl-D-aspartate (NMDA) receptor (NMDAR) openings. In contrast, older neurons (DIV5 to DIV8) displayed clear alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated synaptic currents, while the NMDAR-mediated activity remained small. Between 8 and 14 days in vitro there was a large increase in the density of synaptically activated NMDARs, although there was no significant increase in the density of the NMDAR-mediated current activated by exogenous glutamate. The results indicate that there is a switch in NMDAR targeting from somatic to synaptic regions during the course of the second in vitro week. Finally, our results support the conclusion that the spontaneous synaptic activity displayed in culture depends on ongoing NMDAR-mediated activity, even when the expression of synaptic NMDARs is low.

The American College of Surgeons Commission on Cancer and the American Cancer Society. Adenocarcinoma of the small bowel: review of the National Cancer Data Base, 1985-1995.

BACKGROUND: Small bowel adenocarcinoma (SBA) accounts for 2% of gastrointestinal (GI) tumors and 1% of GI cancer deaths. The objective of this study was to review the National Cancer Data Base (NCDB) to identify case-mix characteristics, patterns of treatment, and factors influencing survival of patients with SBA. METHODS: NCDB data from patients diagnosed with primary SBA between 1985-1995 were analyzed. Chi-square statistics were used to compare differences between groups. Disease specific survival (DSS) was calculated using the life table method for patients diagnosed between 1985-1990; univariate differences in survival were compared using the Wilcoxon statistic, and multivariate analyses were performed using a Cox regression model. RESULTS: There were 4995 SBA cases reported to the NCDB between 1985-1995, 55% of which occurred in the duodenum, 18% in the jejunum, 13% in the ileum, and 14% in nonspecified sites. The overall 5-year DSS was 30.5%, with a median survival of 19.7 months. By multivariate analysis, factors significantly correlated with DSS included patient age, tumor site, disease stage, and whether cancer-directed surgery was performed. CONCLUSIONS: SBA is found most commonly in the duodenum, and patient DSS is reduced at this site compared with those patients with jejunal or ileal tumors. This reduction in survival was associated with a lower percentage of cancer-directed surgery. Patients age > 75 years had a reduced DSS and more duodenal tumors, and were less frequently treated by cancer-directed surgery than their younger counterparts. This study reflects the experience with SBA from a large cross-section of U.S. hospitals, allowing for the identification of prognostic factors and providing a reference with which results from single institutions may be compared.

Direct genetic testing for Smad4 mutations in patients at risk for juvenile polyposis.

BACKGROUND: The identification of germline mutations in juvenile polyposis (JP) families has made presymptomatic genetic testing possible. In this study we report the results of genetic testing in two large JP families and develop an algorithm for the clinical management of these patients. METHODS: DNA was extracted from 55 members of 2 JP kindreds, and the Smad4 mutations in the germline were determined by direct sequencing. All family members were then tested for mutations with use of single-strand conformational polymorphism analysis and were invited for genetic counseling. RESULTS: All 18 affected members of both kindreds had a 4-bp deletion in exon 9 of the Smad4 gene. In 30 patients at risk for JP, 17 had previously had negative endoscopic screening results and 13 had never been screened. Five patients at risk had inherited germline Smad4 mutations. Two carriers have had hematochezia but have not been screened, whereas 3 were asymptomatic. The mean age of carriers was 29.8 years (range 9.1-49.5 years), whereas that of noncarriers was 41.0 years (range 8.1-76.5 years). CONCLUSIONS: Compliance has been a problem with endoscopic screening for JP. With genetic testing non-carriers may no longer require frequent screening endoscopy, whereas gene carriers can be targeted for close endoscopic surveillance and early intervention to prevent the development of gastrointestinal cancers. Direct genetic testing significantly improves the presymptomatic diagnosis of gene carriers in JP families with Smad4 mutations.

Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells.

1. Although glutamate receptors have been shown to be involved in neuronal maturation, a developmental role for kainate-type receptors has not been described. In addition, the single-channel properties of native kainate receptors have not been studied in situ. We have characterized the electrophysiological properties of native kainate receptors of granule cell neurons at two distinct stages in postnatal development, using whole-cell and outside-out patch-clamp recordings in acute cerebellar slices. 2. Kainate-type currents were detected in both immature and mature granule cells. However, noise analysis showed that the apparent unitary conductance of kainate-type channels is significantly higher in proliferating than post-migratory granule cells. The conductance and rectification behaviour of the channels in immature granule cells indicate that they contain unedited GluR5 and GluR6 subunits and are likely to be calcium permeable. 3. Single-channel kainate-type currents were observed in outside-out patches from proliferating granule cells in the external germinal layer. The kinetic behaviour of kainate receptors in immature cells was complex. Openings to multiple conductance levels were observed, although our analysis indicates that the channels spend most of their open time in a 4 pS state.

The risk of gastrointestinal carcinoma in familial juvenile polyposis.

BACKGROUND: Familial juvenile polyposis (JP) is an autosomal dominant condition in which affected individuals develop upper or lower gastrointestinal (GI) juvenile polyps, or both, and have a predisposition to cancer of the gastrointestinal tract. The risk of GI cancer has not been well defined because of the small number of these families and the lack of follow-up. The objective of this study was to determine the prevalence and age at diagnosis of GI polyposis and cancer in a large JP kindred. METHODS: Medical records were reviewed, patients were interviewed, and histories were taken. Pathology reports and slides were reviewed by our pathologists. A database was created for analysis of clinical and pathologic factors. RESULTS: This kindred contains 117 members, 29 of whom have had upper or lower GI polyps or cancer, or both. All those affected have had colonic juvenile polyps or cancer, except for two who died of advanced gastric cancer and never had colonic evaluation. Nine individuals have had both upper and lower GI polyps or cancer. Sixteen of 29 (55%) affected patients have developed gastrointestinal cancer. Eleven (38%) have had colon cancer, and six (21%) have had upper GI cancers. CONCLUSIONS: The risk of gastrointestinal malignancy in affected members of this JP kindred exceeds 50%. The high risk of GI cancer warrants frequent endoscopic screening of both affected and at-risk family members. Screening will soon be facilitated by presymptomatic genetic testing for the identification of gene carriers.