Aims: The purpose of this study was to investigate the clinical feasibility and utility of low-density array analysis on samples obtained from endoscopic ultrasound-guided fine needle aspiration biopsy in locally advanced and/or metastatic pancreatic ductal adenocarcinoma and chronic pancreatitis.Patients and methods: In this prospective multicenter study, we quantified candidate gene expression in biopsies sampled from 44 locally advanced and/or metastatic pancreatic carcinoma and from 17 pseudotumoural chronic pancreatitis using dedicated low-density array microfluidic plates.Results: We first demonstrated that 18S gene expression is stable and comparable in normal pancreas and pancreatic cancer tissues. Next, we found that eight genes (S100P, PLAT, PLAU, MSLN, MMP-11, MMP-7, KRT7, KRT17) were significantly over expressed in pancreatic cancer samples when compared to pseudotumoural chronic pancreatitis (p value ranging from 0.0007 to 0.0215): Linear discriminative analysis identified S100P, PLAT, MSLN, MMP-7, KR77 as highly explicative variables. The area under receiver operating curve establishes the clinical validity of the potential diagnostic markers identified in this study (values ranging from 0.69 to 0.76). In addition, combination of S100P and KRT7 gave better diagnosis performances (Area Under Receiver Operating Curve 0.81, sensitivity 81%, specificity 77%).Conclusion: We demonstrate that molecular studies on EUS-guided FNA material are feasible for the identification and quantification of markers in PDAC patients diagnosed with non-resectable tumours. Using low-density array, we isolated a molecular signature of advanced pancreatic carcinoma including mostly cancer invasion-related genes. This work stems for the use of novel biomarkers for the molecular diagnosis of patient with solid pancreatic masses. Copyright (C) 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.

Introduction: Le diagnostic différentiel entre adénocarcinome pancréatique (CP) et pancréatite chronique (PC) est très difficile. Un ensemble de marqueurs moléculaires spécifiques de l'une ou l'autre des affections ont été isolés de façon rétrospective après études par puce à ADN à partir de pièces opératoires. Le but était de valider de façon prospective et multicentrique si l'analyse de 22 de ces marqueurs au sein du matériel de cytoponction sous échoendoscopie était fiable pour le diagnostic différentiel entre CP et PC.

La mutation de l'oncogène KRAS a été reconnue comment facteur pouvant influencer la survie et la réponse thérapeutique de certains cancers épithéliaux comme le cancer colorectal et le cancer du poumon. Le but était d'évaluer de façon prospective et multicentrique si la mutation de KRAS influençait la survie et la réponse à la chimiothérapie chez des patients porteurs d'un adénocarcinome pancréatique non résécable. Entre Janvier 2005 et Avril 2007, 129 patients (H : 68, F : 61, âge moyen : 66,1 ± 16 ans) porteurs d'un adénocarcinome pancréatique prouvé histologiquement ont été inclus dans 4 centres. Une analyse cytopathologique et la recherche d'une mutation de KRAS (codon-12 et codon-13 - RFLP et séquençage direct) étaient effectuées sur le matériel de ponction-biopsie de la tumeur pancréatique primitive prélevé par échoendoscopie. 79 patients étaient porteurs d'un cancer métastatique (61 %), 50 avaient un cancer localement avancé (n = 31, 24 %) ou étaient inopérables (n = 19, 14 %). 96 patients (74 %) ont reçu une première ligne de chimiothérapie par gemcitabine (17 patients deux lignes, 5 patients trois lignes). Le suivi moyen de la population était de 8,8 ± 5 mois (3 - 18 : médiane 8 mois). Une analyse des codon-12 et -13 du gène KRAS a été possible dans 100 % des cas. Une mutation du codon-12 était présente chez 85 patients (66 %) (pas de mutation du codon 13). Au sein de la population globale il n'y avait pas de différence de survie que le KRAS soit muté (survie à 1 an : 20 %) ou non (survie à 1 an : 25 %) (LogRank - Kaplan-Meier, p = 0,79). De même la mutation de KRAS n'influençait pas la survie au sein des différents sous-groupes : avec et sans chimiothérapie, localement avancé et métastatique (p = 0,33 à 0,9 - aucun effet centre). Indépendamment de la mutation de KRAS la chimiothérapie était le seul facteur de bon pronostic en terme de survie (première ligne p = 0,023, deuxième ligne p = 0,001). Contrairement à d'autres cancers, la mutation de l'oncogène KRAS dans la tumeur n'est pas associée à un plus mauvais pronostic de l'adénocarcinome pancréatique localement avancé ou métastatique. De plus, le statut de KRAS n'influence pas les résultats de la chimiothérapie en termes de survie globale. Ce résultat peut expliquer l'échec des essais thérapeutiques pour le cancer du pancréas par la vaccination anti-KRAS ou par inhibiteurs de farnésyl transférase. Financements SNFGE, IRMAD.

BACKGROUND AND STUDY AIMS: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.PATIENTS AND METHODS: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.RESULTS: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.CONCLUSION: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.

Introduction: Le diagnostic différentiel entre cancer du pancréas (CP) et pancréatite chronique (PC) est très difficile. Le but était d'évaluer de façon prospective et multicentrique si en associant cytopathologie à partir de ponction-biopsie sous EE (PB-EE) et recherche de la mutation de KRAS il était possible d'améliorer le diagnostic différentiel entre CP et PC dans sa forme pseudo-tumorale.

Une mutation ponctuelle activante de l'oncogène k-ras est présente dans 85 à 95% des cancers du pancréas (CP). La précision diagnostique de la ponction-biopsie dirigée sous échoendoscopie (PBEE) pour le diagnostic de masse solide du pancréas varie de 81 à 85% en intention de ponction. La recherche cumulée de la mutation de l'oncogène k-ras pourrait augmenter ce rendement et améliorer le diagnostic différentiel entre cancer et pancréatite chronique pseudotumorale. Le but de cette étude était d'évaluer de façon prospective multicentrique (4 centres français) la valeur diagnostique de l'analyse de l'oncogène k-ras à partir du matériel de PBEE et couplé à la cytopathologie.

BACKGROUND:Peritoneal carcinomatosis from pancreatic cancer has a poor prognosis with a median survival of 3.1 months. This is mainly due to lack of effective treatment. Interleukin 12 (IL12) is a proinflammatory cytokine that has a potent antitumoral effect by stimulating innate and adoptive immunity.AIM:To examine the antitumoral effect and toxicity of intraperitoneal delivery of IL12 using an ex vivo gene therapy approach in a murine model of pancreatic peritoneal carcinomatosis.METHODS:Peritoneal carcinomatosis was generated by direct intraperitoneal inoculation of the pancreatic cancer cell line Capan-1 in athymic mice. Syngenic fibroblasts were genetically modified in vitro to secrete IL12 using a polycistronic TFG murine IL12 retroviral vector coding for both p35 and p40 murine IL12 subunits. Ex vivo gene therapy involved injection of the genetically modified fibroblasts intraperitoneally twice a week for 4 weeks.RESULTS:Treatment of pre-established peritoneal carcinomatosis with fibroblasts genetically modified to express IL12 induced a marked inhibition of tumour growth as measured by comparison of the weights of the intraperitoneal tumour nodules in the treated and control animals (3.52 (SD 0.47) v 0.93 (SD 0.21) g, p<0.05) and improved survival. This effect was associated with infiltration of the peritoneal tumour nodules with macrophages. Peritoneal lavage confirmed enhancement of the innate peritoneal inflammatory activity, with an increased number of activated macrophages and natural killer cells. Moreover, macrophages harvested from animals with peritoneal carcinomatosis and treated with IL12-expressing fibroblasts expressed an activated proinflammatory antitumoral M1 phenotype that included strongly enhanced reactive oxygen species and nitric oxide production. There was no treatment-related toxicity.CONCLUSION:Multiple injections of genetically modified fibroblasts to express IL12 is an effective and well-tolerated treatment for experimental murine pancreatic peritoneal carcinomatosis via activated innate immunity and in particular activated M1 macrophages.

Background and Aim: The majority of patients cannot benefit from the conventional curative treatments that are currently used for hepatocellular carcinoma (HCC), which remains a world health problem. Interleukin (IL)-12 is one of the most potent anti-tumor cytokines. The aim of the present study was to examine the anti-tumor effect and toxicity of intrahepatic delivery of IL-12 using an ex vivo gene therapy approach in a murine model of HCC.Methods: Syngenic fibroblasts or MM45T-Li HCC tumor cells were genetically modified in vitro to express IL-12 using a polycistronic TFG murine IL-12 retroviral vector (TFGmIL-12) coding for both p35 and p40 murine IL-12 subunits. Hepatocellular carcinoma was generated using direct intrahepatic inoculation of the tumor cell line into the left liver lobe of BALB/c mice.Results: Direct liver expression of IL-12 by the injected genetically modified tumor cells induced a marked inhibition of tumor growth. This effect was associated with an early infiltration of macrophages, and lymphocytes forming numerous intralobular foci. There was no significant liver toxicity, as shown by normal biochemical liver tests. At a later time, the intralobular foci were rare and consisted mainly of CD4(+) T cells, while CD8(+) T cells were present in the lobule. Intrahepatic expression of IL-12 did not modify circulating or splenic B lymphocytes or natural killer (NK) cells. The inhibition of tumor growth was maintained in nude mice even when depleted in NK cells. Importantly, in a second model, treatment of established day 7 liver tumors in BALB/c mice using direct intra-tumor injection of syngenic fibroblasts that were genetically modified to express IL-12 significantly reduced tumor size.Conclusion: In conclusion, these data provide evidence that experimental HCC can be efficiently and safely treated using ex vivo IL-12 gene therapy, which seems promising for future clinical studies. (C) 2004 Blackwell Publishing Asia Pty Ltd.

effectiveness of LY293111 in vivo with dynamic monitoring of tumor progression. The welldifferentiated human pancreatic cancer cell line $2-0 I3, with stable expression of enhanced green fluorescent protein was used. Cells (Sx105) in 10 i~l MEM were injected into the duodenal lobe of the pancreas of athymic mice. The surgical procedure and weekly examinations were monitored using a stereo fluorescence microscope combined with an imaging system. Image quality was improved by a reversible skin-flap. We compared 4 groups with 8 mice/group: control (no treatment); LY293111; gemcitabine; LY293111 + gemcitabine. kY293111 was administered daily p.o. and gemcitabina injected 4 times i.p. Animals were euthanized 4 weeks after surgery. A staging system was developed to evaluate the tumor progression according to the TNM-classification. All 32 animals injected with $2-013 cells developed pancreatic cancer. Within 4 weeks, the control animals developed end-stage disease with invasive cancer obstructing the duodenum and bile duct, liver, lung and lymph node metastases, peritoneal carcinomatosis with malignant ascites and cachex~a. All treatment groups showed sigmficant inhibition of tumor growth and metastases. The combined treatment group with gemcitabine & LY293111 showed the greatest tumor suppression according to the scoring system, tumor weight and area. This cancer model allows sensitive detection of tumor growth, metastases and angiogenesis over time, mimics the clinical signs of pancreatic cancer in humans and has proven to be valuable for studying in vivo tumor behaviour and response to therapeutic agents. LY293111, especially in combination with gemcitahine, markedly inhibited cancer growth and prevented liver metastases. In conclusion, LY293111 is a new and promising therapeutic agent for adjuvant as well as palliative therapy of pancreatic adenocarcinoma.

A gain of function mutation resulting in the substitution of leucine for serine at codon 810 (S810L) in the human mineralocorticoid receptor (MR) is responsible for early-onset hypertension that is exacerbated in pregnancy. All steroids, including progesterone, that display antagonist properties when bound to the wild-type MR are able to activate the mutant receptor (MR(L810)). These findings suggest that progesterone may contribute to the dramatic aggravation of hypertension in MR(L810) carriers during pregnancy. However, the steroid(s) responsible for hypertension in MR(L810) carriers (men and nonpregnant women) has not yet been identified. Here we show that cortisone and 11-dehydrocorticosterone, the main cortisol and corticosterone metabolites produced in the distal nephron, where sodium reabsorption stimulated by aldosterone takes place, bind with high affinity to MR(L810). The potency with which cortisone and 11-dehydrocorticosterone bind to the mutant MR contrasts sharply with their low wild-type MR-binding capacity. In addition, cotransfection assays demonstrate that cortisone and 11-dehydrocorticosterone are potent activators of the MR(L810) trans-activation function. Because the plasma concentration of cortisol in humans is about 30-fold higher than that of corticosterone, these findings strongly suggest that cortisone is one of the endogenous steroids responsible for early-onset hypertension in men and nonpregnant women carrying the MR(L810) mutation.

The ability of steroid ligands to inactivate the human mineralocorticoid receptor (MRWT) has been shown to be due to their inability to contact Asn770, a residue of the H3 helix involved in stabilizing contacts with the H11–H12 loop region. However, all steroid ligands that display antagonist properties when bound to MRWT, have been shown to activate a mutant receptor (MRL810) associated with a severe form of hypertension. Biochemical studies revealed that S810L mutation induces a change in the receptor conformation and increases the steroid–receptor complexes stability. From a three-dimensional model of the MR ligand-binding domain, it is likely that the S810L mutation causes a steric hindrance between the side chains of Leu810 (H5) and Gln776 (H3) that provokes a bending of the H3 helix. As a consequence, the positioning of MRWT antagonists within the ligand-binding cavity is modified in such a way that they can activate the mutant MRL810. The results from biochemical studies also revealed that 5α-pregnan-20-one, 4,9-androstadiene-3,17-dione and RU486, unable to bind MRWT, acted as potent MRL810 antagonists.

The binding of mineralocorticoid hormones to the mineralocorticoid receptor is the first step in a cascade of events leading to the stimulation of Na(+) reabsorption by renal cortical collecting duct (CCD) principal cells. The agonist properties of mineralocorticoid hormones are linked to contacts between their 21-hydroxyl group and Asn770, a residue of the ligand-binding domain of the human mineralocorticoid receptor (hMR). Here, we investigate whether the presence of a hydroxyl group at position 11, 17, or 20 could also alter the activity of progesterone (P), a mineralocorticoid antagonist without the 21-hydroxyl group. Both 17 alpha-hydroxyprogesterone (17OHP) and 20 alpha-hydroxyprogesterone (20OHP) antagonized the aldosterone-induced trans-activation activity (IC(50): 17OHP, 10(-7) M; 20OHP, 10(-8) M) of the hMR transiently expressed in COS-7 cells lacking steroid receptors. In cultured mouse mpkCCD(cl4) principal cells, 17OHP and 20OHP also prevented the aldosterone-stimulated amiloride-sensitive component of the short-circuit current (Ams I(sc)), reflecting Na(+) absorption mediated by the epithelial Na(+) channel (ENaC). In contrast, 11 beta-hydroxyprogesterone (11OHP) activated the transiently expressed hMR in COS-7 cells in a dose-dependent manner (ED(50): 10(-8) M) and, like aldosterone, stimulated Ams I(sc) in mpkCCD(cl4) cells. Docking 11OHP within the hMR-ligand-binding domain homology model revealed that the agonist activity of 11OHP is caused by contacts between its 11 beta-hydroxyl group and Asn770. Furthermore, 11OHP was unable to activate the mutant hMR/N770A, in which Ala is substituted for Asn at position 770. These findings demonstrate that in the absence of the 21-hydroxyl group, the 11 beta-hydroxyl group can produce the contact with the hMR-Asn770 required for the hMR activation leading to stimulated Na(+) absorption.

Aldosterone effects are mediated by the MR, which possesses the same affinity for mineralocorticoids and glucocorticoids. In addition to the existence of mechanisms regulating intracellular hormone availability, we searched for human MR splice variants involved in tissue-specific corticosteroid function. We have identified a new human MR isoform, hMRDelta5,6, resulting from an alternative splicing event skipping exons 5 and 6 of the human MR gene. hMRDelta5,6 mRNAs are expressed in several human tissues at different levels compared with wild-type human MR, as shown by real time PCR. Introduction of a premature stop codon results in a 75-kDa protein lacking the entire hinge region and ligand binding domain. Interestingly, hMRDelta5,6 is still capable of binding to DNA and acts as a ligand-independent transactivator, with maximal transcriptional induction corresponding to approximately 30-40% of aldosterone-activated wild-type human MR. Coexpression of hMRDelta5,6 with human MR or human GR increases their transactivation potential at high doses of hormone. Finally, hMRDelta5,6 is able to recruit the coactivators, steroid receptor coactivator 1, receptor interacting protein 140, and transcription intermediary factor 1alpha, which enhance its transcriptional activity. Ligand-independent transactivation and enhancement of both wild-type MR and GR activities by hMRDelta5,6 suggests that this new variant might play a role in modulating corticosteroid effects in target tissues.

Sequence analysis revealed a strong homology between the ligand-binding domain (LBD) of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR). Nevertheless, steroids with bulky C11-substituents bind to hGR, unlike hMR. In this report, a mutant hMR, in which the residue Ala-773 facing the C11 steroid position was replaced by a glycine (A773G), was assayed for its capacity to bind steroids, to interact with receptor coactivators, and to stimulate transcription. The capacity of A773G to bind aldosterone and C11-substituted spirolactones was the same as that of the wild-type receptor. The agonist properties of aldosterone, as well as the antagonist feature of compounds bearing a 11 beta-allenyl group and a C17-ketone function, remain unchanged. In contrast, C11-substituted steroids with a 17 gamma-lactonic ring displayed antagonist properties with hMR and acted as potent agonists with A773G. An agonist-dependent hMR interaction with SRC-1 was observed for both the wild-type and the mutant receptors. The hMR activation process is discussed in the light of the hMR-LBD homology model based on the structural data of the human progesterone receptor LBD.

The crystal structures of ligand-free and agonist-associated ligand-binding domain (LBD) of nuclear receptors (NRs) reveal that the amphipathic helix H12 is folded back toward the LBD core in the agonist-associated conformation, allowing the binding of coactivators. We used alanine scanning mutagenesis to explore the role of the residues of the loop connecting H11 and H12 in the activation of the human mineralocorticoid receptor (hMR), a member of the NRs family. H950A retained the ligand binding and transcriptional activities of the wild-type receptor and interacted with coactivators. In contrast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to the wild- type receptor and caused a receptor conformational change in these mutant hMRs as it does for the wild-type receptor. But the aldosterone-induced transcriptional activity of the mutant hMRs was lower (L952A, E955A, P957A) than that of the wild-type receptor or completely abolished (K953A, V954A) and their interaction with coactivators was impaired (E955A) or suppressed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LBD, we propose that the integrity of the H11-H12 loop is crucial for folding the receptor into a ligand-binding competent state and for establishing the network of contacts that stabilize the active receptor conformation.

Aldosterone exerts its biological effects through binding to mineralocorticoid receptor (MR). Ligand binding induces a receptor transconformation within the ligand-binding domain and dissociation of associated proteins from the receptor. The ligand-activated receptor binds as a dimer to the response elements present in the promoter region of target genes and initiates the transcription through specific interactions with the transcription machinery. The glucocorticoid hormone cortisol binds to the human MR (hMR) with the same affinity as aldosterone, but is less efficient than aldosterone in stimulating the hMR transactivation. The antimineralocorticoid spirolactones also bind to the hMR but induce a receptor conformation that is transcriptionally silent. In this report, we describe the key residues involved in the recognition of agonist and antagonist ligands and propose a two-step model with a dynamic dimension for the MR activation. In its unliganded state, MR is in an opened conformation in which folding into the ligand-binding competent state requires both the heat shock protein 90 and the C-terminal part of the receptor. An intermediate complex is generated by ligand binding, leading to a more compact receptor conformation. This transient complex is then converted to a transcriptionally active conformation in which stability depends on the steroid-receptor contacts.

The ligand binding domains of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR) display a high sequence homology. Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones, are very closely related, leading to the cross-binding of these hormones to both receptors. The present study reports on the mechanism by which hMR and hGR are activated preferentially by their cognate hormones. We found that the ability of corticosteroids to stimulate the receptor’s transactivation function is depending on the stability of the steroid-receptor complexes. In the light of a hMR structural model we propose that contacts through the corticosteroid C21 hydroxyl group are sufficient to stabilize hMR but not hGR and that additional contacts through the C11- and C17-hydroxyl groups are required for hGR.