Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors

<p>Abstract</p> <p>Background</p> <p><it>RASSF1A </it>gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor (PET) but <it>RASSF1A </it>expression has never been studied. The <it>RASSF1 </it>locus contains two CpG islands (<it>A </it>and <it>C</it>) and generates seven transcripts (<it>RASSF1A</it>-<it>RASSF1G</it>) by differential promoter usage and alternative splicing.</p> <p>Methods</p> <p>We studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the (i) methylation status of the <it>RASSF1 </it>CpG islands using methylation-specific PCR and pyrosequencing and (ii) expression of <it>RASSF1 </it>isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR (MSP) and by quantitative methylation-specific PCR (qMSP); pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches.</p> <p>Results</p> <p>MSP detected methylation in 16/20 (80%) PETs and 13/20 (65%) normal pancreas. At qMSP, 11/20 PETs (55%) and 9/20 (45%) normals were methylated in at least 20% of <it>RASSF1A </it>alleles.</p> <p>Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15/20 (75%) cases (<it>P </it>= 0.01). The evaluation of mRNA expression of <it>RASSF1 </it>variants showed that: i) <it>RASSF1A </it>was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET (<it>P </it>= 0.003); ii) <it>RASSF1A </it>methylation inversely correlated with its expression; iii) <it>RASSF1 </it>isoforms were rarely found, except for <it>RASSF1B </it>that was always expressed and <it>RASSF1C </it>whose expression was 11.4 times higher in PET than in normal tissue (<it>P </it>= 0.001). A correlation between <it>RASSF1A </it>expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in <it>RASSF1A </it>expression upon demethylating treatment.</p> <p>Conclusions</p> <p><it>RASSF1A </it>gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm. <it>RASSF1A </it>is always expressed in PET and normal pancreas and its levels are inversely correlated with gene methylation. Isoform <it>RASSF1C </it>is overexpressed in PET and the recent demonstration of its involvement in the regulation of the Wnt pathway points to a potential pathogenetic role in tumor development.</p

Incidental nonfunctioning pancreatic endocrine tumors: clinical and surgical implications

Introduzione: l\u2019attuale diffusione e maggior accessibilit\ue0 delle tecniche imaging negli ultimi anni hanno aumentato l\u2019incidenza di riscontro di neoplasie endocrine non-funzionanti del pancreas (NF-PNETs) asintomatiche. I NF-PNETs incidentali (I-NF-PNETs) solitamente presentano diametro e stadio minori rispetto ai NF-PNETs sintomatici (S-NF-PNETs) ed il riscontro occasionale sembra rappresentare un fattore prognostico favorevole sia per lo stadio di malattia sia per il grading istologico. Vi \ue8 comunque una assenza di dati circa la gestione dei I-NF-PETs potenzialmente non aggressivi. Obiettivi:1) definire il comportamento biologico dei I-NF-PETs sottoposti a resezione chirurgica e 2) valutare una eventuale politica di Follow-Up nella gestione dei I-NF-PETS di stadio I. Metodi: sono stati inclusi nello studio tutti i pazienti con diagnosi confermata all\u2019istologia di NF-PET sporadico sottoposti tra il 1990 ed il 2011 a resezione chirurgica presso il Dipartimento Chirurgico dell\u2019Universit\ue0 di Verona ed il reparto di chirurgia dell\u2019Opedale S. Cuore-Don Calabria di Negrar. E\u2019 stata eseguita una valutazione comparativa delle caratteristiche demografiche, cliniche e patologiche tra I-NF-PETs e S-NF-PETs. E\u2019 stata eseguita l\u2019analisi statistica adeguata per identificare le differenze statisticamente significative del comportamento biologico dei I-NF-PETs versus gli S-NF-PETs. Risultati: Sono stati evidenziati 131 pazienti (42.8%) con diagnosi di I-NF-PET e 175 pazienti (57.2%) con diagnosi di S-NF-PET. Non \ue8 stata riscontrata differenza di sesso tra i due gruppi (p=0.752). L\u2019et\ue0 media \ue8 stata per i maschi: 62 anni (range 24-83) nei I-NF-PETs e 55 anni (range 17 \u2013 78) per gli S-NF-PETs; per le femmine rispettivamente di 55 anni (range 35 \u2013 72) e 53 anni (range 25 \u2013 74), p= 0.223. Gli I-NF-PETs si sono riscontrati pi\uf9 frequentemente a livello del corpo-coda del pancreas (65 casi, 49.6%), mentre gli S-NF-PETs si sono localizzati maggiormente a livello del corpo-coda (56.6%) e della coda (38.3%) (p= 5 mm) and a serotonin immunoreactivity at the immunohistochemical evaluation. From September 2007 to September 2011 a total of 19 patients with I-NF-PNET diagnosis were enrolled. All cases was classified as NET-G1 and median size was 15 mm (range 9 \u2013 20). In all cases, no MPD obstruction was confirmed at preoperative imaging. All this patients refused surgical resection. Currently Follow-Up was available for all patients, with a median follow-up of 22 months (range 6 \u2013 48). All Patients were alive, asymptomatic and with tumor stable in size and no evidence of progression disease. Conclusions: this study shows that patients with incidentally detected NF-PETs represent about 40% of resectable NF-PETs and frequency of incidental diagnosis was increasing in last years. Incidental detection seems to be an important favorable prognostic factor for histopathological features, patients overall survival and disease free survival. Anyway pancreatic surgery have a recognized high rate of perioperative morbidities and for < 20 mm and carefully selected pancreatic neuroendocrine \u201cincidentalomas\u201d a clinical-laboratory and radiographic surveillance might be possible

Valutazione dei risultati nel confezionamento di anastomosi per fistola artero-venosa con diversi materiali di sutura

Nei pazienti con insufficienza renale cronica la dialisi extracorporea rappresenta tuttora la terapia di prima scelta mediante l'utilizzo di una fistola artero-venosa (FAV) ottenuta anastomizzando una vena ad una arteria al braccio. La FAV va però incontro nel 20% dei casi ad una occlusione entro i primi 20 giorni; a tale incidenza si aggiunge un altro 10% di occlusione ad 1 anno. Uno dei principali meccanismi fisiopatologici chiamati in causa per spiegare l'occlusione e la stenosi oltre alla non perfetta tecnica chirurgica, è l'elevata presenza di turbolenza di flusso che favorisce il danno endoteliale. A ciò si aggiunge la reazione dell'endotelio della rima anastomotica al corpo estraneo rappresentato dal filo di sutura. Alla contemporanea presenza di questi due fattori è dovuto il fatto che nell' 80% dei casi la stenosi si realizza a livello dell'anastomosi e del primo tratto di vena efferente

Systematic review of resection of primary midgut carcinoid tumour in patients with unresectable liver metastases.

BACKGROUND: Surgery for small intestinal neuroendocrine tumours (SI-NETs) is limited by metastatic disease in most patients. However, resection of the primary lesion alone has been advocated in patients with unresectable liver metastases. The present systematic review investigated the value of surgical resection of the primary lesion in patients with unresectable metastatic disease. METHODS: MEDLINE was searched for studies reporting the outcome of patients with SI-NETs and unresectable liver metastases where there was an explicit comparison between resection of the primary lesion alone and no resection. The primary outcome was overall survival. Secondary outcomes were progression-free survival, treatment-related mortality and relief of symptoms. RESULTS: Meta-analysis was not possible, but six studies were analysed qualitatively to highlight useful information. Possible confounders in these studies were the inclusion of patients with other primary tumour sites, unknown primary tumour or non-metastatic disease. Bearing in mind these limitations, there was a clear trend towards longer survival in patients who underwent surgical resection in all studies; their median overall survival ranged from 75 to 139 months compared with 50-88 months in patients who did not have resection. The difference between the two groups was statistically significant in three studies. Data on symptomatic improvement were scarce and did not suggest a clear benefit of surgery. Surgery-related mortality seemed low. CONCLUSION: Available data suggest a possible benefit of resection of the primary lesion in patients with unresectable liver metastases, but the studies have several limitations and the results should therefore be considered with caution. Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley &amp; Sons, Ltd

Middle-preserving pancreatectomy for multicentric body-sparing lesions of the pancreas.

The appropriate surgical approach of a pancreatic multifocal disease that skips the body of the gland remains unknown. We sought to analyze a parenchyma-sparing surgical technique consisting of a middle-preserving pancreatectomy (MPP) evaluating feasibility, safety, and oncological and functional outcomes.Between 1999 and 2007, 5 patients affected by pancreatic benign or slow malignant multicentric body-sparing disease underwent MPP.There were 3 men and 2 women with a median age of 35 years (range 28-70 years). The median operative time was 365 minutes (range 330-440 minutes). Postoperative mortality was nil and postoperative morbidity was 1 (20\%). At a median follow-up of 20 months (range 14-118 months) all of the patients are alive and disease-free. Overall, 2 patients developed insulin-dependent diabetes mellitus, as well as exocrine insufficiency. One patient developed only exocrine insufficiency.MPP is a feasible procedure and might reduce the risk of both endocrine and exocrine insufficiency

The determinant factors of recurrence following resection for ductal pancreatic cancer.

Long-term survival for patients with pancreatic carcinoma is low, even following resection. Most patients who undergo curative treatment, develop recurrence usually at the same site of resection or in the liver. Failure seals the fate of the patient. Local recurrence occurs frequently; however, it is rarely a direct cause of death. In fact, most patients die from distant metastases. From a clinical point of view, it is important to distinguish recurrence from relapse. In fact, recurrence can be recognized as the reappearance of the disease in the surgical bed, often due to inadequate surgical clearance. On the other hand, the concept of relapse should be much more related to the appearance of the disease in a distant site. Both underestimated staging of the diagnosis and the biological features of the tumor can cause relapse. Up to now, there have only been a few reviews on the incidence and pattern of failure following resection. Detailed knowledge of the recurring sites of pancreatic carcinoma and study of the factors influencing disease-free survival are significant in developing neoadjuvant, surgical and adjuvant treatment. The aim of this review is to point out the major factors most commonly identified as determinants of both recurrence and relapse

Is Enucleation Safe When the Distance Between the Tumor and the Main Pancreatic Duct Is Less Than 3 mm? Results from a Multi-Institutional Retrospective Study

Background Enucleation of small tumors can prevent pancreatic function impairment although the incidence of postoperative pancreatic fistula is relatively high. It has been suggested that this procedure should be avoided when the distance between the tumor and the main pancreatic duct is less than 3 mm. Objective To evaluate the safety of pancreatic enucleation for tumors distant less than 3 mm from the main pancreatic duct. Methods We reviewed the databases of the Department of Surgery of Marburg University (MU) and “Sacro Cuore” (SCH) Hospital of Negrar (1990-2012). All patients underwent intra-operative ultrasound (US) to measure the distance between the main pancreatic duct and the tumor. Binary logistic regression analysis of predictors of pancreatic fistula was performed. Results Sixty patients underwent enucleation in the two institutions. There were 21 males (35%) and 39 females (65%) with a median age of 50 years. The main reason for surgery was insulinomas (60%) followed by nonfunctioning neuroendocrine tumors (22%), gastrinomas (8%) and other tumors (6%). The median operative time was 137 minutes (IQR: 120-160). The overall rate of pancreatic fistula was 48% whereas the mortality was nil. The rate of pancreatic fistula was similar among the two institutions (55% in the SCH versus 42% in the MU; P=0.305). Overall, 31 patients (52%) had a distance between the tumor and the main pancreatic duct less than 3 mm. Re-exploration was necessary in 5 patients (8%) who had a tumor distant less than 3 mm from main pancreatic duct whereas the rate of grade C pancreatic fistula was similar among the two groups (25% vs. 29%; P=0.257). The only variable associated with a higher risk of pancreatic fistula was the distance between the tumor and main pancreatic duct less than 3 mm (odds ratio: 5.51; P=0.003). Conclusions Although the distance between the main pancreatic duct and tumor less than 3 mm is associated with a higher risk of pancreatic fistula, enucleation remains acceptably safe also in this group of patients. An intra-operative US is always mandatory to improve the post-operative management other than preventing main pancreatic duct injuries

Pancreatic endocrine tumors: improved TNM staging and histopathological grading permit a clinically efficient prognostic stratification of patients

Pancreatic endocrine tumors are rare diseases and devising a clinically effective prognostic stratification of patients is a major clinical challenge. This study aimed at assessing whether the tumor-node-metastasis (TNM)-based staging and proliferative activity-based grading recently proposed by the European NeuroEndocrine Tumors Society (ENETS) have clinical value. TNM was applied to 274 patients with histologically diagnosed pancreatic endocrine tumors operated from 1991 to 2005, with last follow-up at December 2007. According to World Health Organization (WHO) classification, 246 were well-differentiated neoplasms (51 benign, 56 uncertain behavior, 139 carcinomas) and 28 poorly differentiated carcinomas. Grading was based on Ki67 immunohistochemistry. Survival analysis not only ascertained the prognostic value of the TNM system but also highlighted that in the absence of nodal and distant metastasis, infiltration and tumor dimensions over 4 cm had prognostic significance. T parameters were then appropriately modified to reflect this weakness. The 5-year survival for modified TNM stages I, II, III and IV were 100, 93, 65 and 35%, respectively. Multivariate analysis identified TNM stages as independent predictors of death, in which stages II, III and IV showed a risk of death of 7, 29 and 58 times higher than stage I tumors (P<0.0001). Ki67-based grading resulted an independent predictor of survival with cut-offs at 5 and 20%. In conclusion, WHO classification assigns clinically significant diagnostic categories to pancreatic endocrine tumors that need prognostic stratification by applying a staging system. The ENETS-TNM provides the best option, but it requires some modifications to be fully functional. The modified TNM described in this study ameliorates the clinical applicability and prediction of outcome of the ENETS-TNM; it (i) assigns a risk of death proportional to the stage at the time of diagnosis, and (ii) allows a clinically based staging of patients, as the T parameters as modified permit their clinical-radiological recognition. Ki67-based grading discerns prognosis of patients with same stage diseases

Galad Score as a Prognostic Marker for Patients with Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) accounts for more than 75% of primary liver cancers, which are the second leading cause of cancer-related deaths. The GALAD (gender, age, AFP-L3, AFP, and des-carboxy-prothrombin) score is a diagnostic tool developed based on gender, age, alpha-fetoprotein, alpha-fetoprotein L3, and des-gamma-carboxy prothrombin, originally designed as a diagnostic tool for HCC in high-risk patients. Methods: We analyzed 212 patients with and without cirrhosis. The population study was divided into patients with liver cirrhosis without evidence of HCC at the time of serum sample collection for GALAD score determination and patients with liver cirrhosis and a confirmed diagnosis of HCC at the time of serum sample collection for GALAD score determination. Patients were followed up until death or liver transplantation. The association between variables and HCC mortality risk was performed, and the results were presented as hazard ratio (HR). The receiver operating characteristic (ROC) curve was used to assess the performance of the GALAD HCC diagnosis. The survival probability was explored using the non-parametric test, and the equality of survival amongst categories was assessed with the log-rank test. Results: Biomarkers were higher in the HCC group compared to cirrhosis. Kaplan–Meier survival probability analysis for individual GALAD categories revealed that a high GALAD level was associated with decreased survival during follow-up, and the difference between the curves was statistically significant (p = 0.01). Conclusions: Our findings suggest that the GALAD score has promise as a prognostic tool, with implications for improving patient management and treatment strategies for HCC