Transcriptional variations in the wider peritumoral tissue environment of pancreatic cancer.

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology.

hERG1 channels regulate VEGF-A secretion in human gastric cancer: clinicopathological correlations and therapeutical implications.

PURPOSE: hERG1 channels are aberrantly expressed in several types of human cancers, where they affect different aspects of cancer cell behavior. A thorough analysis of the functional role and clinical significance of hERG1 channels in gastric cancer is still lacking. EXPERIMENTAL DESIGN: hERG1 expression was tested in a wide (508 samples) Italian cohort of surgically resected patients with gastric cancer, by immunohistochemistry and real-time quantitative PCR. The functional link between hERG1 and the VEGF-A was studied in different gastric cancer cell lines. The effects of hERG1 and VEGF-A inhibition were evaluated in vivo in xenograft mouse models. RESULTS: hERG1 was positive in 69% of the patients and positivity correlated with Lauren's intestinal type, fundus localization of the tumor, G1-G2 grading, I and II tumor-node-metastasis stage, and VEGF-A expression. hERG1 activity modulated VEGF-A secretion, through an AKT-dependent regulation of the transcriptional activity of the hypoxia inducible factor. Treatment of immunodeficient mice xenografted with human gastric cancer cells, with a combination of hERG1 blockers and anti-VEGF-A antibodies, impaired tumor growth more than single-drug treatments. CONCLUSION: Our results show that hERG1 (i) is aberrantly expressed in human gastric cancer since its early stages; (ii) drives an intracellular pathway leading to VEGF-A secretion; (iii) can be exploited to identify a gastric cancer patients' group where a combined treatment with antiangiogenic drugs and noncardiotoxic hERG1 inhibitors could be proposed.

Ezrin expression is an independent prognostic factor in gastro-intestinal cancers.

BACKGROUND: Ezrin, a member of the ezrin-radixin-moesin (ERM) family of plasma membrane-cytoskeleton linker proteins, has been associated with metastatic behavior. METHODOLOGY: Microarrayed pathological tissues of surgically resected colorectal cancer liver metastasis (CRLM) and whole tissue sections of cancer of the ampulla of Vater (CAV) were analyzed to determine ezrin expression levels and correlation with survival. The requirement of ezrin in invasive capability was assessed using in vitro assays. RESULTS: Surgically resected CAV showing a low ezrin score have a better 5-year disease-specific survival than those showing a high ezrin score (P < 0.0001). Similarly, high ezrin expression at the invasive front of CRLM resulted in poor disease-free survival (P = 0.05). Multivariate analysis demonstrated high ezrin expression to be an independent adverse prognostic factor for CAV (hazard ratio (HR) 15.22 (95 % confidence interval (CI) 1.98-117.03), P < 0.01) and CRLM (HR 6.42 (95 % CI 1.01-52.43), P = 0.05), among other clinically relevant variables such as lymph node metastasis (for CAV) and the presence of extrahepatic disease, large hepatic metastases (>5 cm), and close surgical resection margins (<5 mm) (all for CRLM). In vitro experiments indicated that ezrin expression was vital for cellular processes such as adhesive and invasive activity. SIGNIFICANCE: High ezrin expression indicates an adverse prognosis in primary CAV and CRLM.

Autoantibodies to Ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy with only a 5% 5-year survival rate. Reliable biomarkers for early detection are still lacking. The goals of this study were (a) to identify early humoral responses in genetically engineered mice (GEM) spontaneously developing PDAC; and (b) to test their diagnostic/predictive value in newly diagnosed PDAC patients and in prediagnostic sera. METHODS AND RESULTS: The serum reactivity of GEM from inception to invasive cancer, and in resectable or advanced human PDAC was tested by two-dimensional electrophoresis Western blot against proteins from murine and human PDAC cell lines, respectively. A common mouse-to-human autoantibody signature, directed against six antigens identified by MALDI-TOF mass spectrometry, was determined. Of the six antigens, Ezrin displayed the highest frequency of autoantibodies in GEM with early disease and in PDAC patients with resectable disease. The diagnostic value of Ezrin-autoantibodies to discriminate PDAC from controls was further shown by ELISA and ROC analyses (P < 0.0001). This observation was confirmed in prediagnostic sera from the EPIC prospective study in patients who eventually developed PDAC (with a mean time lag of 61.2 months between blood drawing and PDAC diagnosis). A combination of Ezrin-autoantibodies with CA19.9 serum levels and phosphorylated α-Enolase autoantibodies showed an overall diagnostic accuracy of 0.96 ± 0.02. CONCLUSIONS: Autoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature.

Somatic mutations in exocrine pancreatic tumors: association with patient survival.

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09-4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14-2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33-7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.

Targeting gemcitabine containing liposomes to CD44 expressing pancreatic adenocarcinoma cells causes an increase in the antitumoral activity.

Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.

ICAT is a novel Ptf1a interactor that regulates pancreatic acinar differentiation and displays altered expression in tumours.

The PTF1 (pancreas transcription factor 1) complex is a master regulator of differentiation of acinar cells, responsible for the production of digestive enzymes. In the adult pancreas, PTF1 contains two pancreas-restricted transcription factors: Ptf1a and Rbpjl. PTF1 recruits P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] which acetylates Ptf1a and enhances its transcriptional activity. Using yeast two-hybrid screening, we identified ICAT (inhibitor of ß-catenin and Tcf4) as a novel Ptf1a interactor. ICAT regulates the Wnt pathway and cell proliferation. We validated and mapped the ICAT-Ptf1a interaction in vitro and in vivo. We demonstrated that, following its overexpression in acinar tumour cells, ICAT regulates negatively PTF1 activity in vitro and in vivo. This effect was independent of ß-catenin and was mediated by direct binding to Ptf1a and displacement of P/CAF. ICAT also modulated the expression of Pdx1 and Sox9 in acinar tumour cells. ICAT overexpression reduced the interaction of Ptf1a with Rbpjl and P/CAF and impaired Ptf1a acetylation by P/CAF. ICAT did not affect the subcellular localization of Ptf1a. In human pancreas, ICAT displayed a cell-type-specific distribution; in acinar and endocrine cells, it was nuclear, whereas in ductal cells, it was cytoplasmic. In ductal adenocarcinomas, ICAT displayed mainly a nuclear or mixed distribution and the former was an independent marker of survival. ICAT regulates acinar differentiation and it does so through a novel Wnt pathway-independent mechanism that may contribute to pancreatic disease.

Irrelevance of microsatellite instability in the epidemiology of sporadic pancreatic ductal adenocarcinoma.

BACKGROUND AND AIMS: Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS. METHODS: MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers. RESULTS: Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one. CONCLUSIONS: MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.

Immunohistochemical detection of arginine methylated proteins (MeRP) in archival tissues.

AIMS: To (i) determine whether methylarginine-specific antibodies can be employed for standard immunohistochemical analysis of paraffin-embedded tissues, (ii) analyse methylarginine expression in normal and neoplastic tissues and (iii) correlate methylarginine expression with that of protein arginine methyltransferase (PRMT1), the predominant cellular arginine methyltransferase. METHODS AND RESULTS: Immunohistochemistry of normal and cancer tissues was performed utilizing three commercial polyclonal antibodies: anti-methylarginine-specific antibody (anti-mRG) raised against a methylarginine peptide, Control antibody (anti-RG), a control antiserum raised against a corresponding arginine peptide without any methylated residues and anti-PRMT1. Nuclear and/or cytoplasmic methylarginine expression was detected in all keratinized and non-keratinized epithelia. A preliminary survey of a series of thyroid, pancreatic, colonic and gastric cancers identified a different pattern of methylarginine expression in comparison with normal tissue. A correlation between methylarginine staining and PRMT1 expression was found in all normal and cancer tissues analysed. CONCLUSION: Methylarginine-specific antibodies are capable of recognizing methylarginine proteins (MeRP) in paraffin-embedded tissues. Methylarginine proteins are expressed widely and show differences in subcellular localization in various organs and neoplastic conditions. The efficient detection of methylproteins by standard immunohistochemistry provides a new tool to investigate the role of methylarginine proteins (MeRP) in biological processes including carcinogenesis.

MEN1 in pancreatic endocrine tumors: analysis of gene and protein status in 169 sporadic neoplasms reveals alterations in the vast majority of cases.

Pancreatic endocrine tumors (PETs) may be part of hereditary multiple endocrine neoplasia type 1 (MEN1) syndrome. While MEN1 gene mutation is the only ascertained genetic anomaly described in PETs, no data exist on the cellular localization of MEN1-encoded protein, menin, in normal pancreas and PETs. A total of 169 PETs were used to assess the i) MEN1 gene mutational status in 100 clinically sporadic PETs by direct DNA sequencing, ii) immunohistochemical expression of menin in normal pancreas and 140 PETs, including 71 cases screened for gene mutations, and iii) correlation of these findings with clinical-pathological parameters. Twenty-seven PETs showed mutations that were somatic in 25 patients and revealed to be germline in 2 patients. Menin immunostaining showed strong nuclear and very faint cytoplasmic signal in normal islet cells, whereas it displayed abnormal location and expression levels in 80% of tumors. PETs harboring MEN1 truncating mutations lacked nuclear protein, and most PETs with MEN1 missense mutations showed a strong cytoplasmic positivity for menin. Menin was also misplaced in a significant number of cases lacking MEN1 mutations. In conclusion, the vast majority of PETs showed qualitative and/or quantitative alterations in menin localization. In 30% of cases, this was associated with MEN1 mutations affecting sequences involved in nuclear localization or protein-protein interaction. In cases lacking MEN1 mutations, the alteration of one of the menin interactors may have prevented its proper localization, as suggested by recent data showing that menin protein shuttles between the nucleus and cytoplasm and also affects the subcellular localization of its interactors.

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.

Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested. METHODS: We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines. RESULTS: The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6. CONCLUSION: Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We suggest that the detection of these two phenotypes might help the selection of patients enrolled in virally-mediated gene therapy trials.

Pancreatic endocrine tumors: expression profiling evidences a role for AKT-mTOR pathway.

PURPOSE: We investigated the global gene expression in a large panel of pancreatic endocrine tumors (PETs) aimed at identifying new potential targets for therapy and biomarkers to predict patient outcome. PATIENTS AND METHODS: Using a custom microarray, we analyzed 72 primary PETs, seven matched metastases, and 10 normal pancreatic samples. Relevant differentially expressed genes were validated by either quantitative real-time polymerase chain reaction or immunohistochemistry on tissue microarrays. RESULTS: Our data showed that: tuberous sclerosis 2 (TSC2) and phosphatase and tensin homolog (PTEN) were downregulated in most of the primary tumors, and their low expression was significantly associated with shorter disease-free and overall survival; somatostatin receptor 2 (SSTR2) was absent or very low in insulinomas compared with nonfunctioning tumors; and expression of fibroblast growth factor 13 (FGF13) gene was significantly associated with the occurrence of liver metastasis and shorter disease-free survival. TSC2 and PTEN are two key inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway and the specific inhibition of mTOR with rapamycin or RAD001 inhibited cell proliferation of PET cell lines. CONCLUSION: Our results strongly support a role for PI3K/Akt/mTOR pathway in PET, which ties in with the fact that mTOR inhibitors have reached phase III trials in neuroendocrine tumors. The finding of differential SSTR expression raises the potential for SSTR expression to be evaluated as a marker of response to somatostatin analogs. Finally, we identified FGF13 as a new prognostic marker that predicted poorer outcome in patients who were clinically considered free from disease.

Expression pattern of claudins 5 and 7 distinguishes solid-pseudopapillary from pancreatoblastoma, acinar cell and endocrine tumors of the pancreas.

Solid-pseudopapillary tumor (SPT) of the pancreas is characterized by a discohesive appearance of the neoplastic cells. This has been linked to the displacement of E-cadherin and beta-catenin from their normal membrane location, which prevents adherens junctions to form. The nuclear localization of beta-catenin is also a feature of SPT that helps in differential diagnosis. This latter includes pancreatic endocrine tumor (PET) as SPT may show neuroendocrine differentiation, and pancreatic acinar cell carcinoma (ACC) and pancreatoblastoma (PB) that may often show nuclear beta-catenin staining. However, the role of additional cell-cell adhesion systems remains to be elucidated in SPT, particularly that of claudins that are essential components of tight junctions showing modulated expression in diverse tumor types. We studied 20 SPT, 20 nonfunctioning PET, 7 ACC, 2 PB, and their matched normal pancreas for the immunohistochemical expression of claudin family members 1, 2, 3, 4, 5, and 7, beta-catenin and E-cadherin. All SPT showed intense membrane claudin 5 and cytoplasmic claudin 2 staining, lack of claudins 3 and 4, and positive cytoplasmic claudins 1 and 7 in few cases. Conversely, PET, ACC, and PB showed strong membrane expression of claudin 7 and lack of claudin 5, whereas claudins 1, 2, 3, and 4 showed variable expression among samples. All SPT showed nuclear beta-catenin and lack of E-cadherin membrane staining, whereas PET, ACC, and PB only showed nuclear beta-catenin in 1, 2, and 2 cases, respectively. SPT shows a peculiar claudin expression profile and the highly specific pattern of claudins 5 and 7 differentiates SPT from PET, ACC, and PB.

Prognostic relevance of lymph node ratio and number of resected nodes after curative resection of ampulla of Vater carcinoma.

BACKGROUND: Nodal metastasis is considered a major prognostic factor in patients with ampulla of Vater carcinoma (AVC). No study has investigated the significance of the ratio between metastatic and resected/examined lymph nodes (LNR) in patients with AVC. METHODS: Demographic, operative, and pathology data, including number of resected/evaluated nodes and LNR, were collected from patients who underwent pancreaticoduodenectomy with radical intent for invasive AVC from 1990 to 2005. Survival rates and recurrence patterns were evaluated and predictors were identified. RESULTS: In 90 evaluable patients (51 males, 39 females, median age 62.5 years), 5-year disease-specific survival (DSS) was 61%. The median number of resected/evaluated nodes was 16 (range: 5-47); 50% of the patients had nodal metastases. The 5-year DSS according to LNR was 75%, 49%, 38%, and 0% for LNR = 0, LNR >0 and < or =0.2, LNR >0.2, and < or =0.4, and LNR >0.4 (P = 0.002), respectively. The 5-year DSS was 81% in patients with >16 resected/evaluated nodes compared with 45% in those with < or =16 resected/evaluated nodes (P = 0.001). On multivariate analysis LNR and a number of resected/evaluated nodes >16 were significant predictors of survival; a number of resected/evaluated nodes >16 was also the only independent predictor of recurrence. CONCLUSIONS: After curative resection for AVC, LNR and a cutoff of 16 resected/evaluated nodes are powerful prognostic factors. LNR might represent a major parameter for patient stratification in adjuvant treatment trials.

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine.

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.

Microsatellite instability in gastric cancer is associated with better prognosis in only stage II cancers.

BACKGROUND: The assessment of microsatellite instability (MSI) is not included yet in the routine evaluation of patients with gastric cancer, as controversial data exist regarding its prognostic value. METHODS: We determined the clinical significance of MSI in 510 sporadic gastric cancers, using the mononucleotide markers BAT25 and BAT26. The results were compared with the immunohistochemical expression of the mismatch repair proteins Mlh1 and Msh2. RESULTS: MSI was present in 83 (16%) cancers and correlated with better survival (P < .001). Multivariate analysis showed that the MSI phenotype was an independent factor (P = .005) and added prognostic information to TNM stage, location, and age. The relative risk of death for MSI cancer patients was 0.6 (95% confidence interval [CI], 0.4-0.8). Moreover, when grouped according to stage, only stage II cancers showed a significant effect of MSI status on survival (P = .011; hazard ratio = 0.3; 95% CI, 0.1-0.8). MSI also correlated with older age (P = .002), female gender (P < .001), intestinal histotype (P = .011), lower T stage (P = .018), and less lymph node involvement (P < .001). Finally, comparison of the results of immunohistochemical expression of the mismatch repair proteins Mlh1 and Msh2 with microsatellite analysis showed concordant results in 95% of neoplasms, with a sensitivity of 82% and specificity of 98%. CONCLUSIONS: Microsatellite analysis of gastric cancer has clinical utility in determination of prognosis, but should be determined in only stage II neoplasms in a routine clinical setting. Immunohistochemistry may be considered sufficient, although microsatellite analysis is preferable.

Family history of gastric cancer: a correlation between epidemiologic findings and clinical data.

BACKGROUND: The pathogenetic mechanisms behind gastric cancer are still unclear. Its familial aggregation, on the other hand, has been very well documented by many epidemiologists. Nonetheless, only a limited number of studies have analyzed possible correlations between demographic and clinical data. METHODS: Between January 1988 and August 2004, 541 patients underwent gastric resection with a curative intent at our department; demographic information, laboratory data, imaging, operative notes, and pathology reports were available for all patients. During 2004 we conducted a series of structured interviews with the surviving patients or their closest relatives regarding oncological family history, limited to first-degree relatives. RESULTS: Family history could be obtained in 383 patients (70.8%). Gastric cancer was by far the most frequently associated tumor: 21.9% of the overall number of tumors reported in the family histories were gastric cancers. Patients were also subdivided into those having at least one other family member with stomach cancer (71 patients; 18.5%) and those with no relatives affected by gastric cancer (312 patients; 81.5%). No statistically significant differences between the groups were observed regarding the primary tumor location, size, pTNM classification, and ABO or Rh blood types. However, the intestinal histotype was significantly (P = 0.015) more frequently represented in individuals with at least one family member affected by gastric cancer compared with those with no relatives with stomach cancer (71.8% vs 55.1%, respectively). CONCLUSIONS: Stomach cancer has a relevant degree of familial aggregation and in our series of patients, this was even more pronounced for the intestinal histotype.

Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker.

Several studies have reported conflicting results regarding correlations of the loss of Fhit expression with clinicopathological parameters in gastric cancer. We investigated the immunohistochemical expression of Fhit in 362 cases of sporadic advanced gastric adenocarcinoma. The series included 64 cases with microsatellite instability associated with defective mismatch repair genes. Fhit expression resulted absent in 72% of the tumors analyzed. Absence of Fhit expression was more frequent in cases with diffuse and mixed histotype compared to the intestinal histotype (P=0.009). Absence of Fhit expression also correlated with tumor stage (P<0.001), lymph node involvement (P<0.001), presence of distant metastasis (P=0.033), and increasing histological grade (P=0.005). Retained Fhit expression also correlated with microsatellite instability as 61% of instable tumors had lost Fhit expression compared to 74% of microsatellite stable cancers (P=0.050). While loss of Fhit correlates with poorer survival in univariate analysis, it is not an independent prognostic factor in multivariate analysis and is thus not of clinical utility.

Allelotype of ampulla of Vater cancer: highly frequent involvement of chromosome 11.

PURPOSE: To determine the genetic differences/similarities in ampulla of Vater cancers (AVC) with respect to other pancreatic tumor types. METHODS: We analyzed eight cases of primary AVC by genome-wide allelotyping on DNA obtained from frozen tissue. A total of 372 microsatellite loci were used for each case, for a total of 2,976 microsatellites analyzed. RESULTS: Of the 2,159 informative markers, 400 were allelic losses and 1,759 markers were retained for an average fractional allelic loss of 0.19. Seven cases showed LOH on at least two markers on chromosomal arm 11p, while six cases showed allelic losses on 11q. The high frequency of LOH on chromosome 11 was also confirmed by analysis of an additional 17 paraffin-embedded AVC. Frequent LOH (50% or greater) was also found on chromosome arms 5q, 6q, 9p, 13, 16p, 17p, and 18p. CONCLUSIONS: It can be inferred that the targets of inactivation on chromosomes 5q, 9p, and 17p appear to be APC, p16, and p53, respectively, while the critical target(s) of inactivation at the other frequently lost loci remain to be characterized. The resulting allelotype reveals that distinctive chromosomal alterations are present in these neoplasms, indicating that it is a tumor entity distinct from pancreatic adenocarcinoma.