Marc Barthet

p8 is a new target of gemcitabine in pancreatic cancer cells

Gemcitabine is the only available chemotherapeutic treatment of pancreatic cancers. It is, however, moderately effective, showing a tumor response rate of only 12%. The aim of this work was to identify new pathways involved in the resistance of pancreatic cancer cells to gemcitabine, in the hope of developing new adjuvant strategies to enhance its therapeutic efficacy. Comparison of gene expression patterns of five human pancreatic cancer cell lines showing different degrees of resistance to gemcitabine revealed specific overexpression of several genes in the most resistant. One of them encoded the antiapoptotic p8 protein. We found that (a) knocking down p8 expression in gemcitabine-resistant cells promoted cell death and increased caspase-3 activity; (b) forced overexpression of p8 in gemcitabine-sensitive cells increased their resistance to gemcitabine-induced apoptosis; and (c) gemcitabine down-regulated p8 mRNA expression. These results suggest that, in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8. Hence, targeting the p8-associated pathway could be a new adjuvant therapy improving the response of patients with pancreatic cancer to gemcitabine treatment.

Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response

Backgroundp8 is a DNA-binding protein induced in many tissues in response to LPS treatment. Hence, p8 could be a mediator of LPS-associated effects or, on the contrary, p8 expression may be part of the protective mechanism of the tissues in response to LPS. Finally, p8 expression in response to LPS could also be a simple epiphenomenon.MethodsTo investigate the role of p8 in vivo, we generated p8-deficient mice by gene targeting. Because p8 is a stress protein, we analyzed the response of p8-/- mice to a systemic stress induced by LPS injection. Liver was chosen as model organ to monitor alterations in gene expression.ResultsLPS resulted in higher serum TNF-α concentration and higher mortality rate in p8-deficient mice than in wild-type. Also, liver and pancreas, but not lung, from p8-/- mice showed increased amounts of MPO and HPO. To gain insight into the molecular bases of such susceptibility, we used high density DNA microarrays consisting of ~6000 genes and ESTs to compare gene regulation in response to LPS in p8+/+ and p8-/- livers. In wild-type, 105 genes and 73 ESTs were up-regulated and 232 genes and 138 ESTs down-regulated. By contrast, 212 genes and 125 ESTs were found up-regulated and 90 genes and 85 ESTs down regulated in p8-/- mice. Among them, only 93 (51 induced and 42 repressed) corresponded to the wild-type pattern, demonstrating that p8 deficiency hinders the normal response to LPS, which may account for the increased sensitivity of p8-/-mice to the endotoxin.ConclusionsThe large number of genes showing abnormal regulation after LPS suggests that p8 is an important regulatory factor involved in many cellular defence pathways.

Frequency and Characteristics of Pancreatitis in Patients with Inflammatory Bowel Disease

Background and Aims: Clinical symptoms of inflammatory bowel disease (IBD)-associated pancreatitis are found in ∼2% of patients, but the frequency of the disease could be much higher since IBD-associated pancreatitis could be mainly a silent disease. The aim of this study was to describe the radiological and biological features of IBD-associated pancreatitis and assess its frequency by comparing data from IBD patients with or without a history of pancreatitis. Methods: 79 patients were prospectively enrolled (median age 36 years). Symptoms of pancreatitis had been previously recorded in 30 of them (group P; the other 49 patients (group C) had no history of pancreatitis. Pancreatic ductal changes were investigated by pancreato-MRI. Exocrine function was assessed by the fecal elastase test and by assaying serum amylase, lipase, C-reactive protein, PAP, IgG4 and pancreatic autoantibodies. Results: Increased levels of amylase and lipase occurred in 11% of IBD patients, that frequency being significantly higher in group P (23%) than in group C (4%) (p = 0.01). Low fecal elastase reflecting impaired exocrine function was observed in 30% of patients and again significantly more in group P (50%) than in group C (17%) (p = 0.04). The frequency of elevated values varied from 12% for amylase and lipase to 18% for PAP, 20% for pancreatic autoantibodies and 45% for CRP, without a difference between groups P and C. Silent exocrinopathy was observed in both groups, pancreatic autoantibodies and pancreatic duct alterations being found in 20 and 11% of patients, respectively. Conclusion: Finding pancreatic insufficiency in about 30% of the included patients and in 50% of those with a previous history of pancreatitis suggests that IBD might be associated with chronic pancreatic alteration. Episodes of mild acute pancreatitis observed in some patients are not always due to adverse effects of treatments and can be acute manifestations of the chronic disease.

The reg4 gene, amplified in the early stages of pancreatic cancer development, is a promising therapeutic target.

Background The aim of our work was to identify the genes specifically altered in pancreatic adenocarcinoma and especially those that are altered early in cancer development. Methodology/Principal Findings Gene copy number was systematically assessed with an ultra-high resolution CGH oligonucleotide microarray in DNA from samples of pancreatic cancer. Several new cancer-associated variations were observed. In this work we focused on one of them, involving the reg4 gene. Gene copy number gain of the reg4 gene was confirmed by qPCR in 14 cancer samples. It was also found with increased copy number in most PanIN3 samples. The relationship betweena gain in reg4 gene copy number and cancer development was investigated on the human pancreatic cancer cell line Mia-PaCa2 xenografted under the skin of nude mice. When cells were transfected with a vector allowing reg4 expression, they generated tumors almost twice larger in size. In addition, these tumors were more resistant to gemcitabine treatment than control tumors. Interestingly, weekly intraperitoneal administration of a monoclonal antibody to reg4 halved the size of tumors generated by Mia-PaCa2 cells, suggesting that the antibody interfered with a paracrine/autocrine mechanism involving reg4 and stimulating cancer progression. The addition of gemcitabine resulted in further reduction, tumors becoming 5 times smaller than control. Exposure to reg4 antibody resulted in a significant decrease in intra-tumor levels of pAkt, Bcl-xL, Bcl-2, survivin and cyclin D1. Conclusions/Significance It was concluded that adjuvant therapies targeting reg4 could improve the standard treatment of pancreatic cancer with gemcitabine.

Identification of Genomic Alterations Associated with the Aggressiveness of Pancreatic Cancer Using an Ultra-High-Resolution CGH Array

Background: Genomic alterations present in pancreatic adenocarcinoma have been described only partially. In addition, the relations between these alterations and the aggressiveness of the phenotype remain unknown. Methods: Genomic DNA and total RNA from 5 pancreatic cell lines, of which 2 have an aggressive phenotype and are gemcitabine-resistant (Mia-Paca2 and Panc-1), and 3 less aggressive and gemcitabine-sensitive (Capan-1, Capan-2 and BxPC3), have been purified. DNA abnormalities have been analyzed using an ultra-high-resolution CGH array and mRNA expression was studied with an Affymetrix GeneChip expression array. Results: We identified 573 amplified and 30 deleted genes common to all 5 cell lines. Some of them have already been described, whereas other genes, implicated in signal transduction, apoptosis, cell cycle or cell migration, are described for the first time as being related to this cancer. Comparison of genomic abnormalities between the 2 most aggressive and the 3 less aggressive cell lines led to the identification of 368 genes specifically amplified in the aggressive cell lines. However, no specific gene deletion seems to be associated with the aggressive phenotype. Conclusion: Using a high-resolution approach, we could precisely describe the genomic alterations associated with pancreatic adenocarcinoma and determine those associated with an aggressive phenotype.

P.94 - La ponction sous échoendoscopie guidée par l’élastographie en temps réel : une étude préliminaire

Introduction L’elastographie echoendoscopique est une technologie recente qui permet de visualiser en temps reel les proprietes physiques d’un tissu. Cette etude preliminaire avait pour buts de verifier la faisabilite et la performance diagnostique des ponctions echoendoscopiques sous guidance elastographique. Patients et Methodes Lors d’un essai prospectif, 35 patients ayant une ponction echoendoscopique d’une lesion suspecte de malignite ont ete inclus. Toutes ces ponctions etaient realisees sous une guidance elastographique en temps reel. Les lesions etaient categorisees en 4 types elastographiques ; type 1 homogene monochrome bleue, type 2 homogene monochrome non-bleue, type 3 heterochrome diffuse et type 4 heterochrome focalisee. Lorsque qu’une lesion presentait 2 zones elastographiques bien distinctes (type 4), chacune d’elles etaient ponctionnees separement. Resultats 51 ponctions ont ete effectuees chez 35 patients au niveau d’adenopathies ou lesions pancreatiques suspectes de cancer. Au total, 13 lesions de type 1 (29,5 %), 5 de type 2 (11,4 %), 16 de type 3 (36,3 %) et 5 de type 4 (11,4 %) ont ete ponctionnees. Un diagnostic final a pu etre pose sur 44 ponctions ; 19 benignes et 25 malignes. La ponction guidee par les images elastographiques etait realisable pour toutes ces lesions. Les sensibilite, specificite, valeur predictive positives et negatives pour identifier une lesion maligne a partir des distributions elastographiques comportant un contingent non-deformable bleu significatif (type 1, 3 et 4), etaient de 78 %, 91 %, 95 % et 67 %. Le rendement des 2 ponctions pour les lesions de type 4 etait identique. Conclusion La ponction guidee par l’elastographie en temps reel sous echoendoscopie est realisable mais son avantage par rapport a la ponction echoendoscopique traditionnelle demeure incertain. Un essai portant sur un plus grand nombre de lesions de type 4 (heterochrome focalise) est en cours.

Echoendoscopie interventionnelle : traitement des suppurations anales

RésuméLes lésions ano-périnéales de la maladie de Crohn représentent un problème majeur pour le confort de vie des malades et pour leur prise en charge médico-chirurgicale. Les fistules sont souvent complexes, plus ou moins associées à un abcès. Les conséquences se portent sur l’appareil sphinctérien avec des risques d’incontinence et de sténose. Généralement, la prise en charge débute par un traitement médical (antibiothérapie, immunosuppresseurs, anti-TNF-α…). Le traitement chirurgical classique consiste en un drainage de l’abcès ou une fistulotomie, qui comportent des risques de complications et de séquelles sur l’appareil sphinctérien. Nous avons développé la voie d’abord endosonographique par ponction trans-périnéale au niveau de la fistule avec encollage Beriplast ou au niveau de l’abcès avec aspiration-lavage puis injection locale d’antibiotique. Cette technique présente une faisabilité excellente et sans complications.SummaryAnoperineal lesions in Crohn disease raise a major problem for patients’ quality of life and subsequent medical and surgical management. Fistulas are often complex and more or less associated to an abscess. Consequences on sphincteric complex can lead to incontinence or stenosis. Generally, the management of such pathology starts with medical treatment (antibiotherapy, immunosuppressive drugs, anti-TNF α…). The classical surgical treatment consists of drainage of the abscess or fistulotomy, both bearing risks of complications and sequelae on the sphincteric complex. We developed EUS access by transperineal puncture at the level of the fistula and application of Beriplast fibrin sealant or at the level of the abscess with aspiration-lavage and local injection of antibiotics. This technique is of excellent feasibility and devoid of complications.

4691 Shock wave lithotripsy with a piezo-electric lithotriptor in the management of chronic pancreatitis.

Aim: pancreatic stones are frequently reported to be a factor of failure of endoscopic treatment of chronic pancreatitis. Our series tried to evaluate the efficacy of extracorporeal piezoelectric shock wave lithotripsy (ESWL) to disintegrate pancreatic stones in the main pancreatic duct. Methods- Patients: 51 patients (40 men, 11 women) with a mean age of 51 years, presenting with chronic pancreatitis, have been treated between May 1995 to January 1990. The mean number of sessions of lithotripsy was 4.96 per patient with the piezoelectric lithotriptor EDAP LT02. The mean cumul (power (%) x time (sec) x frequency (shock/sec.) /160) per patient was 70,5 +/- 40,5. The total or uncomplete success was defined by the feasibility ofpancreatic stenting. Results: The fragmentation of stones in the main pancreaticduct was achieved in 83,3 % of cases. The total success was achievedin 25 patients (49%), an uncomplete succcess in 11 patients (21%) and atotal failure in 15 patients (29%). Pancreatic stenting failed in 3/33patients after the ESWL and 10/33 patients before the ESWL. Pain disappearedin 72% of the cases after ESWL. There was a significant relationbetween the fragmentation of stones and the disappearance of pain. A significantrelationship was shown between the cumul value and the fragmentationrate. Conclusion: ESWL with the piezoelectric lithotriptor provides satisfactory results on the disappearance of the pain and on the fragmentation. ESWL seems helpful in the endoscopic treatment of chronic pancreatitis.