Search

The benefit of adjuvant chemotherapy in the early stages of colorectal cancer (CRC) is still disappointing and the prediction of treatment outcome quite difficult. Recently, through a transcriptomic approach, we evidenced a role of PNN and KCNQ1OT1 gene expression in predicting response to fluoropyrimidine-based adjuvant chemotherapy in stage III CRC patients. Thus, the aim of this study was to validate in an independent cohort of stages IIIII CRC patients our previous findings. PNN and KCNQ1OT1 mRNA expression levels were evaluated in 74 formalin-fixed paraffin-embedded tumor and matched normal mucosa samples obtained by stages IIIII CRC patients treated with fluoropyrimidine-based adjuvant chemotherapy. PININ, the protein encoded by PNN, was immunohistochemically evaluated in 15 tumor and corresponding normal mucosa samples, selected on the basis of a low, medium, or high mRNA expression tumor/mucosa ratio. PNN and KCNQ1OT1 mRNA mean expression levels were significantly higher in tumor compared with normal tissues. Patients with high PNN or KCNQ1OT1 tumor mRNA levels according to ROC-based cutoffs showed a shorter disease-free survival (DFS) compared with patients with low tumor mRNA gene expression. Also, patients with tumor mRNA expression values of both genes below the identified cutoffs had a significantly longer DFS compared with patients with the expression of one or both genes above the cutoffs. In a representative large cohort of stages IIIII CRC untreated patients retrieved from GEO datasets, no difference in DFS was observed between patients with high and low PNN or KCNQ1OT1 gene expression levels. These data confirm our previous findings and underscore the relevance of PNN and KCNQ1OT1 expression in predicting DFS in early stages of CRC treated with fluoropyrimidine-based adjuvant chemotherapy. If further validated in a prospective case series, both biomarkers could be used to identify patients who benefit from this treatment and to offer alternative chemotherapy regimens to potential unresponsive patients. In relation to the suggested biological role of PNN and KCNQ1OT1 in CRC, they might also be exploited as potential therapeutic targets.

Insulinoma is an insulin-producing pancreatic neuroendocrine tumor that can be malignant in about 10% of cases. Locoregional invasion, lymph node metastases, or remote metastases are the main criteria of malignant insulinoma. Its incidence in patients with pre-existing diabetes mellitus (DM) is exceptionally rare. In this report, we describe a 66-year-old man with long-standing type 2 DM who presented with recurrent episodes of diaphoresis due to severe hypoglycemia despite the withdrawal of insulin therapy, hypercalcitoninemia, and biochemical and radiological findings suggestive of metastatic malignant insulinoma. Unfortunately, after few days of diazoxide treatment, edema, hypotension, oliguria, and water retention were observed, patient’s clinical status deteriorated rapidly, and he died in our department from acute renal failure.

Five-year overall survival of stage III colorectal cancer (CRC) patients treated with standard adjuvant chemotherapy (ACHT) is highly variable. Genomic biomarkers and/or transcriptomic profiles identified lack of adequate validation. Aim of our study was to identify and validate molecular biomarkers predictive of ACHT response in stage III CRC patients by a transcriptomic approach. From a series of CRC patients who received ACHT, two stage III extreme cohorts (unfavorable vs. favorable prognosis) were selected. RNA-sequencing was performed from fresh frozen explants. Tumors were characterized for somatic mutations. Validation was performed in stage III CRC patients extracted from two GEO datasets. According to disease-free survival (DFS), 108 differentially expressed genes (104/4 up/downregulated in the unfavorable prognosis group) were identified. Among 104 upregulated genes, 42 belonged to olfactory signaling pathways, 62 were classified as pseudogenes (n = 17), uncharacterized noncoding RNA (n = 10), immune response genes (n = 4), microRNA (n = 1), cancer-related genes (n = 14) and cancer-unrelated genes (n = 16). Three out of four down-regulated genes were cancer-related. Mutational status (i.e., RAS, BRAF, PIK3CA) did not differ among the cohorts. In the validation cohort, multivariate analysis showed high PNN and KCNQ1OT1 expression predictive of shorter DFS in ACHT treated patients (p = 0.018 and p = 0.014, respectively); no difference was observed in untreated patients. This is the first study that identifies by a transcriptomic approach and validates PNN and KCNQ1OT1 as molecular biomarkers predictive of chemotherapy response in stage III CRC patients. After a further validation in an independent cohort, PNN and KCNQ1OT1 evaluation could be proposed to prospectively identify stage III CRC patients benefiting from ACHT.

// Ida Landini 1 , Andrea Lapucci 1 , Alessandro Pratesi 2 , Lara Massai 2 , Cristina Napoli 3 , Gabriele Perrone 1 , Pamela Pinzani 4 , Luigi Messori 2, * , Enrico Mini 1, * and Stefania Nobili 3, * 1 Department of Experimental and Clinical Medicine, University of Florence, Firenze, Italy 2 Department of Chemistry “Ugo Schiff”, University of Florence, Firenze, Italy 3 Department of Health Sciences, University of Florence, Firenze, Italy 4 Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy * These authors have contributed equally to this work Correspondence to: Enrico Mini, email: enrico.mini@unifi.it Stefania Nobili, email: stefania.nobili@unifi.it Luigi Messori, email: luigi.messori@unifi.it Keywords: auranofin; tumour drug resistance; human tumour cell lines; drug effects; gene expression Received: March 24, 2017 Accepted: August 08, 2017 Published: October 09, 2017 ABSTRACT The anti-arthritic drug auranofin exerts also potent antitumour activity in in vitro and in vivo models, whose mechanisms are not yet well defined. From an auranofin-sensitive human ovarian cancer cell line A2780, a highly resistant (>20-fold) subline (A2780/AF-R) was developed and characterized. Marked reduction of gold accumulation occurred in auranofin-resistant A2780 cells. Also, moderately higher thioredoxin reductase activity in A2780/AF-R cells was observed while no changes in intracellular glutathione content occurred. Resistance to auranofin was associated with a low level of cross-resistance to some investigational gold compounds as well as to oxaliplatin and other anticancer drugs with different mode of action (i.e. melphalan, vinblastine, doxorubicin, etoposide, and paclitaxel). Reduced gold accumulation was associated to substantial gene expression changes in various influx (e.g. SLC22A1, SLC47A1, SLCO1B1 ) and efflux (e.g. ABCB1, ABCC2, ABCC3 ) transporters. The expression levels of selected proteins (i.e. SLC22A1, SLC47A1, P-gp) were also changed accordingly. These data provide evidence that multiple drug transporters may act as mediators of transport of auranofin and other gold compounds in cancer cells. Further investigation into the molecular mechanisms mediating transport of auranofin and new gold complexes in view of their potential clinical application in the treatment of cancer is warranted.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

Although adjuvant chemotherapy has significantly increased overall survival in resected Stage III colorectal cancer, disease recurrence is still high (30-40%). 20-25% of Stage II patients also develop recurrent disease. Thus, high-risk patients may benefit from chemotherapy. As patient response to standard chemotherapy varies, the study of molecular differences in the expression of pharmacologically relevant genes may help clinicians to understand variability and tailor therapy. The expression of 5-fluorouracil (5-FU) pathway genes in tumors from 53 Stages II-III colorectal cancer patients who underwent 5-FU adjuvant chemotherapy was investigated by reverse transcription quantitative real-time polymerase chain reaction. Patients were dichotomized into high- and low-mRNA expression level groups using median values of gene mRNA levels. Then, a threshold analysis to identify a cut-off distinguishing recurrent- or nonrecurrent-disease was used. A high degree of interpatient variation in relative tumor expression of study genes was observed. Multiple gene correlations were found, which suggest possible coregulation mechanisms. No statistically significant relationship between experimental data and baseline clinical/pathological characteristics or clinical outcome was observed using gene expression median values. Threshold analysis indicated significant inverse relationships between deoxyuridine triphosphatase (DUT), ferrodoxin reductase (FDXR) or tumor protein p53 (TP53) and disease-free survival (DFS) in the entire case series and between DUT or NM23-H1 and DFS in Stage III patients: higher gene expression was associated with shorter DFS. This study provides data on relationships between expression of 5-FU pathway genes and clinical outcome of colorectal cancer patients undergoing 5-FU adjuvant chemotherapy and underscores the predictive role of specific genes. Validation in an independent case series is warranted.

Background Efforts to identify cell sources and approaches for cell therapy of liver diseases are ongoing, taking into consideration the limits recognized for adult liver tissue and for other forms of stem cells. In the present study, we described the first procedure of via hepatic artery transplantation of human fetal biliary tree stem cells in patients with advanced cirrhosis. Methods The cells were immune-sorted from human fetal biliary tree by protocols in accordance with current good manufacturing practice (cGMP) and extensively characterized. Two patients with advanced liver cirrhosis (Child-Pugh C) have been submitted to the procedure and observed through a 12 months follow-up. Results The resulting procedure was found absolutely safe. Immuno-suppressants were not required, and the patients did not display any adverse effects correlated with cell transplantation or suggestive of immunological complications. From a clinical point of view, both patients showed biochemical and clinical improvement during the 6 month follow-up and the second patient maintained a stable improvement for 12 months. Conclusion This report represents proof of the concept that the human fetal biliary tree stem cells are a suitable and large source for cell therapy of liver cirrhosis. The isolation procedure can be carried out under cGMP conditions and, finally, the infusion procedure is easy and safe for the patients. This represents the basis for forthcoming controlled clinical trials. Electronic supplementary material The online version of this article (doi:10.1186/s12876-014-0204-z) contains supplementary material, which is available to authorized users.

The aim of this study was to search for mutations in the human mutS homolog 2 (hMSH2) and human mutL homolog 1 (hMLH1) genes in 25 unrelated Brazilian kindreds with suspected hereditary nonpolyposis colorectal cancer (HNPCC).The families were grouped according to the following clinical criteria: Amsterdam I or II; familial colorectal cancer (CRC); an early age of onset of CRC in the proband only; or with at least one or two relatives who had HNPCC-related cancers; CRC in the proband only. All patients were studied with direct sequencing.Ten mutations were detected (10 of 25 [40%]); of nine different mutations, seven were novel. The hMLH1 gene had a higher mutation detection rate than hMSH2 (8 of 25 [32%] vs. 2 of 25 [8%]). Only 3 of these 10 families fulfilled the Amsterdam criteria. Two different polymorphisms were detected in the hMLH1 gene and four in the hMSH2 gene.The hMLH1 gene had a higher mutation detection rate than hMSH2. The physician who deals with CRC must take into consideration the heredity issue with patients who present with an early age of onset or a familial history of CRC- or HNPCC-related cancers, including gastric cancer, even if they do not fulfill the former Amsterdam criteria.

The two main forms of hereditary colorectal cancer are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). Some families do not meet all the diagnostic criteria for HNPCC or FAP and are classified as familial colorectal cancer (FCC). Little information is available on the spectrum of tumors related to HNPCC and FCC in South America. Objective: To describe the frequency of malignant tumors in a group of Brazilian families with HNPCC or FCC in an Institutional Hereditary Colorectal Cancer Registry. Material and methods: The study included 61 families (29 HNPCC and 32 FCC) between January 1998 and June 2001. HNPCC families were clinically classified according to the Amsterdam Criteria I or II. FCC families were characterized by the presence of at least two individuals with CRC or extra-colonic tumors associated with the HNPCC spectrum, at least one of them being under 50 years of age. Results: In the 29 families with HNPCC, 201 patients with cancer were identified among 1241 individuals (589 men and 652 women). Among the 201 patients 223 tumors were observed: 137 CRC (55 in men and 82 in women) and 86 extra-colonic (37 in men and 49 in women). In the 32 families with FCC, 146 patients with cancer were identified among 1053 individuals (505 men and 548 women); 158 tumors were observed in 146 patients, 75 CRC (33 in men and 42 in women) and 83 extra-colonic tumors (47 in men and 36 in women). The most frequent extra-colonic primary sites among the HNPCC families were: endometrium (26.5%) and breast (26.5%) (women), and stomach (35.1%) (men). Among the FCC families, the most common primary sites were: breast (27.8%) (women), and stomach (44.4%) (men). Conclusion: The high frequency of endometrial and gastric cancer found was expected, since these tumors are part of the HNPCC spectrum, but the high frequency of breast cancer requires further molecular investigation to determine a possible hereditary predisposition associated with hereditary CRC.

About 60% of patients with diffuse large B-cell lymphoma (DLBCL) may be cured by primary chemotherapy with an R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) regimen. Most of the rest will die of the disease, mainly due to the occurrence of tumor drug resistance. Many efforts have been made to explain the molecular mechanisms of drug resistance in patients with cancer, including those with DLBCL. This exploratory study was designed to correlate the mRNA expression levels of candidate genes mainly involved in the doxorubicin pathway (ABCB1, GSTP1, TOPO2α, BCL2, PKCβII) with the outcome of 54 patients with DLBCL undergoing a dose-dense R-CHOP regimen. After multivariate analysis, high GSTP1 (p = 0.003) and TOPO2α (p = 0.02) gene expressions were associated with shorter overall survival and progression-free survival, respectively, suggesting that these genes may represent an unfavorable prognostic factor in the case of R-CHOP treatment. These biomarkers may be useful for selecting patients eligible for personalized chemotherapy after validation in an independent set.

Background & Aims Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. Methods Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. Results Foetal biliary tree cells proliferated clonogenically for more than 1month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. Conclusions Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo . These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life.

Introduction: Standard chemotherapy represented by the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) regimen is successful in about 60% of patients (pts) with diffuse large B-cell lymphoma (DLBCL). Pts who do not benefit from this treatment, due to the development of tumor drug resistance, have a very poor prognosis. Currently, knowledge on reasons of treatment related failures in DLBCL are scanty and predictive biomarker of response are largely unknown. We hypothesized that polymorphisms of gene involved in the pharmacokinetics and pharmacodynamics of drugs included in R-CHOP regimen may play a role in predicting the outcome in DLBCL pts.Thus, we designed a multicentre prospective pharmacogenetic trial aimed at identifying gene polymorphisms potentially predictive of drug efficacy/resistance in DLBCL pts treated with R-CHOP. An interim analysis on the first 80 enrolled ptswas planned and has been performed. Methods: The study included chemonaive DLBCL pts at various stages of disease candidate to an R-CHOP standard treatment. The Ethical Committee of each participating centre approved the pharmacogenetic protocol, and all pts signed a written informed consent. According to the aims of this interim analysis, the impact of single nucleotide polymorphisms (SNPs) on R-CHOP efficacy was evaluated by objective response (OR) rate at the end of treatment. The efficacy of R-CHOP was evaluated according to the Cheson criteria by performing standard hematochemical and instrumental (TC and FDFG-PET) tests and defining complete remission (CR), partial remission (PR), non response or progressive disease (PD). Genomic DNA wasextracted from peripheral blood of 80 pts. Twentysingle nucleotide polymorphisms (SNPs) from18candidate genes (ABCB1, ABCC1, ABCC2, ABCG2, CYBA, CYP2C9, FCGR2A, GSTP1, IL2, MLH1, NCF4, NQO1, NQO2, RAC2, TNF, TOP2A, TP53, TUBB)involved in pharmacokinetics and pharmacodynamics of R-CHOP (www.pharmgkb.org) have been analysed by a genotyping array based on Affimetrix methodology. Univariate analysis was performed to evaluate associations between polymorphisms and clinical/pathological characteristics or OR (Fisher exact test). Multivariate logistic regression analysis was performed to estimate adjusted odds ratios along with the corresponding 95% confidence intervals for the polymorphisms and OR. Results: Median age was 63 years. There were 37 men and 43 women. 47.5 % of pts were in stage I-II,52.5 % of pts in stage III-IV. 27.5% of ptshad bulky disease, 43.8 % of pts had involvement of extranodal site. 47.5% of pts had pathological LDH value. According to the revised IPI, 15 % pts were in the low risk group, 58.7 % in the intermediate risk group, and 26.3 % in the high risk group.Overall, 468 courses of R-CHOP had been administered (mean: 5.85 courses, range: 4-6). 81% of pts had CR to R-CHOP whereas the remaining showed PR (14%) or PD(5%). No statistically significant correlation was found between OR and clinical characteristics of pts.However, stage III-IV pts showed a worst OR than stage I-II pts (77% vs 87% of CR, respectively); pts with bulky disease had worst OR than non-bulky disease pts(73% vs 84.5% of CR, respectively); ptswith R-IPI 3-5 a worst OR than pts with R-IPI 0-2 (71.5% vs 85% of CR, respectively). Univariate and multivariateanalysis identified TOPOII rs13695as a predictor of OR (p=0.042). Pts with CT or TT genotypesshowed worst OR than CC wild-type homozygous pts (odds ratio 3.070, CI95% 1.113-13.457). Also, a statistical trend toward significance was observed for MLH1 rs1800734 polymorphism (p=0.062): ptswith homozygous genotype for the mutant allele showed a better OR than wild-type and heterozygous pt genotypes. Conclusions: No significant relationship between clinical/pathological characteristics and OR was observed. Our preliminary data show that SNPs affecting a gene involved in doxorubicin pharmacodynamics, i.e. the drug target TOPOII, as well asone of the major components of DNA mismatch repair, i.e. MLH1 gene,may predict response in DLBCL pts treated with R-CHOP. These preliminary results from the interim analysis are promising and warrant completion of pt accrual to reach the planned number of cases at the end of our study. Acknowledgments This work was supported by a grant from the Associazione Giacomo Onlus, Castiglioncello (LI), Italy to E.M. and Cassa di Risparmio di Firenze, Firenze, Italy to S.N. Disclosures No relevant conflicts of interest to declare.

Background and objectives Thymidylate synthase (TS) expression levels appear to be related to response to 5-fluorouracil-(5-FU)-based chemotherapy in colorectal cancer (CRC) patients. Three polymorphisms have been proposed as modulators of TS expression: a tandemly repeated sequence (2R/3R) in the 5′ UTR, a SNP (G > C) within the 3R allele and a 6 bp deletion in the 3′ UTR. To evaluate the influence of TS expression and polymorphisms on clinical outcome of 5-FU-treated patients we performed a comprehensive genetic analysis on 63 CRC patients. Methods TS expression levels were analyzed in normal and tumor tissues. TS coding sequence and UTR polymorphisms were investigated on DNA from normal tissue. LOH analysis was performed to determine tumor genotype. Results A difference in disease-free survival (DFS), although not statistically significant, was observed between high and low mRNA expression levels: patients with low levels showed longer DFS. The 2R2R genotype showed significantly lower expression than the 3R3R and 2R3R genotypes in normal tissue. No other TS polymorphism was associated with mRNA expression or clinical outcome. Conclusions The results obtained in this pilot study indicate that the number of 5′ UTR repeats is the major genetic determinant of TS expression. The lack of association with other polymorphisms might be partially explained by the existence of linkage disequilibrium in the TS gene. Our data support the growing evidence that TS control may require multiple mechanisms acting in close coordination with one another and suggest that TS genotyping alone in tumor samples is not sufficient to accurately predict response to 5-FU.

Colorectal cancer is the second leading cause of cancer-related deaths in the world. Despite many therapeutic opportunities, prognosis remains dismal for patients with metastatic disease, and a significant portion of early-stage patients develop recurrence after chemotherapy. Epigenetic gene regulation is a major mechanism of cancer initiation and progression, through the inactivation of several tumor suppressor genes. Emerging evidence indicates that epigenetics may also play a key role in the development of chemoresistance. In the present review, we summarize epigenetic mechanisms triggering resistance to three commonly used agents in colorectal cancer: 5-fluorouracil, irinotecan and oxaliplatin. Those epigenetic biomarkers may help stratify colorectal cancer patients and develop a tailored therapeutic approach. In addition, epigenetic modifications are reversible through specific drugs: histone-deacetylase and DNA-methyl-transferase inhibitors. Preclinical studies suggest that these drugs may reverse chemoresistance in colorectal tumors. In conclusion, an epigenetic approach to colorectal cancer chemoresistance may pave the way to personalized treatment and to innovative therapeutic strategies.

Background Polycystin-1 and -2 (PC-1 and PC-2) are critical components of primary cilia, which act as mechanosensors and drive cell response to injury. PC-1 activation involves the cleavage/processing of PC-1 cytoplasmic tail, driven by regulated intramembrane proteolysis or ubiquitine/proteasome, translocation in the nucleus and activation of transcription factors. Mutations of PC-1 or PC-2 occur in polycystic liver where cholangiocyte proliferation is enhanced. Aim We evaluated the involvement of PC-1 and PC-2 in modulating cholangiocyte proliferation. Methods We investigated rat cholangiocytes induced to proliferate by 17β-oestradiol. Proliferation was evaluated by PCNA immunoblotting or [3H]-thymidine incorporation into DNA. PC-1 silencing was performed by siRNA, while inhibition of regulated intramembrane proteolysis or proteasome by γ-secretase inhibitor, leupeptin or MG115. Results Cholangiocyte proliferation was associated with decreased PC-1 and PC-2 expression, which was inversely correlated with enhanced PCNA. The selective silencing of PC-1 induced activation of cholangiocyte proliferation in association with decreased PC-1 expression. Two different regulated intramembrane proteolysis inhibitors, γ-secretase-inhibitor and leupeptin, and the proteasome inhibitor, MG115, abolished the 17β-oestradiol proliferative effect. Conclusions PC-1 and PC-2 play a major role as modulators of cholangiocyte proliferation suggesting that primary cilia may act as sensors of cell injury driving, when activated, a proliferative cholangiocyte response to trigger the reparative processes.

Cholangiocarcinoma is commonly considered a rare cancer. However, if we consider the hepato-biliary system a single entity, cancers of the gallbladder, intra-hepatic and extra-hepatic biliary tree altogether represent approximately 30% of the total with incidence rates close to that of hepatocellular carcinoma, which is the third most common cause of cancer-related death worldwide. In addition, cholangiocarcinoma is characterized by a very poor prognosis and virtually no response to chemotherapeutics; radical surgery, the only effective treatment, is not frequently applicable because late diagnosis. Biomarkers for screening programs and for follow-up of categories at risk are under investigation, however, currently none of the proposed markers has reached clinical application. For all these considerations, cancers of the biliary tree system should merit much more scientific attention also because a progressive increase in incidence and mortality for these cancers has been reported worldwide. This manuscript deals with the most recent advances in the epidemiology, biology and clinical presentation of cholangiocarcinoma.

Thymidylate synthase (TS) intratumoural expression may be a prognostic marker and predict outcome of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer patients. The TS gene promoter enhancer region contains two different polymorphisms which can influence TS mRNA transcriptional and translational efficiency: a polymorphic tandem repeat sequence (2 or 3 repeats; 2R and 3R) and a single nucleotide polymorphism (SNP), G > C, within the second repeat of the 3R alleles. We studied the relationship between tumoural TS mRNA expression levels and TS gene polymorphisms in the colonic mucosa of 48 colorectal cancer patients. The 3R/3R genotype was characterised by higher TS mRNA levels in the tumour than the 2R/2R-2R/3R genotypes (P = 0.071). Regarding the relationship with the SNP polymorphism, a statistically significant difference in TS gene expression between the 3RG/3RG genotype and 2R/2R-2R/3RC-2R/3RG genotype subset was observed (P = 0.017). No statistically significant correlation was observed between experimental data and baseline clinical-pathological characteristics as well as clinical outcome in the relatively small patient series investigated. This is the first study reporting an association between the TS intra-repeat SNP and gene expression levels in colorectal cancer patients. These results suggest that in 3R/3R patients, the G > C polymorphism may be an important factor in determining TS mRNA expression levels, and warrant further investigation of the role of TS promoter polymorphisms as predictors of sensitivity to 5-FU-based chemotherapy in larger case series.

The putative tumour suppressor gene FHIT (fragile histidine triad) spans the common fragile site FRA3B, which is highly susceptible to breaks and deletions induced by genotoxic agents. Tumours associated with exposure to carcinogens, such as colorectal adenocarcinomas, should be particularly susceptible to alterations in the FHIT gene. We studied the frequency of FHIT alterations and their correlations with clinicopathologic features in sporadic colon carcinomas.FHIT expression was investigated by reverse transcription polymerase chain reaction in 56 primary sporadic colorectal carcinomas. The same tumours and matched normal tissues were also investigated for loss of heterozygosity by using two markers located inside the FHIT gene.Twenty-nine of 56 tumours (51.8%) expressed aberrant FHIT transcripts. Four tumours had absence or nearly undetectable levels of the normal-sized FHIT transcript. Sequencing analysis of the altered transcripts showed FHIT mRNA lacking one or more exons, more frequent deletions of exons 4-5-6 or 4-5-6-7-8. At the genomic level 46.4% (13 of 28) of the cases showed alterations involving FHIT locus. We did not find any correlation between FHIT gene alterations and clinicopathologic characteristics of the tumours.Since the FHIT gene is frequently altered, its role in the molecular pathogenesis of sporadic colon carcinoma deserves further investigation.

Abstract 2478 Diffuse large B cell lymphoma (DLBCL) is one of the most common types of non-Hodgkin's lymphoma. Approximately half of patients will be cured of their disease by primary therapy, including the R-CHOP regimen (rituximab, doxorubicin, cyclophosphamide, vincristine, desamethasone). The remaining die of the disease, mainly because of the occurrence of tumor drug resistance. Many efforts have been made to explain the biochemical and molecular mechanisms involved in resistance to the drugs used in the treatment of cancer patients, including those with DLBCL. A dose-intense therapy regimen (e.g. R-CHOP14) may help to improve the treatment outcome of DLBCL patients. We have carried out a retrospective study aimed at correlating the mRNA expression levels of genes involved in metabolism, mechanisms of action and resistance to doxorubicin (i.e. MDR1, GSTP1, TOPO-2a, Bcl-2, PKC-b2) that represents the backbone of the R-CHOP regimen with treatment outcome data of 54 patients at various stages of disease. The expression of the 5 above mentioned genes was determined in formalin-fixed paraffin-embedded samples from DLBCL using real time RT-PCR. A threshold analysis to identify a cut-off distinguishing recurrent or non-recurrent disease was used. The correlations between gene expression data and clinical/pathological characteristics as well as survival parameters have been evaluated by standard statistical tests. The case series included 32 males and 22 females; 6 patients had follicular lymphoma grade IIIb and 48 diffuse large B cell lymphoma; 19 presented symptoms at diagnosis. Thirty patients showed abnormal LDH values, the IPI was intermediate-high risk or high risk in 14 patients. Forty-six patients (85.2%) obtained a complete remission and 8 (14.8%) a partial response. The median overall survival (OS) as well as the median progression free survival (PFS) have not yet been reached after a median follow-up of 43.6 months. The mRNA expression levels of TOPO-2a and GSTP1 were detectable in all samples, that of PKC-b2 in 52 samples, that of MDR1 and bcl-2 in 34 and 29 samples, respectively. A high degree of interpatient variation in relative tumor expression of the study gene was observed: from 0.008 for TOPO-2a to >100.000 for PKCbII. Threshold analysis indicated significant inverse relationships between PKC-b2 and PFS (p=0.046): higher gene expression was associated with shorter PFS. Conversely, higher expression of ABCB1 was associated with prolonged PFS (p=0.039). This kind of analysis also showed associations between OS and TOPO-2a, GSTP1and PKC-b2: higher gene expression was associated with shorter OS. Overall, our results confirm that the high expression of some genes such as TopoIIa, GSTP1 and PKCβII may represent a prognostic factor in case of an intensified anthracycline-based chemotherapy with immunotherapy. Moreover, our results suggest that intensified immunochemotherapy could affect the role of bcl2, ABCB1, GSTP1 and TopoIIa in predicting tumor response. These results and others from related studies may help to identify gene profiles useful for selecting patients eligible for more intensified or personalized chemotherapy. Prospective larger studies are warranted. Supported by a grant from Associazione Giacomo Onlus, Castiglioncello (LI). Disclosures: No relevant conflicts of interest to declare.

Gastric cancer is one of the leading causes of cancer-related deaths worldwide, although the incidence has gradually decreased in many Western countries. Two main gastric cancer histotypes, intestinal and diffuse, are recognised. Although most of the described genetic alterations have been observed in both types, different genetic pathways have been hypothesized. Genetic and epigenetic events, including 1q loss of heterozygosity (LOH), microsatellite instability and hypermethylation, have mostly been reported in intestinal-type gastric carcinoma and its precursor lesions, whereas 17p LOH, mutation or loss of E-cadherin are more often implicated in the development of diffuse-type gastric cancer. In this review, we summarize the sometimes contradictory findings regarding those markers which influence the progression of gastric adenocarcinoma.

Background and Aims: Vector-free approach developed by Gunther using PCR amplified full-length HBV genomes is one of the best in vitro system in reproducing the in vivo infection conditions. However, a major limitation of the method is the low level of intracellular HBV-DNA production. We investigated the reason(s) for this low efficiency. Methods: We analysed the replication fitness of HBV isolates from 11 untreated acute (3) or chronic (8) hepatitis B patients. Following Gunther’s method, the amplicons obtained by full-length PCR were cut by SapI to obtain linear HBV-DNA monomers competent for intracellular circularization upon transfection into HuH7 cells. Results: Seven (64%) of the 11 genome tested were replication competent in our system. Upon SapI cutting it was not possible by gel electrophoresis to distinguish digested from undigested HBVDNA, as both migrate with the same molecular weight. Direct sequencing of the region encompassing the catalytic site showed that in 19/20 experiments the majority of the HBV-DNA treated by SapI was undigested. Uncut HBV-DNA is considered unable to initiate replication because the Basic Core Promoter and Precore regions remains isolated. Since only a minority of the input DNA was replication competent, we hypothesized that this may be a reason for the low efficiency of the Gunther’s method. To overcome this limit we performed ligase treatment after SapI digestion obtaining the circularization of the monomer in vitro, then the fulllength circular-closed form was separated by gel electrophoresis for transfection. Densitometric quantification showed that in vitro circularization produced 4 times more replicative intermediates than the Gunther’s method. Furthermore, replicative intermediates were detected earlier in cells transfected with circular-closed monomers than with Gunther’s method (24 vs 48 hours). Conclusions: The low HBV replication efficiency of the Gunther’s method can be significantly improved by ligase treatment of the SapI digested full-length HBV-DNA prior transfection.

Cholangiocarcinoma (CCA) is a malignant tumour that arises from biliary epithelium at any portion of the biliary tree. CCA is currently classified as intra-hepatic or extra-hepatic CCA (EH-CCA). Recent evidences suggest that intra-hepatic CCA (IH-CCA) and EH-CCA are biologically different cancers, giving further support to a number of recent epidemiological studies showing large differences in terms of incidence, mortality and risk factors. The purpose of this manuscript is to review recent literature dealing with the descriptive epidemiology and risk factors of CCA with a special effort to compare IH- with EH-CCA.

Il lavoro costituisce un commento all'art. 46 del Testo unico degli enti locali, dedicato all'elezione del sindaco e del presidente della provincia nonché alla formazione della giunta.

Il lavoro costituisce un commento all'art. 74 del Testo unico degli enti locali, dedicato all'elezione del presidente della provincia, tenendo conto anche dell'elezione del sindaco metropolitano.