Differential diagnosis between pancreatic adenocarcinoma (PADC) and pseudotumoral forms of chronic pancreatitis remains difficult. Mutation of KRAS oncogene is present in 75% to 95% of PADC. This ...study aimed to evaluate whether the combined analysis of KRAS mutation with cytopathological findings from endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) might improve discrimination between PADC and chronic pancreatitis.
This prospective multicenter study included 178 patients with solid pancreatic masses (men 104, women 74; mean age 64.5 years). Cytopathological examination and KRAS mutation analysis (codon-12 and codon-13, restriction fragment length polymorphism RFLP and direct sequencing) were performed on EUS-FNAB material. Final diagnoses were obtained on EUS-FNAB analysis and/or a second biopsy and/or clinical follow-up and/or surgery: PADC, n = 129; chronic pancreatitis, n = 27; other pancreatic neoplasms, n = 16; and benign lesions, n = 6.
KRAS status analysis was successful in all EUS-FNAB samples. Codon-12 KRAS point mutation was found in 66% of PADC samples. No case of chronic pancreatitis displayed KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone for diagnosis of PADC versus chronic pancreatitis were 83%, 100%, 100%, 56% and 86%, respectively. When KRAS mutation analysis was combined with cytopathology, these values reached 88%, 100%, 100%, 63% and 90% respectively.
Although the value of KRAS analysis in addition to EUS-FNAB is limited for distinguishing pancreatic mass lesions, when chronic pancreatitis presented as a pseudotumor a negative finding (wild-type KRAS), was useful in strongly suggesting a benign lesion.
The knowledge of the binding sites of G protein-coupled
cholecystokinin receptors represents important insights
that may serve to understand their activation processes
and to design or optimize ...ligands. Our aim was to identify
the amino acid of the cholecystokinin-A receptor (CCK-AR)
binding site in an interaction with the sulfate of CCK,
which is crucial for CCK binding and activity. A three-dimensional
model of the CCK-AR-CCK complex was built.
In this model, Arg197 was the best candidate residue for
a ionic interaction with the sulfate of CCK. Arg197 was
exchanged for a methionine by site-directed mutagenesis.
Wild-type and mutated CCK-AR were transiently expressed
in COS-7 cells for pharmacological and functional analysis.
The mutated receptor on Arg197 did not bind the agonist radioligand
125I-BH-Thr, Nle-CCK-9; however, it
bound the nonpeptide antagonist 3H-SR27,897
as the wild-type receptor. The mutant was ≅1,470-
and 3,200-fold less potent than the wild-type CCK-AR to
activate G proteins and to induce inositol phosphate production,
respectively. This is consistent with the 500-fold lower
potency and 800-fold lower affinity of nonsulfated CCK
relative to sulfated CCK on the wild-type receptor. These
data, together with those showing that the mutated receptor
failed to discriminate nonsulfated and sulfated CCK while
it retained other pharmacological features of the CCK-AR,
strongly support an interaction between Arg197 of the CCK-AR
binding site and the sulfate of CCK. In addition, the mutated
CCK-AR resembled the low affinity state of the wild-type
CCK-AR, suggesting that Arg197–sulfate interaction
regulates conformational changes of the CCK-AR that are
required for its physiological activation.
To develop an early diagnostic test for pancreatic cancer based on the identification of K-ras mutations in pure pancreatic juice collected during endoscopic retrograde pancreatography.
Prospective ...study with masked comparison. The standard criteria for the diagnosis of pancreatic cancer were pancreatography or surgery (or both) and histopathology, with follow-up ranging from 6 to 40 months.
Referral center.
24 patients with no pancreatic disease (group 1); 29 patients with nontumoral pancreatic disease (group 2); and 22 patients with pancreatic tumor (group 3). Endoscopic ductal aspiration of cells or brush cytology was done on patients having endoscopic retrograde pancreatography for diagnostic or therapeutic reasons.
Confirmation of mutation rates in patients with pancreatic cancer.
K-ras gene analysis was done by polymerase chain reaction-mediated restriction fragment length polymorphism analysis and direct sequencing. All patients from groups 1 and 2 (n = 53) had a normal sequence for the K-ras 12th codon (group 1, 0% 95% CI, 0% to 14%; group 2, 0% CI, 0% to 12%). Mutations were seen in 17 of the 22 patients in group 3 (77% CI, 55% to 92%). Two of the 17 had no evidence of pancreatic cancer when K-ras was first studied. One had chronic abdominal pain and the other presented with acute pancreatitis. Both were initially free of any pancreatic mass, but they developed tumors 18 and 40 months, respectively, after the K-ras mutations were identified.
Identification of K-ras mutations in samples of pancreatic juice may be useful in differentiating between pancreatic cancer and noncancerous pancreatic diseases. K-ras mutation can precede clinical evidence of pancreatic cancer, but the clinical implications of this finding need further study.
Recently, we reported that the mutation of His207 to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, ...S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364–371). To characterize the functional group in CCK that interacts with His207, we first substituted His207 to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening ofl-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp8 of CCK with His207. The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp8 of CCK to other amino acid residues. This interaction was further confirmed by mutating His207 to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.
Background/Aims: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, ...especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. Methods: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). Results: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. Conclusions: The combination of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases.
Background and Aims: Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular ...carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis.
Methods: Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled 33P‐αATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes).
Results: A two‐way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up‐ and down‐expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down‐expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down‐expressed in all MN samples whereas it was over‐expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time–polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected.
Conclusion: We identified a molecular signature of MN characterized by a down‐expression of several genes. One of them, Klf6 was found to be down‐expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.
Exocrine pancreas from different species behaves differently in response to the presence of intact or digested nutrients in the duodenum. A failure of cholecystokinin (CCK) release after a meal has ...been shown among patients with exocrine pancreatic insufficiency. This abnormality could be restored by the administration of pancreatic extracts, suggesting that digested rather than intact nutrients are responsible for the release of CCK and subsequently gallbladder contraction in humans. The aim of this study was to determine the specific role of different lipidic stimuli in humans. Seven male patients (mean age, 52 years) with pancreatic insufficiency secondary to chronic pancreatitis were selected. Pancreatic insufficiency was considered severe in five of them (lipase output, < 1,000 IU/min) and moderate in another two (lipase output, > 1,000 and < 2,300 IU/min). Plasma CCK (by bioassay), gallbladder contraction (by ultrasound), and enzyme output (chymotrypsin) in response to duodenal administration of either oleic acid as free fatty acids or 20% Intralipid as triglycerides were measured in each patient with at least a 48-h interval between each test. In all these patients with pancreatic insufficiency, duodenal perfusion of free fatty acids generated a more pronounced (91 +/- 11 vs. 49 +/- 21 pM) and faster (15 vs. 30 min) (p < 0.05) CCK release than triglycerides. Furthermore, gallbladder contraction was more efficient when free fatty acids instead of triglycerides were administered in the duodenum (86 +/- 5 vs. 69 +/- 4%) at 10 min (p < 0.05) and (73 +/- 8 vs. 51 +/- 5%) at 15 min (p < 0.03). Among patients with measurable residual pancreatic function, enzyme outputs were shown to be higher during free fatty acid than triglyceride perfusion. In humans, free fatty acids rather than triglycerides, when present in the duodenum, stimulate CCK release and gallbladder contraction. In patients with moderate pancreatic insufficiency this phenomenon may increase residual enzymatic secretion. These results allow us to encourage the development of enzymatic preparations as acid-resistant lipases that cause a fast release of free fatty acids in the duodenum.
The dual performance, both economic and environmental, is a major challenge for the wine industry, which remains highly dependent on the use of pesticides. Fungicide control alone accounts for 80% of ...inputs in France. A DSS (DeciTrait®) gathering all the information necessary for the implementation of a low pesticides protection against the main fungal diseases (downy mildew - powdery mildew - black-rot - grey mould) was conceived in a collaborative approach within a CASDAR project. This DSS is easy to use and offers the user a protection strategy at the plot level based on the information and knowledge collected. The bioassays carried out within the CASDAR project clearly demonstrate the potential for reduction of phytosanitary inputs allowed by this DSS, for reducing both the number of treatments and the dose applied.
La double performance, économique et environnementale, constitue un enjeu majeur pour la filière viticole qui demeure très dépendante de l’utilisation des pesticides. A elle seule, la lutte fongicide représente 80% des intrants. Un OAD (DeciTrait®) regroupant l’ensemble des informations nécessaires à la mise en œuvre d’une protection économe en intrants vis-à-vis des principales maladies cryptogamiques (mildiou - oïdium - black-rot - botrytis) a été conçu dans une démarche collaborative ausein d’un projet CASDAR. Cet OAD est simple à utiliser et propose à l’utilisateur une stratégie de protection personnalisée sur la base des informations et connaissances recueillies. Les essais biologiques conduits au sein du projet CASDAR mettent clairement en évidence le potentiel deréduction des intrants phytosanitaires permis par cet OAD, tant sur la réduction du nombre de traitements que sur la dose appliquée.
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