Louis Buscail

Targeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: Hopes and realities

Mutation of the KRAS oncogene in pancreatic cancer is responsible for permanent activation of the P21 RAS protein and the cascade of signalling pathways. Consequently, multiple cellular processes, such as transformation, proliferation, invasion, and survival are activated. The aim of this review was to present all potential clinical applications of targeting KRAS in terms of diagnosis and management of pancreatic adenocarcinoma. Quantitative polymerase chain reaction technology provides reliable assessment of KRAS mutations, both in tissues and from fine-needle aspiration biopsies. Numerous studies report that the combination of endoscopic ultrasound-guided cytopathology and a KRAS mutation assay can improve the positive and differential diagnosis of pancreatic cancer, differentiating between benign versus malignant solid pancreatic cancer, and reducing false-negative results compared to cytopathology alone. In addition, the presence of a KRAS mutation is frequently associated with a worse prognosis, both in cases of advanced and resected tumours. However, the KRAS mutation assay is not as efficient at predicting a response to both anti-epidermal growth factor receptor treatments and/or chemotherapy. Targeting of KRAS to treat pancreatic adenocarcinoma has been applied at different stages of RAS molecular intracellular processes: at the transcription level with antisense or interference RNA, at the posttranslational level with inhibitors of farnesyl transferase or anti-RAS vaccination peptides, and to target multiple signalling pathways using inhibitors of mitogen-activated protein kinase, phosphoinositide 3-kinase, AKT, mammalian target of rapamycin, RAF. Despite some encouraging results at pre-clinical and phase I stages, no significant clinical benefits have been observed. Combinatory approaches with standard chemotherapy will be welcome.

The SV2 variant of KLF6 is down-regulated in hepatocellular carcinoma and displays anti-proliferative and pro-apoptotic functions

BACKGROUND & AIMS KLF6 protein is a transcription factor that plays important functions in hepatocellular carcinoma (HCC), which is one of the leading causes of death by cancer worldwide. Previous studies showed the existence of three splice variants of KLF6, termed SV1, SV2, and SV3. An increased SV1/KLF6 mRNA ratio in HCC was already described. In this study, we aimed to investigate the expression of the SV2 variant in HCC samples and its role in hepatic cells. METHODS We measured the expression of the SV2 variant in HCC and adjacent tissue samples by q-RT-PCR. We established IHH and HepG2 stable cell lines over-expressing the SV2 variant and measured cell growth and apoptotic rate. RESULTS We observed a reduced expression of the SV2 variant in HCC samples versus surrounding tissues and normal liver. Interestingly, our findings demonstrate that the over-expression of the SV2 variant in IHH and HepG2 cells leads to a significant reduction of proliferation associated with cell death by apoptosis. We further demonstrate that the SV2 expression leads to an induction of the cell-cycle-controlling p21(CIP/WAF1) and the pro-apoptotic Bax genes, mediated by the p53 protein. We show further that the SV2 expression in IHH and HepG2 cells induces their sensitivity to the anti-cancer drug, gemcitabine. CONCLUSION We reveal a reduced expression of the SV2 variant of KLF6 in HCC samples and describe anti-proliferative and pro-apoptotic functions for this variant in hepatic cells.

The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.

Prospective evaluation of the aetiological profile of acute pancreatitis in young adult patients.

BACKGROUND The aetiologies of acute pancreatitis in young adult patients are poorly known. AIMS To prospectively evaluate the causes of acute pancreatitis in patients aged less than 35 years. METHODS Overall, 309 consecutive patients admitted to our centre for acute pancreatitis received first-line investigations, including medical history, standard laboratory tests, abdominal ultrasound and computed tomography. If no aetiology was found, second-line investigations were performed, including endoscopic ultrasound, magnetic-resonance cholangiopancreatography and genetic testing in cases of idiopathic pancreatitis. RESULTS Overall, 66 patients aged between 16 and 35 years were included. After first-line investigations, 49% of cases of acute pancreatitis remained idiopathic. Second-line investigations reduced this rate to 21%. The frequency of aetiologies for acute pancreatitis significantly differed in adults aged ≤ 35 compared to those aged >35 years: biliary aetiology was less frequent (23% versus 43%, p=0.003) as well as alcohol-related (8% versus 24%, p=0.01); drug-induced was more common (16% versus 4%, p=0.0007), as well as cannabis-related (13% versus 1%, p<0.0001), or genetic (10% versus 1.5%, p=0.003). CONCLUSIONS The aetiologies of acute pancreatitis significantly differed in adults aged less than 35 years when compared to older patients. Thus, use of medications, exposure to cannabis, and genetic mutations should be actively sought in these patients.

Cirrhotic patients with portal hypertension-related bleeding and an indication for early-TIPS: A large multicentre audit with real-life results

BACKGROUND The Baveno VI consensus meeting concluded that an early TIPS must be considered in high-risk cirrhotic patients presenting with variceal bleeding (VB) (Child B + active bleeding at endoscopy or Child C10-13 patients). Whether this therapeutic approach is feasible in a real-life setting remains unclear. AIMS To determine (1) the proportion of patients eligible for early-TIPS among cirrhotic patients with VB, (2) the proportion of these patients who underwent early-TIPS placement and the main reasons for discarding TIPS, and (3) the outcomes of patients who experienced early-TIPS placement in a large, national, prospective, multicentre audit including academic and non-academic centres. MATERIALS AND METHODS All French centres recruiting gastrointestinal bleeding were invited to participate. All consecutive patients with cirrhosis and PHT-related bleeding were included. RESULTS 964 patients were included (58 centres: 26 academic, 32 non-academic; patient characteristics: male sex, 77%; age, 59.6 ± 12.1 years; aetiologies of cirrhosis (alcoholic,viral/other, 67%/15%/18%); source of bleeding (EV/GV/other, 80/11/9%); active bleeding at endoscopy 34%; Child A 21%/B 44%/C 35%. Overall, 35% of the patients were eligible for early-TIPS, but only 6.8%, displaying less severe cirrhosis underwent early-TIPS placement. The main reason for discarding TIPS was a lack of availability. The actuarial probability of survival at one year was significantly increased in early-TIPS patients (85.7±0.07% vs 58.9±0.03%, p=0.04). The severity of liver disease was the only parameter independently associated with improved one-year survival. CONCLUSION In this real-life study, one-third of the cirrhotic patients admitted for VB fulfilled the criteria for early-TIPS placement, whereas only 7% had access to TIPS. TIPS was restricted to patients displaying less severe cirrhosis. The severity of liver disease was the only parameter that influenced survival.

Endoscopic ultrasound-guided fine-needle aspiration plus KRAS and GNAS mutation in malignant intraductal papillary mucinous neoplasm of the pancreas

Background: KRAS and GNAS mutations are common in intraductal papillary mucinous neoplasia of the pancreas (IPMN). The aims of this study were to assess the role of pre-therapeutic cytopathology combined with KRAS and GNAS mutation assays within cystic fluid sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to predict malignancy of IPMN. Patients and methods: We prospectively included 37 IPMN patients with clinical and/or imaging predictors of malignancy (men: 24; mean age: 69.5 years). Cytopathology (performed on cystic fluid and/or IPMN nodules), KRAS (Exon 2, codon 12) and GNAS (Exon 8, codon 201) mutations assays (using TaqMan® allelic discrimination) were performed on EUS-FNA material. The final diagnosis was obtained from IPMN resections (n = 18); surgical biopsies, EUS-FNA analyses, and follow-up (n = 19): 10 and 27 IPMN were benign and malignant, respectively. Results: Sensitivity, specificity, positive and negative predictive values, and accuracy of cytopathology alone to diagnose IPMN malignancy were 55 %, 100 %, 100 %, 45 %, and 66 %, respectively. When KRAS-mutation analysis was combined with cytopathology these values were 92 %, 50 %, 83 %, 71 %, and 81 %, respectively. GNAS assays did not improve the performances of cytopathology alone or those of cytopathology plus a KRAS assay. Conclusions: In patients with a likelihood of malignant IPMN at pre-therapeutic investigation, testing for KRAS mutations in cystic fluid sampling by EUS-FNA improved the results of cytopathology for the diagnosis of malignancy whereas GNAS mutation assay did not.

The results of seton drainage combined with anti‐TNFα therapy for anal fistula in Crohn's disease

Combined infliximab and sphincter‐sparing surgery can be effective in perianal fistula associated with Crohns disease (CD). This study aimed to assess the efficacy of local surgery combined with infliximab on sustained fistula closure and to identify predictive factors for response after this combined treatment.

P587 PROGNOSTIC VALUE OF AFP LEVELS IN PATIENTS WITH HEPATOCELLULAR CARCINOMA: PROSPECTIVE EVALUATION OF 715 CONSECUTIVE PATIENTS PRESENTED AT OUR MULTIDISCIPLINARY COMMITTEE FROM 2006 TO 2011

P586 APPEARANCE OF COMBINED HEPATOCELLULAR CHOLANGIOCARCINOMA IN CIRRHOSIS AT CONTRAST ENHANCED IMAGING TECHNIQUES E. Sagrini, M. Iavarone, S. Vavassori, F. Stefanini, M. Maggioni, I. Pettinari, A. Pecorelli, M. Colombo, L. Bolondi, F. Piscaglia. Digestive Disease and Internal Medicine, S. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Unit of Gastroenterology 1, UO Anatomia Patologica, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy E-mail: elisabetta.sagrini@gmail.com

Commentary: Pancreatic cancer: is the worst to come?

The paper by Anne-Marie Bouvier et al., brings scientific confirmation to a clinical impression. What we know, say and write is that pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death in Western countries. In a recent North American analysis on deaths, projection of the ‘top cancer killers’ has, due to demographic changes, placed pancreatic cancer as the second cause of death by cancer in 2030 after lung cancer. In addition, and as stated by Bouvier et al. in their manuscript, ‘very recent predictions suggest that the burden of pancreatic cancer is expected to rise over the next 15 years regardless of age and sex in European regions as well as in other regions defined according to the World Health Organization’. It is a fact that the prognosis remains poor despite advances in diagnosis using high resolution imaging such as endoscopic ultrasound with fine needle aspiration biopsy, and treatments with new regimens of chemotherapeutic protocols (gemcitabine, FOLFIRINOX and nabpaclitaxel). Indeed, a vast majority of patients (85%) are diagnosed with tumours and/or metastases already locally advanced, because they lack specific symptoms and early markers for this otherwise highly aggressive disease. For these patients, the overall survival has doubled in the past 15 years (median survival passed from 6 to 12 months), but the 5-year survival remains less than 3%. When considering different birth cohorts (i.e. from 1920 to 1950), the probability of developing or of dying from pancreatic cancer before age 75 has increased in France. One might think that this increasing incidence is due to increasing life expectancy and/or to improved diagnosis. However, the trends are real, the incidence of pancreatic cancer is increasing and the age at diagnosis will certainly decrease in the future. Even if the trends are not similar in some other countries of Europe, it is well established that pancreatic cancer incidence follows the industrialization of Western countries. Some pre-neoplastic lesions and predisposing diseases have been identified, such as intraductal papillary mucinous tumours of the pancreas, chronic pancreatitis (especially in its hereditary origin) or hereditary cancer syndromes (Peutz Jeghers, familial atypical multiple mole melanoma syndrome, familial pancreatic cancer syndrome, BRCA2 family history . . .) but, on the whole, they account for no more than 5% to 10% of pancreatic cancer cases. In other words, the follow-up of these lesions or patients as it is organized nowadays will not modify the prognosis of the pancreatic cancers that arise de novo in the vast majority of cases. In addition, there is no high-risk population that could benefit from screening. However, even if we could isolate this high-risk sub-group, there is as yet no specific tumour marker available for pancreatic ductal adenocarcinoma. Numerous case-control and cohort studies have been conducted and some risk factors have been inconsistently identified, such as tobacco, obesity, diabetes and exposure to toxic substances. A recent meta-analysis relying on more than 100 studies concluded that tobacco smoking (‘strong’