Barbara Bournet

let-7 MicroRNA Transfer in Pancreatic Cancer-Derived Cells Inhibits In Vitro Cell Proliferation but Fails to Alter Tumor Progression

Pancreatic ductal adenocarcinoma (PDAC) is still the fourth leading cause of cancer-related deaths in Western countries, with increasing incidence. Neither effective prognostic markers nor therapies exist for this cancer. MicroRNAs are potent inhibitors of protein translation, and aberrantly expressed in many cancers. Because let-7 microRNA targets the K-ras oncogene, we aimed to characterize let-7 expression and function in PDAC in vitro and in vivo. Let-7 expression was quantified by real-time RT-PCR from resected tumors and matching adjacent tissue, and in endoscopic ultrasound-guided fine needle aspiration material from patients with PDAC. Let-7 is detected by reverse transcription in situ PCR in a PDAC tissue microarray. PDAC-derived cells were transfected with plasmid-based synthetic microRNAs or by lentiviral transduction, in vitro and in vivo. Let-7 microRNA expression is strongly reduced in PDAC samples, as compared with adjacent tissue. Let-7 is present in normal acinar pancreatic cells, and lost in poorly differentiated cancer samples. In addition, let-7 expression was repressed in patients with PDAC not eligible for surgery. Restoring let-7 levels in cancer-derived cell lines strongly inhibits cell proliferation, K-ras expression, and mitogen-activated protein kinase activation, but fails to impede tumor growth progression after intratumoral gene transfer or after implantation of Capan-1 cells stably overexpressing let-7 microRNA. We describe here for the first time the extensive loss of expression of let-7 in PDAC. In addition, this study provides the initial steps for a microRNA replacement therapy for this cancer.

The Silencing of MicroRNA 148a Production by DNA Hypermethylation Is an Early Event in Pancreatic Carcinogenesis

BACKGROUND The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is accounted for by the absence of early diagnostic markers and effective treatments. MicroRNAs inhibit the translation of their target mRNAs. The production of microRNAs is strongly altered in cancers, but the causes of these alterations are only partially known. DNA hypermethylation is a major cause of gene inactivation in cancer. Our aims were to identify microRNAs whose gene expression is inactivated by hypermethylation in PDAC and to determine whether this hypermethylation-mediated repression is an early event during pancreatic carcinogenesis. We also sought to investigate whether these differentially methylated regions can serve as a diagnostic marker for PDAC. METHODS MicroRNA production was measured by microarray hybridization and reverse-transcription quantitative PCR. The level of DNA methylation was measured by bisulfite mapping and semiquantitative methylation-specific PCR. RESULTS We identified 29 microRNAs encoded by genes whose expression is potentially inactivated by DNA hypermethylation. We focused our study on microRNA 148a (miR-148a) and found its production to be repressed, not only in PDAC samples but also in preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions. More importantly, we found that hypermethylation of the DNA region encoding miR-148a is responsible for its repression, which occurs in PanIN preneoplastic lesions. Finally, we show that the hypermethylated DNA region encoding miR-148a can serve as an ancillary marker for the differential diagnosis of PDAC and chronic pancreatitis (CP). CONCLUSIONS We show that the hypermethylation of the DNA region encoding miR-148a is responsible for its repression in PDAC precursor lesions and can be a useful tool for the differential diagnosis of PDAC and CP.

Peroral transgastric/transduodenal necrosectomy: success in the treatment of infected pancreatic necrosis.

Objective:To assess the results and complications of an endoscopic transgastric/transduodenal approach as a possible alternative to conventional surgery. Summary Background Data:Infected organized pancreatic necrosis carries a high mortality despite antibiotic therapy and numerous conventional and laparoscopic surgical techniques of debridement. The advent of natural orifice transluminal endoscopic surgery (NOTES) provides a possible alternative approach. Methods:Between 2004 and 2007, patients with infected organized pancreatic necrosis were referred for endoscopic necrosectomy as their initial treatment of choice. Accessibility was confirmed by CT and endoscopic ultrasound. Access to the cavities was transgastric or transduodenal, after passing the endoscope inside the retroperitoneal cavity all necrotic and purulent material was evacuated under direct endoscopic vision. Results:Thirteen patients (12 men, mean age: 55 years, range: 38–66 years) underwent endoscopic necrosectomy. Two patients had complementary percutaneous drainage for endoscopically inaccessible cavities. Resolution infection was the rule in all cases. Infection recurred in 4 patients and a necrotic cavity persisted in 1 patient; all were managed by further endoscopic necrosectomies (total = 23 necrosectomy sessions; mean, 1.8 per patient; range, 1–3). Mean duration of each session was 3.5 hours (range, 2.5–4 hours). Endoscopic treatment was eventually successful in all patients with gradual diminution of the necrotic cavities on CT images. Average duration of follow-up was 19.5 months (range, 2–56 months) with no recurrence of the infectious process and no surgery was required for any patient. Complications included bleeding (n = 3) and transient aggravation of sepsis (n = 3). No mortality occurred. Conclusions:This technique is highly effective and safe in the treatment of infected organized pancreatic necrosis. Results are achievable and sustainable with a limited number of sessions.

Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with KRAS mutation assay to distinguish pancreatic cancer from pseudotumoral chronic pancreatitis

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.

Long-term clinical and imaging follow-up of nonoperated branch duct form of intraductal papillary mucinous neoplasms of the pancreas.

Objectives The aim of our study was to perform a 10-year imaging and clinical prospective follow-up of patients with nonoperated branch duct (BD) intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Methods Forty-nine patients with BD-IPMN who displayed a low probability for malignancy were followed up including a clinical component and a series of imaging techniques such as computed tomography, magnetic resonance cholangiopancreatography, and endoscopic ultrasonography. Results After a mean follow-up period of 77 months, 77.5% of patients remained free of symptoms. An increase in the size and number of BD cysts without mural nodules and with no significant increase of main duct size occurred in 18 patients at an average interval of 47 months. Five patients were operated on owing to recurrent pancreatitis and/or an increase in the size of either cysts or the main duct (mean time delay after diagnosis: 20 months). Pathologically, they were diagnosed as benign adenoma (n = 1) or borderline (n = 4). Conclusions Our long-term clinical and imaging follow-up indicated that none of the patients with BD-IPMNs developed malignancy. Therefore, BD-IPMNs with no signs of malignancy should be managed conservatively. We propose that following a 2-year patient follow-up, biannual imaging follow-ups could be sufficient. Abbreviations IPMN - intraductal papillary mucinous neoplasm of the pancreas, BD - branch duct, MPD - main pancreatic duct, EUS - endoscopic ultrasound, CT scan - computed tomographic examination, MRCP - magnetic resonance cholangiopancreatography

Interventional Endoscopic Ultrasound in Pancreatic Diseases

During the last 15 years, endoscopic ultrasound (EUS) has become an important imaging procedure for diagnosis and management of pancreatic diseases. The clinical interest of EUS is now enhanced by interventional procedures. Noteworthy, fine-needle aspiration biopsy is one of the most important contributions of EUS, in particular for the investigation of patients with pancreatic cancer and cystic tumors. EUS-guided fine-needle aspiration appears to be a safe and reliable technique to obtain tissue from pancreatic masses with a low risk of complications. EUS became also a therapeutic procedure, especially applied for celiac plexus neurolysis, pseudocyst drainage, and pancreaticogastrostomy. Further developments are expected by improvement of needle devices such as pancreatic pseudocyst drainage kits. In the future, EUS might be also a support for local application of new treatments of pancreatic tumors, such as gene or cellular therapy products. In this review, we discuss the current clinical applications of interventional EUS and the future development for diagnosis and management of pancreatic diseases.

Clinical fate of branch duct and mixed forms of intraductal papillary mucinous neoplasia of the pancreas

Aims:  The aim of the present study was to assess the clinical fate of, and to gain new insights into, branch duct and mixed (predominantly main duct type) forms of intraductal papillary mucinous neoplasia of the pancreas (IPMN).

MicroRNAs as emerging biomarkers and therapeutic targets for pancreatic cancer.

Despite tremendous efforts from scientists and clinicians worldwide, pancreatic adenocarcinoma (PDAC) remains a deadly disease due to the lack of early diagnostic tools and reliable therapeutic approaches. Consequently, a majority of patients (80%) display an advanced disease that results in a low resection rate leading to an overall median survival of less than 6 months. Accordingly, robust markers for the early diagnosis and prognosis of pancreatic cancer, or markers indicative of survival and/or metastatic disease are desperately needed to help alleviate the dismal prognosis of this cancer. In addition, the discovery of new therapeutic targets is mandatory to design effective treatments. In this review, we will highlight the translational studies demonstrating that microRNAs may soon translate into clinical applications as long-awaited screening tools and therapeutic targets for PDAC.

Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with a KRAS mutation assay using allelic discrimination improves the diagnosis of pancreatic cancer.

Goals and Background: Mutation of the KRAS oncogene is present in 75% to 95% of pancreatic cancer tissues. This study aimed to evaluate whether endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA), combined with analysis of the KRAS mutation, improves the diagnosis of pancreatic cancer in cases of inconclusive or doubtful cytopathologic analysis. Patients and Methods: We prospectively included 186 patients with a pancreatic mass (103 men; mean age: 62 y). Cytopathology and KRAS mutations, using TaqMan MGB allelic discrimination, were performed on EUS-FNA material. A final diagnosis was obtained from EUS-FNA analysis and/or a subsequent biopsy if necessary, and/or surgery, and follow-up: these were pancreatic adenocarcinoma (n=104), other malignant pancreatic tumors (n=22), and benign lesions (n=60, including 35 cases of chronic pancreatitis). Results: Inconclusive or doubtful (low-grade dysplasia or atypia) cytopathology was found in 68 cases. Of these, 29 patients who had adenocarcinoma were subsequently diagnosed, including 19 cases with a former KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone to diagnose adenocarcinoma were 73%, 100%, 100%, 75%, and 85%, respectively. When KRAS mutation analysis was combined with pathology, these values reached 88%, 99%, 99%, 89%, and 93%, respectively. The performance of EUS-FNA to diagnose malignancy was similarly improved after the KRAS-mutation assay (negative predictive value increased from 67% to 88%; accuracy increased from 85% to 94%). Conclusions: EUS-FNA plus KRAS-mutation analysis, using allelic discrimination, is accurate and improves the diagnosis of pancreatic adenocarcinoma when pathology is inconclusive or doubtful.

Salivary MicroRNA in Pancreatic Cancer Patients

Background Pancreatic cancer is the fourth leading cause of cancer death in Western countries, with the lowest 1-year survival rate among commonly diagnosed cancers. Reliable biomarkers for pancreatic cancer diagnosis are lacking and are urgently needed to allow for curative surgery. As microRNA (miRNA) recently emerged as candidate biomarkers for this disease, we explored in the present pilot study the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery, precancerous lesions, inflammatory disease or cancer-free patients as a potential early diagnostic tool. Methods Whole saliva samples from patients with pancreatic cancer (n = 7), pancreatitis (n = 4), IPMN (n = 2), or healthy controls (n = 4) were obtained during endoscopic examination. After total RNA isolation, expression of 94 candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm. Human-derived pancreatic cancer cells were xenografted in athymic mice as an experimental model of pancreatic cancer. Results We identified hsa-miR-21, hsa-miR-23a, hsa-miR-23b and miR-29c as being significantly upregulated in saliva of pancreatic cancer patients compared to control, showing sensitivities of 71.4%, 85.7%, 85,7% and 57%, respectively and excellent specificity (100%). Interestingly, hsa-miR-23a and hsa-miR23b are overexpressed in the saliva of patients with pancreatic cancer precursor lesions. We found that hsa-miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis as compared to the control group, with sensitivity of 100% and 75%, and specificity of 100% and 80%, respectively. Last hsa-miR-216 was upregulated in cancer patients as compared to patients diagnosed with pancreatitis, with sensitivity of 50% and specificity of 100%. In experimental models of PDAC, salivary microRNA detection precedes systemic detection of cancer cells markers. Conclusions Our novel findings indicate that salivary miRNA are discriminatory in pancreatic cancer patients that are not eligible for surgery. In addition, we demonstrate in experimental models that salivary miRNA detection precedes systemic detection of cancer cells markers. This study stems for the use of salivary miRNA as biomarker for the early diagnosis of patients with unresectable pancreatic cancer.