Félix Lluís

K-ras Mutations in DNA Extracted From the Plasma of Patients With Pancreatic Carcinoma: Diagnostic Utility and Prognostic Significance

PURPOSE Previous studies have demonstrated the presence of K-ras mutations in the plasma of patients with pancreatic carcinoma. However, the diagnostic utility and the prognostic significance of this finding have never been addressed. PATIENTS AND METHODS Forty-four consecutive patients with histologically confirmed primary pancreatic ductal adenocarcinoma were included. A control group of 37 patients with chronic pancreatitis, 10 patients with other tumors of the pancreatic area, nine patients with acute pancreatitis, and four healthy volunteers was also included. Plasma DNA was isolated and K-ras codon-12 mutations were analyzed by means of restriction fragment length polymorphism-polymerase chain reaction and single-strand conformation polymorphism techniques. Patients were followed up to establish their clinical outcome. RESULTS The mutant-type K-ras gene was found in plasma DNA samples of 12 (27%) of 44 patients with pancreatic ductal adenocarcinoma; this finding was related to the tumor stage (P = .05), mainly in the presence of distant metastases (P = .02). In addition, K-ras mutations were detected in the plasma DNA of two (5%) of 37 patients with chronic pancreatitis. In the subset of patients with pancreatic masses, the sensitivity and specificity of plasma K-ras analysis for pancreatic adenocarcinoma were 27% and 100%, respectively. Finally, pancreatic carcinoma patients with the mutant-type K-ras gene in plasma DNA exhibited a shorter survival time than patients with the wild-type gene (P<.005), and plasma K-ras mutations were identified as the only independent prognostic factor (odds ratio, 1.51; 95% confidence interval, 1.02 to 2.23). CONCLUSION Plasma K-ras analysis is a highly specific, low-sensitivity approach that has diagnostic and prognostic clinical implications in patients with pancreatic carcinoma.

Endogenous cholecystokinin enhances postprandial gastroesophageal reflux in humans through extrasphincteric receptors

BACKGROUND & AIMS Exogenous cholecystokinin (CCK) decreases lower esophageal sphincter (LES) pressure and increases transient LES relaxations (TLESRs) in humans. The aims of this study were to determine whether endogenous CCK increases gastroesophageal reflux in humans and whether this is a direct effect on the LES. METHODS Esophageal pH, LES pressure, and gallbladder volume were measured in 8 healthy volunteers after ingestion of a 181-kcal meal alone or adding 12 g cholestyramine to increase endogenous CCK release. In 7 additional volunteers, the effect of cholestyramine was studied during intravenous perfusion of saline or the CCK-A receptor antagonist loxiglumide. In circular LES strips from 9 transplant donors, we measured the effect of CCK-8 (10(-11) to 3 x 10(-8) mol/L) on basal tension and on electrical field-induced relaxation. RESULTS Cholestyramine increased gallbladder emptying, reflux episodes, TLESRs, and time of esophageal pH of <4. Loxiglumide inhibited postprandial gallbladder emptying, reflux episodes, TLESRs, and time of pH of <4 and prevented the decrease in LES pressure induced by cholestyramine. In vitro, CCK-8 contracted LES strips through a tetrodotoxin-insensitive pathway but did not modify electrical field-induced LES relaxations. CONCLUSIONS Endogenous CCK enhances postprandial gastroesophageal reflux in humans by increasing the rate of TLESRs and reduces postprandial LES pressure. These actions seem mediated by extrasphincteric CCK-A receptors that override a direct contractile effect of CCK on the LES muscle.

Validation of the determinant-based classification and revision of the Atlanta classification systems for acute pancreatitis.

BACKGROUND & AIMS Two new classification systems for the severity of acute pancreatitis (AP) have been proposed, the determinant-based classification (DBC) and a revision of the Atlanta classification (RAC). Our aim was to validate and compare these classification systems. METHODS We analyzed data from adult patients with AP (543 episodes of AP in 459 patients) who were admitted to Hospital General Universitario de Alicante from December 2007 to February 2013. Imaging results were reviewed, and the classification systems were validated and compared in terms of outcomes. RESULTS Pancreatic necrosis was present in 66 of the patients (12%), peripancreatic necrosis in 109 (20%), walled-off necrosis in 61 (11%), acute peripancreatic fluid collections in 98 (18%), and pseudocysts in 19 (4%). Transient and persistent organ failures were present in 31 patients (6%) and 21 patients (4%), respectively. Sixteen patients (3%) died. On the basis of the DBC, 386 (71%), 131 (24%), 23 (4%), and 3 (0.6%) patients were determined to have mild, moderate, severe, or critical AP, respectively. On the basis of the RAC, 363 patients (67%), 160 patients (30%), and 20 patients (4%) were determined to have mild, moderately severe, or severe AP, respectively. The different categories of severity for each classification system were associated with statistically significant and clinically relevant differences in length of hospital stay, need for admission to the intensive care unit, nutritional support, invasive treatment, and in-hospital mortality. In comparing similar categories between the classification systems, no significant differences were found. CONCLUSION The DBC and the RAC accurately classify the severity of AP in subgroups of patients.

DNA copy number changes and evaluation of MYC, IGF1R, and FES amplification in xenografts of pancreatic adenocarcinoma.

We analyzed eight samples of xenografted human pancreatic tumors and two metastases developed in mice by comparative genomic hybridization (CGH). The most recurrent changes were: gains on chromosomes 8 (8q24-qter; 7/8 cases), 15 (15q25-q26; 6/8 cases), 16 (16p in 6/8 cases; 16q in 5/8 cases), 20 (20q; 6/8 cases), and 19 (19q; 5/8 cases); and losses on chromosomes 18 (18q21; 6/8 cases), 6 (6q16-q21 and 6q24-qter; 5/8 cases each), and 9 (9p23-pter; 5/8 cases). The two metastases maintained the aberrations of the original pancreatic tumor plus gain of 11q12-q13 and 22q. Loss of heterozygosity analysis was carried out for 10p14-pter, a region that was lost in 3/8 samples. All of them presented allelic imbalance for all the informative loci. Fluorescence in situ hybridization and Southern analysis were performed to test some candidate oncogenes in 8q24 (MYC) and 15q25-qter (IGF1R and FES). Two of seven tumors showed high-level amplification of MYC relative to the centromere (> 3-fold), another two tumors had low-level amplification (1.5- to 3.0-fold), and one displayed 5.5 MYC signals/cell. In relation to the FES gene, low-level amplification was found in three tumors. Southern analysis showed five cases with a low-level amplification of IGF1R. Our data suggest that either few extra gene copies may be enough for cancer progression or other genes located in these regions are responsible for the amplifications found by CGH.

Clinical usefulness of K-ras gene mutation detection and cytology in pancreatic juice in the diagnosis and screening of pancreatic cancer.

Background The significance of K-ras codon 12 mutation in pancreatic juice is still unclear. Although considerable controversy surrounds this question, the diagnostic utility of K-ras in patients with clinical suspicion of pancreatic cancer (PC) and in PC-risk patients remains unknown. Objective To study prospectively the utility of the K-ras gene mutation and cytology in the diagnosis and screening of PC, and to assess its contribution to clinical decision making. Methods Pancreatic juice samples obtained from 90 patients were evaluated prospectively. Group I (n = 40) comprised patients with clinical suspicion of PC; group II (n = 50) comprised 49 patients with chronic pancreatitis and one patient proceeding from a PC family screening. The K-ras mutation was detected by means of artificial restriction fragment length polymorphisms (RFLP) in DNA after polymerase chain reaction (PCR) amplification. Results In group I, of those patients with a definitive diagnosis of PC, malignant cells were found in 27% and K-ras mutation in 44%. In five cases, molecular analysis contributed to diagnosis (4/11 with negative cytology and 1/2 with insufficient cytological material). K-ras mutation revealed an early tumour in one patient, and was the only sample available for diagnosis in another. In group II, the K-ras gene mutation was detected in 8/49 patients (16%) with chronic pancreatitis, one of whom developed PC (2%). Conclusions K-ras mutation analysis of pancreatic juice may complement cytological evaluation in the diagnosis of PC, in spite of its limited contribution to clinical decision making. The presence of K-ras mutation in chronic pancreatitis classifies a subgroup of PC-risk patients who should be evaluated carefully by long-term follow-up.

Orthotopic models of human pancreatic cancer

Orthotopic transplantation of solid tumor fragments of human tumors in nude mice reproduces their pattern of local growth and distal dissemination. While lymphatic, hepatic or peritoneal dissemination can be reproduced, perineural invasion is absent. Early passages (less than 3) of xenografts show a high degree of stability regarding K‐ras, p53 and p16 gene status. On the other hand, advanced passages of tumors acquire additional alterations in the p15 and Smad4 genes. Mutations in K‐ras, p53, p15 and Smad4 genes can be acquired, in this model system, in the more advanced stages of pancreatic tumor dissemination. Finally, it is also possible to standardize local growth of these tumors as well as its dissemination pattern giving us a preclinical tool to evaluate the anticancer activity of new drugs.

Peptide YY inhibits nutrient-, hormonal-, and vagally-stimulated pancreatic exocrine secretion.

Peptide YY (PYY) is a recently isolated gut peptide that is found primarily in the mucosal endocrine cells of the terminal ileum, colon, and rectum of several mammalian species, including humans. The purpose of this study was to characterize the effect of PYY on pancreatic exocrine secretion in six conscious dogs prepared with pancreatic and gastric fistulas. In control experiments, pancreatic exocrine secretion was stimulated by either intrave-nous (i.v.) administration of secretin (100 ng/kg/h), cholecystokinin-8 (50 ng/kg/h), neurotensin (5 μg/kg/h), or 2-deoxy-D-glucose (75 mg/kg); or by the intraduodenal infusion of hydrochloric acid (4 mEq/h), a mixture of amino acids (phenylalanine + tryptophan at 5 mmol/h), sodium oleate (9 mmol/h), or a liquid meal. On separate days, PYY (12.5, 25,50, 100, 200, or 400 pmol/kg/h) was given intravenously in combination with one of the above pancreatic se-cretagogues. Intravenous PYY at 200 and 400 pmol/kg/h inhibited secretin-stimulated pancreatic bicarbonate output significantly (p <0.05). Pancreatic bicarbonate and protein responses to all pancreatic secretagogues were re-duced significantly (p <0.05) by PYY at 400 pmol/kg/h. Intravenous adminis-tration of atropine (0.6 mg bolus, followed by 0.02 mg/kg/h) did not abolish the ability of PYY to inhibit secretin-stimulated pancreatic bicarbonate secretion. This study demonstrates that PYY can inhibit nutrient-, hormonal-, and va-gally-stimulated pancreatic exocrine secretion in the dog; its mechanism of action appears to be independent of cholinergic innervation.

Neuroendocrine potential of the colon and rectum

T he stomach and small bowel have been extensively studied as sources of gastrointestinal (GI) hormones, whereas the colon has been considered infrequently in assessments of the gut functioning as an endocrine organ (1,~). The colon and rectum, however, contain peptides and amines that are both stored in numerous mucosal endocrine cells, which suggests a hormonal role, as well as in neurons and nerve terminals, which suggests a neurotransmitter function (3). Furthermore, the results of standard experimental techniques in GI endocrinology, such as intraluminal perfusion with nutrients, show the colon to be a source of endocrine effects (4-10). In this review, we describe mucosal endocrine cells of the colon and summarize the hormonal actions of their peptides, which explain the endocrine effects that may originate from the colon. We review colonic neuropeptides and summarize recent studies on the characterization and ontogeny of colonic peptides and endocrine cells, as well as their effect as local regulators of colonic absorption or secretion, and motility. Finally, we list and describe those conditions and diseases that affect neuroendocrine elements of the colorectum, such as malabsorption, inflammatory bowel disease, Hirschsprung’s disease, and carcinoid tumors.

Effect of Pancreastatin on Pancreatic Endocrine and Exocrine Secretion

Pancreastatin is a novel peptide that was recently purified from extracts of the porcine pancreas. The present study shows that pancreastatin (10−9-10−8 M) can stimulate release of insulin from both the isolated perfused rat pancreas and from cultured rat islet cells in the presence of a low, noninsulinotropic concentration of glucose (4.2 mM). Pancreastatin (10−9M) can also inhibit release of insulin stimulated by a high concentration of glucose (16.7 mM). Pancreastatin, at 10−8 M, can enhance glucose (8.3 mM) induced release of insulin in the static islet cell incubation. In addition, pancreastatin (10−9-10−8 M) can inhibit, in a dose-dependent fashion, cholecystokinin (CCK)-8 stimulated release of amylase from dispersed guinea pig pancreatic acini. Pancreastatin alone, however, did not affect basal release of amylase. Our study shows that pancreastatin can exert a direct effect on both pancreatic endocrine and exocrine secretion.

Update of the Atlanta Classification of Severity of Acute Pancreatitis: Should a Moderate Category Be Included?

Background: Persistent and multiple organ failure (POF and MOF) are predictive of death in acute pancreatitis (AP). Local complications without organ failure are associated with morbidity but a low risk of mortality. Aim: To design a three-category classification of AP severity and to compare it with the Atlanta Classification (AC) in terms of morbidity and mortality. Method: Severe AP was defined as death, POF (>48 h) or MOF. Moderate AP was defined as the presence of acute collections and/or pancreatic necrosis. Mild AP was defined by exclusion. We compared this classification with AC in 144 episodes of AP. Results: In the three-category classification, severe AP was associated with significantly more frequent intensive care unit admission, invasive treatment and mortality than moderate and mild AP (p < 0.01). Severe AP patients required longer hospital stay and more nutritional support than mild AP patients (p < 0.01). Patients with moderate AP had significantly longer hospital stay and more need for nutritional support than patients with mild AP (p < 0.01). Five patients died, all of them with MOF and/or POF. Conclusions: A three-category classification distinguishes three homogeneous groups of severity.