Allelic alterations in well-differentiated neuroendocrine tumors (carcinoid tumors) identified by genome-wide single nucleotide polymorphism analysis and comparison with pancreatic endocrine tumors.

Well-differentiated neuroendocrine tumors (WDNT, carcinoid tumors) are uncommon indolent neoplasms. The genetic alterations of these tumors are not well characterized. We used genome-wide high-density single nucleotide polymorphism (SNP) array analysis to detect copy number alterations in 29 WDNTs, including seven lung, seven nonileal gastrointestinal, and 15 ileal tumors, and compared with allelic imbalances in 15 pancreatic endocrine tumors (PETs). Most frequent allelic imbalances in WDNTs were losses of chromosome 18 in 10 tumors (34%), chromosome 21 or 21q in six (21%), chromosome 13 or 13q in five (17%) and chromosome 16 or 16q in four (14%) tumors, and amplification of chromosome 20 or 20p in seven (24%) tumors. We also found one tumor with loss of heterozygosity of chromosomes 10 and 15 without copy number loss. These allelic imbalances were associated with primary site of tumor: loss of chromosome 18 was present exclusively in ileal WDNTs (P = 0.001), and loss of chromosome 21 or 21q was more frequent in nonileal gastrointestinal WDNTs (P = 0.02). The tumors with loss of chromosome 21 were larger compared to tumors without loss (P = 0.03). Chromosomal aberrations were less common in WDNTs from lung and gastrointestinal tract compared to PETs (P = 0.001). Our study shows that genome-wide allelotyping using SNP array is a powerful new tool for the analysis of allelic imbalances in WDNTs, and some of these alterations are tumor site-dependent and are different than in PETs.

Allelic alterations in pancreatic endocrine tumors identified by genome-wide single nucleotide polymorphism analysis.

Pancreatic endocrine tumors (PETs) are uncommon and the genetic alterations in these indolent tumors are not well characterized. Chromosomal imbalances are frequent in tumors but PETs have not been studied by high-density single nucleotide polymorphism (SNP) array. We used genome-wide high-density SNP array analysis to detect copy number alterations using matched tumor and non-neoplastic tissue samples from 15 patients with PETs. In our study, whole or partial loss of chromosomes 1, 3, 11, 22 was present in 40, 47, 53, 40% of tumors respectively, and gain of chromosomes 5, 7, 12, 14, 17, and 20 was present in 47, 60, 47, 53, 53, and 47% of tumors respectively. One tumor had loss of heterozygosity of chromosome 3 and another of chromosome 22 without copy number alterations, suggesting uniparental disomy due to non-disjunction and deletion or to chromosomal recombination. Chromosomal aberrations of the autosomal chromosomes were correlated with chromosomal loss or gain of other chromosomes (r>0.5, P<0.5). About 60% of PETs had high allelic imbalances (AI) defined by more than four chromosomal aberrations, and 40% of tumors had low AI. The PETs with high AI were larger: the mean tumor size with high AI was 5.4 +/- 3.1 cm compared with 2.3 +/- 1.3 cm for low AI (P = 0.03). Our study shows that genome-wide allelotyping is a powerful new tool for the analysis of AI in PETs.

Hypomethylation of LINE-1 and Alu in well-differentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors).

Neuroendocrine tumors including carcinoid tumors and pancreatic endocrine tumors are uncommon, and the genetic alterations in these indolent tumors are not well characterized. We studied global hypomethylation by analyzing long interspersed nucleotide elements (LINE)-1 and Alu methylation using pyrosequencing in 35 neuroendocrine tumors and corresponding normal tissue. The tumor samples were less methylated than normal tissue at LINE-1 (P=0.04) and Alu (P=0.001). The mean relative tumor hypomethylation (difference in methylation between normal tissue and in tumor) was 11.5+/-10.0 for LINE-1 and 5.8+/-6.4 for Alu, and were correlated with each other (correlation coefficient 0.6, P=0.001). Relative tumor hypomethylation of LINE-1 was higher in ileal carcinoid tumors than in non-ileal carcinoid tumors and pancreatic endocrine tumors (P=0.047), and tumors with lymph node metastasis (P=0.02), chromosome 18 loss (P=0.001) and RAS-association domain family 1, isoform A gene methylation (P=0.02). Alu methylation in tumors was inversely correlated with methylation of O(6)-methyl-guanine methyltransferase gene (P=0.02). Our study shows that hypomethylation is more common in carcinoid tumors than in pancreatic endocrine tumors and is associated with clinicopathologic features, and genetic and epigenetic alterations in these tumors, including lymph node metastasis.

Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection.

Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices (whole milk, apple juice, ground beef, pastry, and raw eggs). These novel assays used paramagnetic bead-based electrochemiluminescent technology in which biotinylated serotype-specific antibodies were bound to streptavidin-coated paramagnetic beads. The beads acted as the solid support and captured analyte from solution. Electrochemiluminescent detection relied on the use of ruthenium chelate-labeled anti-serotype antibodies and analysis with a BioVeris M-Series M1R analyzer. The sensitivities of the assays in clinically relevant matrices were 50 pg/ml for serotypes A and E, 100 pg/ml for serotype B, and 400 pg/ml for serotype F. The detection limits in selected food matrices ranged from 50 pg/ml for serotype A to 50 to 100 pg/ml for serotypes B, E, and F. The antibodies used for capture and detection exhibited no cross-reactivity when tested with the other serotypes. When purified native toxin was compared with toxins complexed to neurotoxin-associated proteins, no significant differences in assay response were noted for serotypes A, B, and F. Interestingly, the native form of serotype E exhibited reduced signal and limit of detection compared with the complexed form of the protein. We suspect that this difference may be due to trypsin activation of this particular serotype. The assays described in this article demonstrate limits of detection similar in range to the gold standard mouse bioassay, but with greatly reduced time to data. These rapid sensitive assays may have potential use in clinical settings, research studies, and screening of food products for botulinum toxins.

Epigenetic alterations in neuroendocrine tumors: methylation of RAS-association domain family 1, isoform A and p16 genes are associated with metastasis.

Well-differentiated neuroendocrine tumors including pancreatic endocrine tumors and carcinoid tumors are uncommon neoplasms that have site-specific differences in clinicopathological features, clinical course and genetic alterations. The epigenetic alterations in these tumors are not well characterized. We therefore compared methylation of the RAS-association domain family 1, isoform A (RASSF1A), p14, p16 and O6-methyl-guanine methyltransferase genes in neuroendocrine tumors from 47 patients including 16 pancreatic, 15 nonileal and 16 ileal neuroendocrine tumors. Methylation of the RASSF1A gene was present in 57% of tumors, p14 in 49%, p16 in 26% and O6-methyl-guanine methyltransferase in 13% of tumors. Ileal neuroendocrine tumors lacked methylation of O6-methyl-guanine methyltransferase gene (P = 0.04). RASSF1A methylation was associated with histopathologic type of tumors (P = 0.03) and lymph node metastasis (P = 0.004), and p16 methylation with older patient age (P = 0.002) and liver metastasis (P = 0.04). Two or more genes were methylated in 53% of tumors, one gene was methylated in 30% of tumors, and all four genes were unmethylated in 17% of tumors. Methylation of one or more gene was associated with older age of patients (P = 0.01), and methylation of two or more genes was associated with liver metastasis (P = 0.044). Our study shows that in neuroendocrine tumors epigenetic alterations vary by tumor subsite and clinicopathologic features, including age of onset, histopathologic type and metastasis status.

Comparison of genetic alterations in neuroendocrine tumors: frequent loss of chromosome 18 in ileal carcinoid tumors.

Carcinoid tumors and pancreatic endocrine tumors are uncommon neuroendocrine neoplasms, and their genetic alterations are not well characterized. These tumors have site-specific differences in neuroendocrine characteristics, clinical course and genetic alterations. We compared clinicopathological features and loss of heterozygosity of chromosomes 11q, 16q and 18, and BRAF gene mutations in 47 patients with neuroendocrine tumors including 16 with pancreatic endocrine tumors, 15 with nonileal carcinoid tumors and 16 with ileal carcinoid tumors. Patients with carcinoid tumors had more frequent history of alcohol consumption compared to patients with pancreatic endocrine tumors (P=0.02), and patients with ileal carcinoid tumors more frequently had liver metastasis compared to patients with nonileal carcinoid tumors and pancreatic endocrine tumors (P=0.02). Allelic loss of chromosome 11q was present in 21% of tumors, chromosome 16q in 13%, and chromosome 18 in 30%. These alterations differed with the anatomical subsite of tumor: allelic loss of chromosome 18 was present in 69% of ileal carcinoid tumors, 13% of nonileal carcinoid tumors and 6% of pancreatic endocrine tumors (P=0.001). In contrast to pancreatic endocrine tumors and nonileal carcinoid tumors, all 11 ileal tumors with loss of chromosome 18 had complete loss of both chromosomal arms. No BRAF mutations were identified. Complete allelic loss of chromosome 18 was associated with smaller tumor size (P=0.02). Our study indicates that genetic alterations vary by tumor subsite and clinicopathologic features, and ileal carcinoid tumors have distinctive clinicopathologic and genetic profiles.

BRAF mutations in aberrant crypt foci and hyperplastic polyposis.

Patients with hyperplastic polyposis have multiple hyperplastic polyps (HPs) and increased risk of colorectal carcinomas. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesions in colorectal carcinogenesis. We evaluated BRAF mutations by DNA sequencing in 53 ACF from patients with sporadic colorectal carcinomas and familial adenomatous polyposis, in 18 sporadic HPs from patients with resected colorectal cancer, and in 70 HPs, 4 serrated adenomas, 3 admixed hyperplastic-adenomatous polyps, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status was compared with clinicopathological features and other genetic alterations by marginal logistic regression. BRAF mutation was present in only 2% of ACF and 6% of sporadic HPs. In contrast, BRAF mutation was present in 43% of HPs (P = 0.01 versus sporadic HPs), 75% of serrated adenomas, 33% of admixed hyperplastic-adenomatous polyps, 30% of tubular adenomas, and 33% of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status in patients with multiple/large HPs and/or hyperplastic polyposis correlated with HPs from the same patient (odds ratio, 5.8; P = 0.0002) but associated with younger age (odds ratio, 0.83; P = 0.006 compared to older age), with a large HP (odds ratio, 22.5; P = 0.01 compared with patients with multiple HPs), with location of HPs in the right colon (odds ratio, 3.0; P = 0.03), and with methylation of the p16 gene and the MINT31 locus [odds ratio, 12.2 (P = 0.0001) and 4.4 (P = 0.02), respectively]. Our study shows that BRAF mutation status is heterogeneous among patients with multiple/large HPs and/or hyperplastic polyposis, suggesting differences in pathogenesis of HPs that indicate subsets within this phenotype.

Differential effects of oligomeric and fibrillar amyloid-beta 1-42 on astrocyte-mediated inflammation.

Activated glia, as a result of chronic inflammation, are associated with amyloid-beta peptide (Abeta) deposits in the brain of Alzheimer's disease (AD) patients. In vitro, glia are activated by Abeta inducing secretion of pro-inflammatory molecules. Recent studies have focused on soluble oligomers (or protofibrils) of Abeta as the toxic species in AD. In the present study, using rat astrocyte cultures, oligomeric Abeta induced initial high levels of IL-1beta decreasing over time and, in contrast, fibrillar Abeta increased IL-1beta levels over time. In addition, oligomeric Abeta, but not fibrillar Abeta, induced high levels of iNOS, NO, and TNF-alpha. Our results suggest that oligomers induced a profound, early inflammatory response, whereas fibrillar Abeta showed less increase of pro-inflammatory molecules, consistent with a more chronic form of inflammation.

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax.

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.