Randomized sham-controlled trial evaluating efficacy and safety of endoscopic gastric plication for primary obesity: The ESSENTIAL trial.

OBJECTIVE: Evaluate safety and efficacy of the pose™ procedure for obesity treatment. METHODS: Subjects with Class I to II obesity were randomized (2:1) to receive active or sham procedure, after each investigator performed unblinded lead-in cases. All subjects were provided low-intensity lifestyle therapy. Efficacy end points were the mean difference in percent total body weight loss (%TBWL) at 12 months between randomized groups, and responder rate achieving ≥5% TBWL. The primary safety end point was incidence of reported adverse events. RESULTS: Three hundred thirty-two subjects were randomized (active, n = 221; sham, n = 111); thirty-four subjects were included in the unblinded lead-in cohort. Twelve-month results were mean TBWL 7.0 ± 7.4% in lead-in, 4.95 ± 7.04% in active, and 1.38 ± 5.58% in sham groups, respectively. Responder rate was 41.55% in active and 22.11% in sham groups, respectively (P < 0.0001); mean responder result was 11.5% TBWL. The differences observed between active and sham groups for co-primary end points were statistically significant (P < 0.0001); however, super superiority margin as set forth in the study design was not met. No unanticipated adverse events or deaths occurred. Procedure-related serious adverse event rates were 5.0% (active) and 0.9% (sham), P = 0.068. CONCLUSIONS: The pose procedure was safe and resulted in statistically significant and clinically meaningful weight loss over sham through 1 year.

Detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer.

Mutated KRAS2 commonly can be detected in the plasma/serum of patients with pancreatic or colorectal cancers possessing this mutated gene. Positive assays are more common in patients with higher stage tumors but some smaller cancers can also be detected; occasionally, patients with large tumors have negative assays. Because relatively few patients with low-stage tumors have been evaluated, more studies in patients with smaller tumors are needed to further define the clinical usefulness of these assays. The reasons for variable results, particularly in patients with larger tumors, is unclear, although a variety of factors may be involved. More sensitive assays need to be developed that will increase the detection rates, although the problem of producing false positives must be minimized. The presence of mutated KRAS2 sequences in the plasma/serum seems to be quite specifically associated with the presence of cancer containing this mutated gene. This is an important feature of KRAS2 as a tumor marker. Preliminary studies in patients with pancreatic cancer suggest that assays for mutated KRAS2 can complement the commonly used CA19-9 assay and provide additional clinically useful information. The results from currently completed studies on the detection of mutated KRAS2 in patients with colorectal and pancreatic cancer are promising, and the potential usefulness of KRAS2 as a clinically important tumor marker should encourage future research.

Metabolism of [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-13C]succinate) in rat hepatocytes.

Hepatocytes prepared from overnight-fasted rats were incubated for 120 minutes in the presence of 2.5 mmol/L [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) or glycerol-1,2,3-tris(methyl[2,3-13C]succinate). The identification and quantification of 13C-enriched metabolites by a recently developed method for the deconvolution of nuclear magnetic resonance (NMR) spectra with multiplet structures and constraints documented a virtually complete recovery of [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) in 13C-labeled glycerol, lactic acid, and glucose. In hepatocytes exposed to [1,3-13C]glycerol-1,2,3-tris(methylsuccinate), glucose was symmetrically labeled, with the vast majority of hexose molecules being enriched with 13C on both C1 and C3 and/or C6 and C4. The respective abundance of glucose isotopomers labeled either on both C3 and C4 or on only 1 of these 2 C atoms indicated that the triose phosphates generated from [1,3-13C]glycerol represented 44% +/- 1% of the total amount of triose phosphates incorporated into the hexose. In hepatocytes exposed to glycerol-1,2,3-tris(methyl[2,3-13C]succinate), the recovery of [2,3-13C]succinate, [2,3-13C]fumarate, and either double- or single-labeled malate, lactate, alanine, and glucose accounted for about half the initial 13C content of the ester. The majority of the glucose molecules were now labeled in both C, and C2 or C6 and C5, with a preferential labeling of C6-C5 relative to C1-C2, the paired C6/C1 and C5/C2 ratios averaging 1.33 +/-0.04. These findings show that glycerol-1,2,3-tris(methylsuccinate) is efficiently and extensively metabolized in hepatocytes. They reinforce the concept that the asymmetry of glucose 13C-labeling by triose phosphates generated from Krebs cycle intermediates is modulated by the availability of glycerol-derived triose phosphates. Lastly, the present study indicates that the latter triose esters, under the present experimental conditions which do not aim at duplicating the physiological in vivo situation, are largely directly channelled in the gluconeogenic pathway, with only a limited intrahepatic contribution of the "indirect" pathway involving their back-and-forth interconversion to and from pyruvate.

Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls.

The amount of non-cell-associated DNA free in blood plasma from pancreatic cancer patients usually exceeds that from healthy donors. We have evaluated the plasma DNA by gel electrophoresis and measured the variation in length of soluble DNA fragments by electron microscopy in plasma from three patients with pancreatic cancer and from three healthy controls. Whereas electrophoresis of nick-translated DNA isolated from plasma obtained from healthy controls showed autoradiographic bands at sizes equivalent to whole-number multiples (1-5x) of nucleosomal DNA (185-200 bp), in the samples obtained from pancreatic cancer patients, stronger ladder patterns appeared. Likewise, strand length distributions of DNA (DNA-SL) in the two groups differ. The DNA-SL distribution data include 2,752 measurements made from cancer patient plasma and 3,291 for control plasma. The shortest DNA-SL measured approximately 30 nm (approximately 88 bp calculated at 0.34 nm/bp) and the largest approximately 28,000 nm (>80,000 bp), with 50% of all lengths measuring between 100 and 900 nm long. The average plasma DNA-SL in controls (311 nm; median, 273 nm) exceeded that in cancer patients (231 nm; median, 185 nm). Small excesses of DNA at approximately 63, approximately 126, approximately 189, approximately 252, and approximately 315 nm, corresponding to small multiples of lengths associated with nucleosomes, were more prominent in the cancer patient plasma than in the healthy control plasma. This study provides evidence indicating differences in non-cell-associated DNA in plasma between cancer patients and healthy controls and indicates that a significant amount of this DNA is probably derived from apoptosis in neoplastic and/or normal cells.

Analysis of the allele-specific PCR method for the detection of neoplastic disease.

PCR assays for the presence of mutant K-ras or p53 sequences are potentially useful as sensitive tests for tumor diagnosis. The technical challenge is to design assays sensitive enough to detect a few molecules of mutant DNA yet sufficiently specific that a false positive signal is not produced by a 10(5)- or 10(6)-fold excess of normal DNA. We determined the detection limit of allele-specific PCR (ASA) as a function of the particular mismatch involved using all 12 possible mismatches in two different DNA sequence contexts (K-ras codon 12 and p53 codon 273). Depending on the identity of the mismatch, mismatched template was amplified 10(2)-10(4)-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represented > 1-0.01% of the total DNA from that locus. Peptide nucleic acid (PNA) clamping was used to improve the K-ras ASA assay. Selective amplification of mutant sequences was achieved using a PNA complementary to the normal sequence to inhibit the amplification of wild-type DNA. PNA clamping followed by ASA resulted in significant improvement in sensitivity and specificity, permitting the detection of tumor DNA diluted with a 300,000-fold excess of normal human DNA.

Promoting worksite smoking control policies and actions: the Community Intervention Trial for Smoking Cessation (COMMIT) experience. The COMMIT Research Group.

BACKGROUND: As an important aspect of the COMMIT trial, worksite smoking-control consultations and supports were provided to employers in 11 diverse, moderate-sized communities. After a 4-year intervention period (1989-1992), impacts on worksite policies, support resources for smokers, and employee perceptions were assessed in these communities and in 11 matched Comparison communities. METHODS: Data from two surveys are reported here. In each of the 22 COMMIT communities, a sample of worksites within each of four size strata were surveyed to determine worksite policies, activities, and resources regarding smoking. Data from employees were obtained from independent community-wide surveys of community residents. RESULTS: Overall, 44% of the worksites surveyed reported having smoke-free policies, with no differences between Intervention and Comparison communities. Thirty-seven percent of Intervention community work-sites reported offering smoking cessation resources or assistance for employees during the period of the study, compared to 31% of Comparison community worksites (P = 0.04). Employees in Intervention communities, relative to those in Comparison communities, reported greater awareness of stop-smoking resources, but equivalent increases in worksite smoking bans. CONCLUSION: Although the level of worksite smoking-cessation activities was higher in Intervention than in Comparison communities, there remains a substantial need to increase the level of such activities and to integrate such activities with restrictive smoking policies.

A comparison of polymerase chain reaction examination of cerebrospinal fluid and conventional cytology in the diagnosis of lymphomatous meningitis.

BACKGROUND: Inherent limitations of conventional cytology often result in a failure to diagnose lymphomatous meningitis in cerebrospinal fluid (CSF) specimens from patients who actually have the disease. The development of polymerase chain reaction (PCR) techniques for the diagnosis of lymphoma based on the detection of clonal rearrangements of the immunoglobulin or T-cell receptor genes offers an alternative, DNA-based test for the diagnosis of lymphoma in the CSF. METHODS: In this retrospective study, 31 CSF specimens from 21 patients were examined by a PCR technique that can detect clonal immunoglobulin gene rearrangements. Twenty-four of the specimens came from 14 patients who eventually had definitive histologic or cytologic diagnoses of B-cell lymphoma. The other seven patients had other neurologic diagnoses, including two patients with reactive lymphocytosis, three with glioblastoma, one with metastatic carcinoma, and one with multi-infarct dementia. The results of the PCR examinations were compared with cytologic evaluation of the same CSF specimens. RESULTS: Five of seven specimens from patients with central nervous system lymphoma that were suspicious for, but not diagnostic of, lymphoma by conventional cytology were positive by PCR. Of 13 specimens from patients with lymphoma that showed no cytologic evidence of malignancy, 5 were positive by PCR. Two of four specimens for which conventional cytology showed definitive evidence of lymphoma were positive by PCR. Two specimens from patients with a reactive lymphocytosis showed a polyclonal pattern by PCR. Specimens from patients with other neurologic diseases were negative by PCR even when cytologically malignant (glioblastoma) cells were present in the specimen. CONCLUSIONS: PCR examination of CSF is practical, complements conventional cytology, and sometimes provides the correct diagnosis when conventional cytology yields only ambiguous results.

PCR-detection of tumor-derived p53 DNA in cerebrospinal fluid.

The sensitivity of polymerase chain reaction (PCR)-based methods for the detection of DNA offers opportunities for tumor diagnosis from the small amounts of tumor-derived DNA released into body fluids. Tumor-derived DNA can be distinguished from DNA derived from non-neoplastic cells by the presence of tumor specific genomic alterations, such as mutations in the p53 gene. This case report describes the use of allele-specific PCR (A-PCR) to detect a C-->T transition in p53 codon 273 in DNA extracted from the cerebrospinal fluid (CSF) of a patient whose glioblastoma contained the same mutation. The results of this study were confirmed by a second independent A-PCR reaction that detected the corresponding G-->A transition on the opposite strand. The specificity of the A-PCR protocol was demonstrated by negative controls, including pooled human placental DNA and the patient's non-tumor DNA, and by the use of A-PCR primers to detect all four possible bases at the site of the mutation. The methodology used in this study is suitable for use as a diagnostic clinical test. Because about half of all human tumors contain p53 mutations, PCR examination of CSF for the presence of mutant p53 sequences may be useful in the diagnosis of recurrent or metastatic tumors. Patients with known carcinoma of the breast or lung might be particularly benefited by this test.

Detection of tumor-derived DNA in cerebrospinal fluid.

The sensitivity of PCR-based methods for the detection of DNA offers opportunities for tumor diagnosis from the small amounts of tumor-derived DNA released into body fluids. We report the detection of tumor DNA in the cerebrospinal fluid (CSF) of two patients with intracranial neoplasms. One patient had a metastatic breast carcinoma which contained amplified HER-2/neu genes, and amplified HER-2/neu gene sequences were present in her CSF. The other patient had a glioblastoma which contained amplified epidermal growth factor receptor (EGFR) genes, and amplified EGFR gene sequences were present in her CSF. This report demonstrates that CSF sometimes contains tumor-derived DNA and suggests that PCR examination of CSF DNA may be diagnostically useful.

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Age and smoking-adjusted lung cancer incidence in a Utah county with a steel mill.

In a recent study of urban air pollution, a Utah county with a steel mill was compared with a county without a steel mill. The result was that 38% of respiratory cancer deaths could be attributed to the air pollution emanating from the mill. Rates for smoking in this previous study were not adjusted, but assumed rats were similar in both counties. We used smoking information obtained from an ongoing radon and lung cancer case-control study to adjust for smoking, and no difference was found in incidence rates of respiratory cancer in the county with the steel mill, compared with the other urban counties and the rural counties among male and female nonsmokers and male smokers. There was a slight excess of lung cancer among female smokers in the county with the steel mill when compared with the other urban counties (rate ratio [RR] = 1.3, 95% confidence interval [95% CI] = 1.0-1.6), but there was no effect in nonsmoking women. We conclude that the findings of the previous study can be explained by differences in smoking rates between the county with the steel mill and the other counties.

Involvement of the RAF1 locus, at band 3p25, in the 3p deletion of small-cell lung cancer.

The ability to establish long-term cell lines of small-cell lung cancer (SCLC) has provided an in vitro model for the disease. We report on the characterization of 10 new human SCLC cell lines established from 34 cytopathologically positive specimens. Based on morphologic and biochemical characterization, growth properties, and expression of MYC and neuroendocrine properties, eight cell lines were categorized as "classic" and two cell lines as "variant". Cytogenetic examination revealed loss of all or part of 3p in all nine SCLC cell lines analyzed. The smallest deletion in common was found at 3p21-3p25. Restriction fragment length polymorphism (RFLP) analyses with probes for 3p were performed for correlation with karyotypic data and supported the cytogenetic findings. In 21 SCLC specimens (cell lines and tumor tissue) with normal DNA, used for comparison, we observed loss of heterozygosity at RAF1 (3p25) in ten of ten informative pairs by using two RFLPs from the RAF1 locus. In addition, loss of heterozygosity was noted in nine of 10 pairs examined with DNF15S2 (3p21) and four of four with D3S3 (3p14). Analysis of cell lines and tumor specimens that lacked paired normal tissue showed a homozygous pattern with the RAF1 probes in all 18 cases. Northern blots revealed significant expression of RAF1 in all cell lines tested. The transcript size was normal. The cytogenetic and RFLP data suggest that the RAF1 locus at 3p25 is involved in the chromosomal deletion of SCLC.

Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice.

The putative retinoblastoma gene (Rb) is a tumor suppressor gene which is believed to cause retinoblastomas when both alleles are inactivated, leading to lack of the encoded Mr 110,000-116,000 phosphoprotein. Inactivation of the Rb gene has also been found in several other tumor types, including small cell lung cancer (SCLC). Absence of the 4.7 kilobase mRNA has been found to be frequent in SCLC, and it has been reported that the Rb Mr 110,000-116,000 protein product is always absent, even in tumors expressing Rb mRNA. Using Western blotting technique with a monoclonal antibody directed against the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors established from 23 patients were studied. Seven of the tumors did not express Rb protein, whereas expression was detectable in 13. Three tumors were not investigated for protein expression. Only two tumors expressed Rb mRNA without detectable Rb protein expression. Gross DNA alterations were found in four tumors, of which only one expressed Rb mRNA. Our results demonstrated frequent absence of Rb mRNA and protein in SCLC, but apparently normal Rb mRNA and protein were both expressed in more than one-half of the tumors.

Molecular mapping of deletion sites in the short arm of chromosome 3 in human lung cancer.

We used 10 restriction fragment length polymorphism (RFLP) probes spanning the length of the short arm of chromosome 3 (3p) to map deletion sites in human lung cancer. Two approaches were used. 1) When a patient's tumor and normal tissue were available, loci with allelic heterozygosity in the normal tissue were tested for loss of alleles at 3p. 2) When the corresponding normal tissue was not available, the frequency of heterozygosity at each locus in a panel of tumors was compared to the corresponding published frequencies in nontumor tissue of healthy individuals or patients with lung cancer. In 14 small cell lung carcinomas (SCLC) with normal DNA for comparison, allele loss was found at all heterozygous loci, with one exception at a locus near the 3p centromere (D3S4). In the total of 53 SCLCs, which included tumors without paired normal tissue, frequency of heterozygosity was significantly reduced in all 10 3p loci. Three loci, DNF 15S2, RAF1, and D3S18, were homozygous in all tumors in the SCLC panel. These loci, which are in regions 3p21 and 3p25, may thus be involved in the origin or evolution of SCLC. We also investigated 24 non-SCLC tumors. In this panel, frequency of heterozygosity was significantly reduced at seven of the 10 loci tested. Comparison of the results shows that the pattern of allele loss on 3p is different in SCLC and non-SCLC, suggesting a difference in pathogenesis at the genetic level.

Insertion of the v-Ha-ras oncogene induces differentiation of calcitonin-producing human small cell lung cancer.

Human small cell lung cancers (SCLC) and cell lines derived therefrom are phenotypically heterogeneous concerning neuroendocrine differentiation. Unlike most SCLC tumors and cell lines that express poorly differentiated neuroendocrine phenotypes, the SCLC cell line DMS 53 exhibits mature endocrine differentiation features, including unusually high expression of the gene for the peptide hormone, calcitonin (CT). We now report that introduction of the viral Harvey ras (v-rasH) oncogene into DMS 53 cells via retroviral infection, with resultant constitutive expression, results in increased features of neuroendocrine differentiation. 7-10 d after infection the cells demonstrated altered morphology, increased CT secretion, increased CT gene expression, markedly diminished cellular proliferation, and nearly abolished methylcellulose cloning efficiency. This response of DMS 53 cells to v-rasH is unlike the tumor progression effects we have previously observed in other SCLC lines. Significantly, the differentiation response that follows expression of the virally introduced v-rasH oncogene in DMS 53 cells is similar to that of neoplastic neuroendocrine cell lines derived from adrenal pheochromocytes and thyroid C cells. The effects of constitutive v-rasH expression in DMS 53 SCLC cells and other neuroendocrine cell lines suggest an important role for rasH or related genes in neuroendocrine differentiation.

Mapping and characterization of an X-linked processed gene related to MYCL1.

A DNA sequence with homology to the myc family of proto-oncogenes has been characterized and found to be a processed gene related to L-MYC (MYCL1). This processed gene (MYCL2) was isolated by cross-hybridization to an oligonucleotide probe synthesized from the C-MYC (MYC) sequence in a highly conserved region of the myc gene family. Sequence analysis of MYCL2 revealed an open reading frame of 1194 bp with no intervening sequences and strong homology to the recently published DNA sequence of MYCL1. Southern and Northern blot analyses of DNAs and RNAs from small cell lung carcinomas confirmed its MYCL1 homology. Mapping of MYCL2 by somatic cell hybrids places this sequence on the long arm of the X chromosome in bands q22----q28.